Book of Abstracts - Divisione di Spettrometria di Massa

Book of Abstracts

MASSA 2022

20-22 giugno 2022 – Carlentini (SR)

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Committees

Scientific Committee

Gianluca Bartolucci University of Florence

Giuliana Bianco University of Basilicata

Cecilia Bergamini ARPAE Bologna

Donatella Caruso University of Milano

Riccardo Flamini CREA-VE, Conegliano (TV)

Roberta Galarini IZS Umbria e Marche, Perugia

Gianluca Giorgi University of Siena

Emanuela Gregori Ist. Sup. Sanità, Roma

Valentina Lazazzara E. Mach Foundation, S. Michele all'Adige (TN)

Fulvio Magni University of Milano Bicocca

Giorgio G. Mellerio University of Pavia

Paola Montoro University of Salerno

Luciano Navarini Illy caffè, Trieste

Organizing Committee

Salvatore Guccione

University of Catania

Rosario Pignatello University of Catania

Milena Rizzo University of Catania

Francesco Paolo Bonina University of Catania

Matteo Pappalardo University of Catania

Carmelo Puglia University of Catania

Calogero Statella Tenuta delle Terre Nere

Cristina Tomasella University of Catania

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MASSA 2022 is patronaged by:

MASSA 2022 is kindly supported and sponsored by:

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Scientific Program

MONDAY, JUNE 20, 2022

2:00 p.m. Registration

3:00 p.m. Opening remarks and greetings from the Rector (University of Catania)

Applicazioni della spettrometria di massa per lo studio ed il controllo dei materiali

polimerici

Chairs: Luciano NAVARINI &

Riccardo FLAMINI

3:30 p.m. PL1 Soft MS of synthetic polymers: a focus on bioplastics characterization

Sabrina Carola Carroccio (CNR-IPCB - Catania)

4:10 p.m. KN1

High resolution-mass spectrometry for safety assessment of food packaging

materials

Federica Bianchi (University of Parma)

4:40 p.m. OR1

TGA-GC-MS and MALDI-TOF mass spectrometry: two promising tools for

the characterization of ultrathin polymer films

Riccardo Chiarcos (University of Piemonte Orientale)

4:55 p.m. OR2

Optimization of polymer processing for the reduction of volatile organic

compounds (VOCs)

Francesco Prandi (University of Bologna)

5:10 p.m. Consiglio direttivo della DSM

7:30 p.m. Welcome party

TUESDAY, JUNE 21, 2022

La spettrometria di massa per lo studio di sostanze naturali

Chairs: Paola MONTORO &

Giuliana BIANCO

9:00 a.m PL2

Lipidomic analysis in food: The role of a detailed elucidation of intact lipids in

functional foods for investigating on nutritional aspects

Paola Dugo (University of Messina)

9:40 a.m OR3

Metabolomics in the study of Foeniculum vulgare. From the characterization

of bioactive compounds to cell metabolomics studies for the confirmation of

their activities

Maria Assunta Crescenzi (University of Salerno)

9:55 a.m OR4

Untargeted metabolomics approach to understand grapevine communication

mediated by volatile organic compounds against downy mildew

Sara Avesani (University of Trento)

10:10 a.m OR5

The development of a manually curated database of the metabolic proteins of

Triticum aestivum (TriMet_DB)

Antonella Di Francesco (University of Catania)

10:25 a.m OR6 LC-MS for the Analysis of Psychoactive Substances in Archaeological Finds

Flaminia Vincenti (Sapienza University of Rome)

10:40 a.m Coffee Time

11:00 a.m OR7

Determination of Carbazole Alkaloids in Murraya Koenigii leaves by means

LC-MRM/PI/IDA/EPI analysis

Eduardo Viteritti (University of Teramo)

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11:15 a.m OR8

Comprehensive two-dimensional liquid chromatography coupled to mass

spectrometry for determination of the polyphenolic profile of food and natural

products

Francesco Cacciola (University of Messina)

11:30 a.m

OR9

Quantification of glyphosate in milled and brown rice in LC-ICP-MS/MS

Paolo Scardina (Agilent Technologies Italia S.p.A.)

11:45 a.m OR10

Raw materials in food manufacturing: a complexity ascertained by high-

resolution mass spectrometry.

Maria Assunta Acquavia (University of Basilicata)

12:00 p.m. OR11

Use of native-mass spectrometry to elucidate the oligomeric state and

substrate-binding affinity of FrlB, a bacterial deglycase

Angela Di Capua (The Ohio State University, Columbus, OH, USA)

12:15 p.m. OR12

Assaying vegetable specimens by FT-ICR mass spectrometry, an election

technique in metabolomic studies

Alba Lasalvia (Sapienza University of Rome)

12:30 p.m. Lunch and Poster session

La spettrometria di massa nelle Scienze della vita

Chairs: Fulvio MAGNI &

Donatella CARUSO

2:00 p.m. PL3 Analytical tools for metabolomics and applications

Giuseppe Paglia (University of Milano-Bicocca)

2:30 p.m. OR13

Proteomic profile of extracellular vesicles secreted by astrocytes using high

resolution mass spectrometry

Maria Gaetana Pittalà (University of Catania)

2:45 p.m. OR14

Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS)

Profiling of Commercial Enocianina and Evaluation of Their Antioxidant and

Anti-Inflammatory Activity

Giovanna Baron (Università degli Studi di Milano)

3:00 p.m. OR15 Emerging per- and polyfluoroalkylated substances (PFASs) in wild boar liver

Simone Moretti (IZS of Umbria and Marche “Togo Rosati”- Perugia)

3:15 p.m. OR16

Plasma proteome investigation of COVID-19 patients with different outcomes

through an untargeted label-free LC-MS/MS approach

Lisa Pagani (University of Milano-Bicocca)

3:30 p.m. OR17

A novel Spatial Multi-omics mass spectrometry imaging workflow to assist

clinical investigations

Vanna Denti (University of Milano-Bicocca)

3:45 p.m. Coffee Time

4:05 p.m. OR18

The detection of new products by HPLC-HRMS validates a novel stoichio-

kinetic model for the reaction between the 2,2-diphenyl-1-picrylhydrazyl

radical (DPPH•) and common antioxidants

Lucrezia Angeli (University of Bolzano)

4:20 p.m. OR19

MS-based approaches in drug development: elucidation of RNA-ligand-

protein interactions for the investigation of bis-3-chloropiperidines targeting

TAR

Alice Sosic (University of Padova)

4:35 p.m. OR20

Discovery and Characterization of a novel Lipid Class via LC-ESI-MS & MSn

analysis: the case of Acyl-Glucuronosylglycerols

Giovanni Ventura (University of Bari)

4:50 p.m. OR21

Quali-quantitative determination of toxic carbonyl compounds in the emissions

of both heated tobacco products (HTP) and conventional cigarettes

Simone Ronsisvalle (University of di Catania)

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5:05 p.m. Assemblea soci DSM

8:30 p.m. Social dinner

WEDNESDAY, JUNE 22, 2022

50 anni della DSM

Chair: Gianluca GIORGI

9:00 a.m. PL4 The first 50 years of GSM – DSM

Giorgio Giacomo Mellerio (University of Pavia)

9:30 a.m. KN2 Mass Spectrometry in Sicily: a Historical Overview

Leopoldo Ceraulo (University of Palermo)

10:00 a.m. Coffee Time

Validazione, qualità del dato e miscellanea

Chairs: Cecilia BERGAMINI &

Roberta GALARINI

10:20 a.m. PL5

Current state and future perspective on validation of Mass Spectrometry

methods.

Marios G. Kostakis (EURACHEM, University of Athens, Grece)

11:00 a.m. OR22

Development and validation of a rapid and simple analytical method for the

simultaneous analysis of pyrrolizidine alkaloids and related N-oxides in

beehive products by salting out assisted liquid liquid combined with LC-

tandem mass spectrometry

Rita Celano (University of Salerno)

11:15 a.m. OR23

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) in honey by LC-Q-

Orbitrap: method development and validation study

Carolina Barola (IZS of Umbria and Marche “Togo Rosati”- Perugia)

11:30 a.m. OR24

Material screening of electronic devices by ICP-MS and HR-ICP-MS: towards

a low background for astroparticle physics experiments

Francesco Ferella (Gran Sasso National Laboratories, L'Aquila)

11:45 a.m. OR25

Different applications of the Liquid Chromatography coupled to Isotope Ratio

Mass Spectrometry (LC-IRMS)

Matteo Perini (Fondazione Edmund Mach, San Michele All’Adige-TN)

12:00 p.m. OR26

Validation of analytical method for the determination of cortisol in hair by

UHPLC-MS/MS

Chiara Maccari (University of Parma)

12:15 p.m. OR27

Fast and sensitive high-throughput antineoplastic drugs surface contamination

monitoring by ultra-high performance liquid chromatography coupled with

tandem mass spectrometry.

Donato Squillaci (University of Firenze)

12:30 p.m. OR28 Rapid analysis and interpretation of metabolomics SWATH acquisition

Mario Armelao (SCIEX Italia)

12:45 p.m. Closing remarks

1:00 p.m. Lunch

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ORALS

Applicazioni della spettrometria di massa per lo studio ed il controllo dei

materiali polimerici

Chairs: Luciano NAVARINI & Riccardo FLAMINI

PL1

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Soft MS of synthetic polymers: a focus on bioplastics characterization

S. C. Carroccio

CNR-Institute for Polymers Composites and Biomaterials (IPCB), Via P. Gaifami 18, 95126 Catania (Italy)

Summary: The impressive development in the characterization of polymeric materials, and specifically

Bioplastics, introduced by the discovery of MALDI TOF Mass Spectrometry, were herein highlighted. In

particular, molar masses determination and distribution, structural changes, and degradation phenomena

involved during the lifetime of bioplastics were taken into account and discussed.

Keywords: Mass Spectrometry, MALDI-TOF, Bioplastics characterization

1. Introduction In the last two decades, soft mass Spectrometry and

in particular matrix-assisted laser

desorption/ionization time-of-flight (MALDI-

TOF) transformed the practice of polymer

characterization in all-polymer chemistry labs

since MS was no longer restricted to the analysis of

small molecules. Soft MS is applicable to

determine average molar masses (MMs), structural

changes, and all types of degradation phenomena

involved in a polymer lifetime, starting from their

synthesis to the final disposal.

In this talk, salient aspects of MALDI

characterization in the field of bioplastics will be

reported and discussed. A focus on measurable

parameters, not achievable from traditional MS,

such as, the chemical formula, architectures, MMs,

and chain end groups, will be evidenced by

considering bioplastics materials.

Finally, the advancement obtained by using

MALDI in determining properties of bioplastics

for applications such as processing, in-service and

final disposal will be reported. Especially the

benefits achievable by applying MALDI-TOF to

the study of polymers degradation phenomena are

impressive. The study involves the collection of

MALDI spectra of samples subjected to external

stimuli, to observe the structural changes induced

by them, relating the latter with the properties of

degraded materials.

If compared with previous studies based on

conventional techniques, the results obtained for

bioplastics analyzed by MALDI are highly

informative, yielding precise information on the

size, structure, and end groups of molecules

originating during the exposure to the external

factors

Figure 1. MS analysis of macromolecular products derived from degradation processes

Reference

1. P. Rizzarelli, S. Carroccio; Analytica Chimica Acta, 808 (2014), pp 18-43.

KN1

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High resolution-mass spectrometry for safety assessment of food packaging

materials

F. Bianchi, N. Riboni, M. Mattarozzi, A. Cavazza, M. Careri

University of Parma, Department of Chemistry, Life Sciences and Environmental Sustainability, Parco Area delle

Scienze 17/A, 43124 Parma, Italy

Summary: An overview of different high resolution mass spectrometry-based approaches for the analysis of

contaminants from food contact materials is presented

Keywords: High resolution-mass spectrometry; Food contact materials; Targeted and untargeted analysis

1. Introduction

Safety of materials in contact with food is an issue

of great concern since different chemicals could

migrate from packaging into food, thus raising

health concern and/or producing changes on both

taste and odour of foodstuffs. Both intentionally

added substances among which antioxidants,

stabilizers, processing aids and non-intentionally

added substances (NIAS) [1] can be released from

the packaging material, compromising food safety.

In this context, accurate and sensitive analytical

methods and a harmonized system for their

application in analytical laboratories are required.

Migration tests are required in order to guarantee

consumers’ safety, food preservation and

organoleptic properties.

Gas chromatography-mass spectrometry and more

recently liquid chromatography coupled to high-

resolution mass spectrometry (LC-HRMS) have

been successfully applied for the determination of

intentionally and non-intentionally-added

compounds both from extracts and directly from

the packaging materials. In particular, HRMS has

advanced as the most powerful tool for the

unambiguous determination of the investigated

analytes [2,3]. In this context, untargeted strategies

represent a major challenge for food safety

assessment, being able to provide a comprehensive

fingerprinting of the packaging material as well as

the identification of new markers [4,5].

Rapid HRMS methods have also been devised for

screening the presence of contaminants released

from food packaging. Taking into account that

sample treatment could affect material integrity

leading to degradation of polymers along with the

release of additives and untargeted compounds, the

capabilities of ambient ionization sources coupled

to HRMS have been exploited for direct molecular

characterization of food contact materials with

minimal or no sample preparation, thus avoiding

chromatographic separation, reducing sample

handling, costs and analysis time [5,6].

Finally, MS imaging approaches, using properly

developed ionization surfaces [7,8], can enable

mapping of the spatial distribution of components

on sample surfaces, thus leading to a better

understanding of the localization of packaging

components for an improved product quality and

safety.

Examples related to the evaluation of the migration

of contaminants from existing or newly developed

materials, including alternative packaging derived

from substances of natural origin will be discussed.

Finally, the fundamental role of chemometrics for

data processing will be critically presented.

References

1. Commission regulation (EU) No 10/2011 of 14 January 2011 on plastic materials and articles intended to come in

contact with food. Official Journal of the European Commission.

2. P. Miralles, V. Yusà, Y. Sanchís, C. Coscollà, Molecules, 26 (2021) 7640.

3. C. W. Klampf, Trends Anal. Chem. 50 (2013) pp. 53–64.

4. A. Cavazza, C. Bignardi, M. Grimaldi, P. Salvadeo, C. Corradini, Food Res. Int. 139 (2021) 109959

5. M. Mattarozzi, M. Milioli, C. Cavalieri, F. Bianchi, M. Careri, Talanta, 101 (2012) pp. 453-459.

6. A. Issart, S. Godin, H. Preud'homme, K. Bierla, A. Allal, J. Szpunar, Anal. Chim. Acta 1058 (2019) pp. 117

7. Q. Xu, R. Tian, C. Lu, Anal. Chem. 93 (2021) pp. 13703−13710.

8. A. Penna, M. Careri, N. D. Spencer, A. Rossi, J. Am. Soc. Mass Spectrom. 26 (2015) pp. 1311-131.

OR1

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TGA-GC-MS and MALDI-TOF mass spectrometry: two promising tools for the

characterization of ultrathin polymer films

R. Chiarcos, V. Gianotti, D. Antonioli, M. Laus

Università del Piemonte Orientale (UPO)

Summary: Two different approaches for the determination of molecular weights and chemical compositions

of polymers anchored by one end to inorganic substrates to form ultrathin films are here presented. A TGA

oven hyphenated with a GC-MS system is used in one case, whereas a MALDI-TOF spectrometer in the

other approach.

Keywords: Ultrathin films, polymers, mass spectrometry.

1. Introduction Ultrathin films consisting of synthetic polymers

anchored by one terminal to an inorganic substrate

are generally indicated as polymer brushes and are

currently employed in an extremely broad range of

applications, from antifouling surfaces to

dispersants for colloidal systems. The most

common approach to obtain polymer brushes is

named grafting to reaction and is based on the

reaction of polymers containing a reactive terminal

group with the substrate.

Recently, equimolar blends of two polymers with

the same chemical composition but different

molecular weights were also grafted to produce

ultrathin brushes [1]. The big challenge is to

determine the brush composition, i.e. the molar

fractions of the two polymers in the final brush, in

order to check if the lightest polymer reacts

preferentially with the substrate, as reported in

literature [2].

The hyphenated apparatus consisting of a

ThermoGravimetrical Analysis (TGA) oven and a

Gas Chromatography-Mass Spectrometry system

allows to determine the compositions of polymer

brushes obtained by grafting a blend of two

polymers, one of which partially deuterated.

Moreover, brush compositions were also indirectly

determined by analysing the ungrafted part of the

polymer blend by MALDI-TOF analyses [3]. In

fact, starting from an equimolar blend, a

preferential grafting of the lightest polymer

produces consequently a depletion of the same

polymer in the unreacted residue of the sample.

With this second approach the deuterium labelling

is not required.

In this talk, both the two approaches will be

discussed, using as a reference system a blend of

two hydroxy- terminated polystyrene samples.

2. Results A hydroxy-terminated deuterated polystyrene with

an average molecular weight of 5200 g/mol

(PSd5.2OH) and a hydroxy-terminated polystyrene

with an average molecular weight of 13000 g/mol

(PS13OH) were mixed together in an equimolar

blend (molPSd5.2OH/molPS13OH = 1). The blend was

spin-coated on a silicon oxide substrate (1 cm x 1

cm) to give a polymer film thick ~30 nm and the

grafting to reaction between the polymers and the

substrate was promoted by thermal annealing at

190° C for 2100 s. The unreacted molecules were

removed by toluene washes and collected for the

MALDI-TOF analyses. The thickness of the

grafted brush was determined by ellipsometry and

the sample was then analyzed by TGA-GC-MS.

In Figure 1a the scheme of the TGA-GC-MS

apparatus is reported [4]. The polymer brush is

annealed from 20 to 1100° C in the TGA oven to

promote the molecule degradation by

depolymerisation (the polymer is reduced to its

monomers). The monomers are then transferred to

the GC-MS using helium as gas carrier and injected

each minute. The MS is setted in the Single Ion

Monitoring (SIM) mode to acquire the signals

related to styrene (m/z = 104) and deuterated

styrene (m/z =112). The collected chromatograms

are reported in Figure 1b.

The areas of the chromatograms related to styrene

and deuterated styrene are indicative of the

monomer amounts. By combining these data with

the thickness of the grafted brush, it is possible to

calculate the brush composition as

molPSd5.2OH/molPS13OH = 1.56 ± 0.03. Considering

that the starting blend has a molar ratio of 1, the

enrichment of the lightest polymer is definitely

confirmed.

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Fig. 1. Scheme of the TGA-GC-MS apparatus (a). Superimposed chromatograms of styrene (red) and deuterated

styrene (blue) collected from the grafted brush.

A double check can be made by analysing the

unreacted molecules collected after the toluene

washes using MALDI-TOF mass spectrometry.

The toluene solution was dried under vacuum and

solubilized again in a matrix/cationizing agent

solution. In details, DCTB and AgTFA were used

as matrix and cationizing agent, respectively. In

Figure 2 an exemplary spectrum is reported. Since

the integral area of each polymer is related to its

amount and considering the decrease in sensitivity

related to the mass increase, the composition of the

unreacted residue of the grafting to reaction can be

calculated using an opportune calibration curve.

Fig. 2. MALDI-TOF spectrum of the unreacted residue. The integral areas related to the two components of the blend

are highlighted

From the compositional data of the unreacted

residue, the specular composition of the brush can

be determined. In the reported case a molar ratio of

molPSd5.2OH/molPS13OH = 1.54 ± 0.04 was obtained,

which is very similar to the direct result obtained

from the TGA-GC-MS analyses.

Finally, the MALDI-TOF approach was applied on

polystyrene blends without the requirement of

isotopic labelling.

References

1. D. Antonioli, R. Chiarcos, V. Gianotti, M. Terragno, M. Laus, G. Munaò, G. Milano, A. De Nicola, M. Perego;

Polym. Chem., 12 (2021), pp 6538-6547.

2. L. Michalek, K. Mundsinger, C. Barner-Kowollik, L. Barner; Polym. Chem., 10 (2019), pp 54-59.

3. R. Chiarcos, D. Antonioli, V. Ospina, M. Laus, M. Perego, V. Gianotti; Analyst, 146 (2021), pp 6145-6155.

4. V. Gianotti, D. Antonioli, K. Sparnacci, M. Laus, T. J. Giammaria, M. Ceresoli, F. Ferrarese Lupi, G. Seguini, M.

Perego; J. Chromatogr. A, 1368 (2014), pp 204-210

OR2

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Optimization of polymer processing for the reduction of volatile organic

compounds (VOCs)

F. Prandi, D. Caretti

Department of Industrial Chemistry “Toso Montanari”, University of Bologna,

Viale del Risorgimento 4, 40136 Bologna (Italy)

Summary: Control of volatile organic compounds (VOCs) for the development of sustainable packaging

Keywords: VOCs, processing, food packaging

1. European regulations and optimization of analytical techniques The EU circular economy Action Plan lays the

foundations to a new plastics economy, where the

design and production of polymers and plastic

products fully respect reuse, repair and recycling

needs and more sustainable materials are

developed and promoted. In this context, it is very

important to investigate the aspect of volatile

pollutants in polymeric materials for food

packaging production.

According to the most recent European directive 1,

any organic compound with a boiling temperature

lower than 250°C at atmospheric pressure must be

considered as a volatile organic compound (VOC).

These substances can migrate from the matrix in

which they are contained, such as food packaging.

In the case of food contact materials, regulation

(EC) 1935/2004 2 provides a harmonized EU legal

framework and establishes the general principles of

safety and inertness for all materials intended to

come in contact with food. Therefore, the design of

any product that will have such use must be

suitable to minimize the release by the polymeric

material of all the substances mentioned in the

European directives.

In food contact materials, it is particularly

important to monitor the presence of non-

intentionally added substances (NIAS).

NIAS can enter the supply chain of FCMs/FCAs at

any level, e.g., during chemical syntheses of raw

materials as well as manufacture, transport, and

recycling. NIAS thus have different origins and can

be grouped into by-products, degradation products

and contaminants.

We investigated several methods of VOCs

extraction and gas chromatographic analysis for

quali-quantitative studies of these compounds.

Specifically, GC-MS analysis was performed on

pristine and recycled HDPE samples subjected to

headspace extraction, analyzing volatiles

substances.

2. Development of sustainable processing In this study, the compression moulding process (a

technology developed by SACMI Imola S.C.) was

evaluated from the point of view of volatile organic

compounds profile. Compression moulding offers

several advantages, including a low extrusion

temperature and low mechanical stress applied to

the polymeric material. Compression molding tests

were conducted with pristine and recycled HDPE,

and then polymeric materials were characterized to

investigate the initial composition of VOCs and the

effect of process parameters on these pollutants.

The results show that the passage in extruder, in the

condition used for subsequent compression

moulding, implies a decrease in volatile

compounds, and the subsequent compression

molding increases this effect. The process

examined leads to a high rate of devolatilization,

particularly of compounds that exhibit a low odor

detection threshold.

References

1. Directive 2004/42/CE of the European Parliament and of the Council of 21 April 2004 on the limitation of emissions

of volatile organic compounds, EUR-Lex, Document 32004L0042F. Takens, D. Rand and L.S. Young editors,

Dynamical Systems and Turbulence, Warwick 1980, pp 36-38.

2. Regulation (EC) No 1935/2004 of the European Parliament and of the Council of 27 October 2004 on materials and

articles intended to come into contact with food, EUR-Lex, Document 32004R1935

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ORALS

La spettrometria di massa per lo studio di sostanze naturali

Chairs: Paola MONTORO & Giuliana BIANCO

PL2

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Lipidomic analysis in food: The role of a detailed elucidation of intact lipids in

functional foods for investigating on nutritional aspects

P. Dugo1, F. Rigano1, D. Donnarumma1, L. Mondello1,2,3

1 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy

3 Chromaleont s.r.l., c/o Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University

of Messina, Messina, Italy 4 Department of Sciences and Technologies for Human and Environment, University

Campus Bio-Medico of Rome, Italy

Keywords: lipidomic analysis; functional foods; linear retention indices

1. Introduction The growing demand in natural matrices,

representing a source of dietary and nutraceutical

molecules, placed, as direct consequence, the

urgent need for the development of suitable

analytical methods able to provide a

comprehensive characterization of both

“conventional” and “unconventional” products. In

the last decades, lipidomic has emerged as a

cutting-edge approach among omics- techniques,

since lipids revealed to be essential molecules in

the regulation of metabolic pathways. To this

regard, the content of essential fatty acids (EFAs),

as well as nutritional indices such as the levels of

omega-3 and omega-6 FAs and their ratio are

essential parameter to evaluate the beneficial

properties of food products. In addition, the

investigation of complex lipids in their native

forms is proved to be crucial to obtain additional

information about lipids role and on FA

arrangement into each species.

2. Results For this reason, the present study is aimed to a

detailed elucidation of intact lipids in different

functional foods, including the profiling of

microalgae, hemp products and the wastes of

the fish industry.

Phospholipids and triacylglycerols were the

most representative lipid classes. However,

mono- and diacylglycerols, pigments and

carotenoids were also detected, representing

an added value for the investigated matrices.

From an analytical point of view, the use of a

recently introduced linear retention index

(LRI) approach [1] in LC paved the way for

the automatization of the identification

process in LC. Furthermore, the use of high

resolution chromatographic techniques (that is

UHPLC), even combined with selective

tandem MS operation mode allowed for the

determination of the entire lipidome with high

sensitivity. Finally, the UHPLC-MS/MS

platform was coupled to a preparative

workstation to fully automatize the analytical

work-flow [2]. This also entailed the

miniaturization of the lipid extraction

procedure, which was compared with a

conventional manual procedure, resulting in

quite similar quali-quantitative profiles.

References 1. F. Rigano, M. Oteri, M. Russo, P. Dugo, L. Mondello; Analytica Chemistry, 90 (2018), pp 3313-3320.

2. F. Rigano, P. Arena, D. Mangraviti, D. Donnarumma, P. Dugo, P. Donato, L. Mondello, G. Micalizzi, Journal of

Separation Science, 44 (2021), pp 1571-1580.

OR3

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Metabolomics in the study of Foeniculum vulgare. From the characterization of

bioactive compounds to cell metabolomics studies for the confirmation of their

activities.

M. A. Crescenzi 1,2, G. D’Urso 1, S. Piacente 1, H. Gallart-Ayala3, J. Ivanisevic3 and P.

Montoro 1.

1 Department of Pharmacy, University of the study of Salerno, via Giovanni Paolo II, 132, I-84084 Fisciano, SA, Italy. 2 PhD Program in Drug Discovery & Development, Pharmacy Department, University of the Study of Salerno.

3 Metabolomics Platform, Faculty of Biology and Medicine, University of Lausanne, Switzerland.

Summary: Cultivation of F. vulgare produces huge amounts of waste. Nowadays alternative ways to reuse

them are sought, considering vegetable waste as a source of metabolites with important biological activities.

The aim of this work was to carry out a metabolic study of fennel waste extract and evaluate its biological

activities.

Keywords: by-product, bioactive compounds, metabolomic analysis.

1. Introduction Foeniculum vulgare belongs to the Umbelliferae

family. Its cultivation creates high amount of by-

products rich in metabolites with potential

biological activities such as antioxidant, apoptotic

and anti-inflammatory [1,2]. In this study, the

potential to retrain fennel waste into bioactive

metabolites is investigated following the workflow

of Figure 1.

2. Experimental LC-ESI/LTQOrbitrap/MS analysis of Tiziano

winter variety of fennel waste was used to analyse

the metabolite profiles of different parts (bulb,

leaves, stems, and little stems) and a Multivariate

Data Analysis was developed to discriminate

different parts of F. vulgare. To identify the fennel

waste part richest in phenolic compounds, a

quantitative analysis was set up by using Tandem

Mass Spectrometry in Multiple Reaction

Monitoring (MRM) mode, monitoring two products

of fragmentation, expanding the analysis to two

other varieties of different seasonality, Preludio

(summer) and Pegaso (spring). LC-ESI/LTQ-Orbitrap/MS chromatograms of

extracts of three varieties of F. vulgare were

subjected to chemometric analysis, Principal

Component Analysis (PCA), to have a better

interpretation of data and to also identify marker

metabolites for each variety.Once the plant

matrices were chemically characterized, the

antioxidant activity of fennel extracts was studied

using the TEAC and DPPH spectrophotometric

assays; subsequently, using a fluorometric assay,

we assessed the potential anti- inflammatory

activity of some metabolites present in fennel

evaluating the inhibition of COX-1 and COX-2

isoforms. A cell metabolomics analysis has been

performed to deepen the anti-inflammatory activity

of fennel waste. A targeted quantitative analysis

using UPLC interfaced to Q-Trap Mass

Spectrometry by using MRM (Multiple Reaction

Monitoring) was developed and then applied on

cells stimulated both with fennel extracts and with

metabolites isolated from F. vulgare, studying

alteration in sphingolipids and eicosanoids

metabolism

3. Results Fennel waste is rich in phenolic acids

(neochlorogenic acid and feruloyl quinic acid),

glycosylated and glucuronates flavonoids

(quercetin glucoside, quercetin glucuronide,

isorhamnetin glucuronide), phenylpropanoids

(coumaroyl quinic acid) and some oxylipins first

identified in F. vulgare [3].

Multivariate statistical analyses allowed to

identify marker metabolites both among the four

waste parts analysed (bulb, stem, little stem and

leaf) and also among the three different varieties

of fennel (Tiziano, Pegaso and Preludio). The

study showed that the leaf is the most promising

part among fennel waste, having a higher content

of active compounds and showing greater

antioxidant activity. The major metabolites of F.

vulgare were found to be inhibitors of the COX-

1 and COX-2 isomers. In particular, they have a

lower IC50 value for COX 2 than for COX-1.

Therefore, at certain concentrations the

metabolites of fennel waste resulted able to

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specifically inhibit the COX-2 isoform that is

activated in inflammatory processes, limiting so

the side effects that occur from the inhibition of

COX-1.

In vitro metabolic studies on cells would seem to

confirm the potential anti-inflammatory activity

of fennel waste; alterations in the metabolism of

compounds involved in inflammatory processes

were highlighted.

Fig. 1. Research workflow

4.Conclusions

Fennel waste studied with a metabolomic approach

has been found to contain biologically active

metabolites, including anti-inflammatory

compounds. suggesting that including it in the

human diet or as a nutraceutical product or as a

functional food can have beneficial effects on

human health.

References 1. S. B. Badgujar, V. V. Patel and A. H. Bandivdekar; Biomed Res Int, 2014, 842674.

2. C. Kure, J. Timmer and C. Stough; Frontiers in pharmacology, 2017, 8, 117-117.

3. M.A. Crescenzi, G. D’Urso, S. Piacente and P. Montoro; Foods, 2021, 10(8), 1893.

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Untargeted metabolomics approach to understand grapevine communication

mediated by volatile organic compounds against downy mildew

S. Avesani1,2,3, V. Lazazzara3, M. Oberhuber2, P. Robatscher2, M. Perazzolli1,3

1 Center Agriculture Food Environment (C3A), University of Trento, Via E. Mach 1, 38098 San Michele all'Adige,

Italy 2Laboratory for Flavours and Metabolites, Laimburg Research Centre, Laimburg 6, Pfatten (Vadena), 39040

Auer (Ora), Italy 3Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38098 San Michele

all'Adige, Italy

Summary: Volatile organic compounds (VOCs) play crucial roles in the communication of plants with other

organisms and are mediators of plant defense against pathogens. The objective of this work is to understand

the mechanism of grapevine communications mediated by VOCs against grapevine downy mildew (caused by

Plasmopara viticola), using an untargeted metabolomics approach.

Keywords volatile organic compounds, downy mildew, metabolomics, grapevine

1. Introduction Plants can produce a wide variety of volatile

organic compounds (VOCs), which can play

crucial roles in the regulation of plant responses

against pathogens1. Different modes of action

against phytopathogens have been attributed to

VOCs, such as induction of plant resistance and

direct inhibition of pathogen growth [1]. Recent

studies showed that the amount of some volatile

terpenes was higher in resistant than susceptible

grapevine genotypes upon Plasmopara viticola

inoculation, indicating their possible involvement

in resistance mechanisms against this pathogen

[2,3]. This work aims at identifying the metabolic

response of VOC-treated grapevine leaves and the

potential activation of VOC-mediated resistance

mechanisms using an untargeted metabolomics

approach.

2. Experimental Susceptible grapevine leaf disks were treated with

a pure terpene, or with water (control) at one day

before and one hour after Plasmopara viticola

inoculation (P. viticola-inoculated) or water

application (mock-inoculated). Downy mildew

severity was assessed six days post inoculation

(dpi) as a percentage of the leaf disk surface

covered by sporulation [3]. Leaf samples were

collected at two time points (one and six dpi) and

an untargeted metabolomics approach was

applied using ultra-high pressure liquid

chromatography- electrospray ionization - high

resolution- quadrupole-time of flight-mass

spectrometry (UHPLC-ESI-Q-TOF-MS) analysis

[4,5]. The Q TOF was operated in both positive

and negative ion modes [5]. Raw data were

processed using an in-house R script and

statistical analysis was performed with the

MetaboAnalyst online platform

(https://www.metaboanalyst.ca/MetaboAnalyst/ho

me.xhtml) [4]. Features with significant changes

in abundance were identified according to the

Volcano Plot analysis, using a t-test (adjusted p-

value ≤ 0.05) and a fold-change higher than two in

at least one comparison [6]. Putative annotations

and metabolic pathways of annotation features

were obtained by KEGG

(https://www.kegg.jp/kegg/) with Vitis vinifera as

a reference organism using the webserver

MassTRIX(http://masstrix3.helmholtzmuenchen.d

e/masstrix3/start.html)[4].

3. Results The assessment of disease severity confirmed that

the pure volatile terpene reduced downy mildew

severity on susceptible grapevine leaf disks.

Principal component analysis applied on the

features (specified by retention time and mass to

charge ratio) discriminated samples according to

VOC treatment and time point, indicating global

metabolite changes in VOC-treated leaf disks. The

annotation of selected features and the respective

metabolic pathways will highlight grapevine

defense responses activated by VOC treatments.

4. Conclusions The untargeted metabolomics approach will

identify the metabolic response of grapevine leaves

to pure VOCs and will help to improve the

knowledge of grapevine defense mechanisms

activated by VOCs against pathogens.

http://www.metaboanalyst.ca/MetaboAnalyst/ho

http://www.metaboanalyst.ca/MetaboAnalyst/ho

http://www.kegg.jp/kegg/)

http://masstrix3.helmholtzmuenchen.de/masstrix3/start.html

http://masstrix3.helmholtzmuenchen.de/masstrix3/start.html

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References

1. E. Quintana‐Rodriguez, A. T. Morales‐Vargas, J. Molina‐Torres, R. M. Ádame‐Alvarez, J. A. Acosta‐Gallegos,

M. Heil; Journal of Ecology, 103.1 (2015), pp 250-260.

2. A. Algarra Alarcon, V. Lazazzara, L. Cappellin, P.L. Bianchedi, R. Schuhmacher, G. Wohlfahrt, I. Pertot, F.

Biasioli, M. Perazzolli; Journal of Mass Spectrometry, 50.8 (2015), pp 1013-1022.

3. V. Lazazzara, C. Bueschl, A. Parich, I. Pertot, R. Schuhmacher, M. Perazzolli; Scientific reports, 8.1 (2018), pp

1- 14.

4. M. Adrian, M. Lucio, C. Roullier-Gall, M. C. Héloir, S. Trouvelot, X. Daire, B. Kanawati, C. Lemaître-Guillier,

B. Poinssot, R. Gougeon, P. Schmitt-Kopplin; Frontiers in Plant Science, 8 (2017), p 101.

5. K. Billet, M. A. Malinowska, T. Munsch, M. Unlubayir, S. Adler, G. Delanoue, A. Lanoue; Plants, 9.8 (2020),

p 1008.

6. S. Krishnan, A. T. Queiroz, A. Gupta, N. Gupte, G. P. Bisson, J. Kumwenda, K. Naidoo, L. Mohapi, V. Mave,

R. Mngqibisa, J. R. Lama, M. C. Hosseinipour, B. B. Andrade, P. C. Karakousis; Frontiers in immunology, 12

(2021) p 2060.

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The development of a manually curated database of the metabolic proteins of

Triticum aestivum (TriMet_DB)

A. Di Francesco1, A. Cucina1, M. G. G. Pittalà1, A. Lanzoni1, C. Mills2, R. Saletti1, S.

Foti1, V. Cunsolo1

1 Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania,

Viale A. Doria 6, 95125, Catania, Italy 2Manchester Institute of Biotechnology, University of Manchester, Manchester M13 9PL, UK

Summary: We report the development of a manually-curated database, the TriticuMet Database, of the

metabolic proteins of Triticum aestivum. This database was used to interpret mass spectrometry data acquired

on the metabolic proteins extracted from the T. aestivum genotype MEC.

Keywords: Proteome analysis, high resolution mass spectrometry, manually-curated database

1. Introduction It is well known that MS-based approaches require

a functionally annotated genome of the specie

under investigation and curated and not redundant

protein sequence databases. The first draft

sequencing of the entire genome of common wheat

(Triticum aestivum), has been completed only

recently and the genomic data have not been

converted into the corresponding protein sequences

[1]. Consequently, because the lacking of

exhaustive and complete database, the MS-based

proteomic studies aimed to the identification of

wheat proteins, their content and distribution, have

encountered many difficulties [2]. On the light of

this evidence, many researchers have moved to

develop exhaustive manually-curated databases to

support proteomic studies. As example, recently, a

manually-curated database (GluPro V1.0) of gluten

proteins, comprising 630 unique full-length protein

sequences that are representative of the different

types of gliadin and glutenin components, has been

compiled [3]. In this context, we report the

development of a manually-curated database,

TriMet_DB, of the metabolic proteins of Triticum

aestivum. Then, the performance of the TriMet_DB

were evaluated in the identification of the

metabolic proteins extracted from of the Triticum

aestivum genotype MEC. The results were

compared with those obtained by searching MS

data against the Swiss-Prot.

2. Experimental Grain of the bread making wheat (Triticum

aestivum) cultivar MEC was grown at CREA-CI,

Foggia. 200 mg of wheat flour were extracted in 2

mL of extraction buffer (0.4 M NaCl, 0.067 M

K2HPO4, pH 7.6). The insoluble fraction was

spinned down, for 15 min at 4°C. The pellet

material was separated and the extraction

procedure was repeated twice. The supernatants

from these extractions were pulled and the

concentration of the extract was determined by

fluorimetric assay using the Qubit Protein Assay

kit with the Qubit 1.0 Fluorometer (ThermoFisher

Scientific, Milan, Italy). Finally, 50 µg of each

sample were reduced, alkylated and digested by

porcine trypsin. Mass spectrometry data were

acquired on a Thermo Fisher Scientific Orbitrap

Fusion Tribrid® (Q-OT-qIT) mass spectrometer

(Thermo Fisher Scientific, Bremen, Germany) in

triplicate runs. MS data were processed using

PEAKS X de novo sequencing software (v. 10.0,

Bioinformatics Solutions Inc., Waterloo, ON

Canada). Data were searched against the manually-

curated TriMet database and against the SwissProt

database restricted to the reviewed T. aestivum

entries.

3. Results The aim of this work was to generate the first

version of an annotated, and non-redundant

resource of wheat (T. aestivum) metabolic protein

entries that would facilitate analysis by MS data. In

particular, to compile our database, we used, as

starting step, two groups of “query sequences”: i)

the 335 reviewed entries of the metabolic proteins

of T. aestivum present in the Swiss-Prot database

(release May 2021), and ii) the 484 proteins of the

species phylogenetically closest to T. aestivum,

identified in a recent work (Di Francesco et al.,

2019) in the metabolic fraction of wheat, by a

shotgun approach [4]. Each “query sequence” entry

was BLAST searched against the TrEMBL section

of Uniprot database to find as many unreviewed

wheat sequences as possible showing the highest

percentage of sequence identity respect to the

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“query sequence”. This step allowed to collected

many protein family (Pfam) clusters that were

checked to eliminate redundant, fragment, or

duplicate sequences. Finally, to compile the first

draft of the TriMet database, all Pfam clusters are

grouped in a FASTA file format that can be directly

read by programs used for mass spectrometry data

interpretation.

To evaluate the performance of the manually-

curated TriMet database, the MS data obtained by

a shotgun analysis of an extract of the metabolic

proteins of wheat (Triticum aestivum) flour of the

cultivar MEC were used. Then, the TriMet

database search results were compared with those

achieved by searching MS data against the T.

aestivum Swiss-Prot_DB. In detail, by searching in

the T. aestivum Swiss-Prot_DB 65 proteins were

identified. On the contrary, using the manually-

curated TriMet database, it was possible to identify

306 protein entries. Of which, 41 proteins were in

common to both the database searches, whereas 24

and 265 were exclusively identified by the T.

aestivum Swiss-Prot_DB and TriMet_DB,

respectively.

4. Conclusions One of the main purposes of many proteomics

analyses is to identify as many proteins as possible

with high quality. Therefore, protein sequence

databases play a fundamental role in the majority

of the currently applied mass-spectrometry-based

proteomics workflows. In this respect, wheat

proteomics is up to date limited because the first

draft sequencing of its entire genome has been

completed only recently, and even today an

exhaustive, well-annotated and complete database

of wheat proteins is lacking. Here we developed a

manually-curated database, named TriMet_DB, of

metabolic wheat (T. aestivum) proteins, containing

4232 entries. The approach used to compile the

manually-curated database permitted the

cataloguing (i.e. protein family classification, and

gene code attribution) of all the protein entries

selected, including the high number of hypothetical

and uncharacterized entries selected by the

TrEMBL section. By means MS-based shotgun

approach the TriMet_DB was tested. In particular,

it was possible to achieve a deeper description of

the protein content of the metabolic fraction

extracted by the bread making wheat (Triticum

aestivum) cultivar MEC, including the Gene

Ontology. Future work will be focused on

expanding the database to include other wheat

species such as Triticum durum, and to incorporate

data from currently available genome sequences of

Chinese Spring, used as standard for Triticum

aestivum, with the aim to facilitate the

identification and functional study of metabolic

proteins of wheat.

References

1. International Wheat Genome Sequencing Consortium (IWGSC); Science, 345 (2014), pp 1251788.

2. M. Rossignol, J. B. Peltier, H. P. Mock, A. Matros, A. M. Maldonado, J. V. Jorrín; Proteomics, 6 (2006), pp 5529-48.

3. S. Bromilow, L. A. Gethings, M. Buckley, M. Bromley, P. R. Shewry, J. I. Langridge, E. N. C. Mills; Journal of

Proteomics, 163 (2017), pp 67-75.

4. A. Di Francesco, R. Saletti, V. Consolo, B. Svensson, V. Muccilli, P. DeVita, S. Foti; Journal of Proteomics, 211

(2020), pp 103530.

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LC-MS for the Analysis of Psychoactive Substances in Archaeological Finds

F. Vincenti1,2, M. Pallotta1, A. Ciccola1, I. Serafini1, M. Croce1,2, G. Di Francesco1, C.

Montesano1, G. Favero3, R. Curini1, M. Sergi4

1Department of Chemistry, Sapienza University of Rome, 00185 Rome, Italy 2Department of Public Health and Infectious Disease, Sapienza University of Rome, 00185 Rome, Italy

3Department of Environmental Biology, Sapienza University of Rome, 00185 Rome, Italy 4Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo,

Italy

Summary: The use of psychoactive substances has been well known since ancient times. It’s known that some

populations used these substances during magical or religious rites. This work shows how the combination of

targeted and semi-untargeted investigations made possible to clarify the use of opium alkaloids by the Dauni

inhabitants.

Keywords: Opium Alkaloids, Cultural Heritage, Dauni

1. Introduction The curative and dangerous properties of many

natural psychoactive substances, , were well known

in antiquity; for example Papaver Somniferum, one

of the most ancient, has been already known since

the Sumerian civilization, and the extraction of

psychoactive substances from plants, in particular

opium, was known and practiced by ancient

Egyptians in Bronze Age, handed down to ancient

Greeks and reached up to the Romans, in which the

first cases of dependence are highlighted [1].

The use of these substances was linked to food and

medicine; moreover, it probably dominated the

rituals due to their symbolic meanings related to

fertility, healing, death.

A particular case is represented by Dauni,

inhabitants of the historical-geographical district of

northern Puglia, which in ancient times (with

Peucezia and Messapia) constituted the Apulia.

This civilization left us a rich ancient collection of

stelae and vases belonging to the VIII – VI century

BC. These ancient vases show pictorial and

morphological references that suggest the use of

opioids by Dauni, probably in a religious and

healing context.

Fig. 2. Urns from the Ceci Macrini private collection

The aim of the work is the development of a

method for the determination of opium alkaloids in

complex matrices, such us archaeological

ceramics, to find evidences of these theories,

combining forensics and cultural heritage through

mass spectrometry.

2. Materials and methods

Urns had been buried for years. Given the valuable

nature of the sample, a sampling technique less

invasive as possible have been implemented. If a

residue of the earth was present, a first sampling

was carried out by means of the grattage technique,

scraping away the residual earth without affecting

the urns itself. Secondly, two different samples

were performed with swab, alcoprep (a commercial

swab containing 2-propanol) and cotton pads

soaked in different solvents such as ethanol and

water. Given the viability of the sample, a versatile

analyte extraction and clean-up method has been

developed, the technique chosen is PLE followed

by dLLME clean-up [2]. The extract obtained was

analysed simultaneously in semi-targeted and

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untargeted modes. An MRM-IDA-EPI method was

developed using a Q-Trap 6500 triple quadrupole

instrument, the samples were analysed in semi-

targeted mode, focusing attention on the detection

of natural opium alkaloids. The untargeted analysis

was conducted using a Q-Exactive Orbitrap to

obtain the complete profile of all natural

compounds that might be present in the vessels.

3. Results and discussion

Even without reference analytical standards, thanks

to the creation and the application of a suspect

screening method based on MRM-IDA-EPI, it was

possible to carry out an initial screening of all the

samples. The method allows to obtain the low

resolution fragmentation pattern of the compounds.

To increase the information confidence, data

obtained from mass spectrometry were coupled

with a prediction study of the retention times of all

suspect analytes performed by a predictive model,

a Quantitative Structure Retention

Chromatographic Relationship (QSRR) one.

Simultaneously, the samples were analysed in high

resolution mass spectrometry with the aim of

obtaining a generic profile of all the compounds

possibly present in the sampled vessels. Carrying

out the two different types of surveys made it

possible to obtain the richest information possible.

The strengths of the two techniques have been

highlighted.

Thanks to the application of the presented method

several opium alkaloids were found in different

urns. In details, morphine, thebaine, quinine and

codeine were present in 7 different manufact. By

means of the untargeted analyses it was possible to

isolate different natural compound, such us

eucalyptol or roccellic acid, deriving from several

medicinal plant commonly used for the preparation

of infusions and oils.

4. Conclusion By applying the two different presented methods to

real samples with targeted and untargeted analyses,

the presence of hallucinogen and antipyretic

substances, such us morphine, codeine,

scopolamine quinine was verified.

Results obtained, allow us to confirm the

hypothesis according to which, the Daunia

population used the opium poppy, and also of the

plants of the Solenaceae and Chincona family,

during several religious and magical rituals [3].

Acknowledgment

The authors would like to thank Dr. Lorello

Macrini, Dr. Marco Macrini for allowing

investigations on the artifacts belonging to their

private collection and Dr. Laura Leone for the

historical and archaeological contribution provided

in order to complete the work

References 1. P. Nencini, Substance Use & Misuse 32(14), 1997, pp. 2111-9

2. F. Vincenti, et al. V. Andreu, Y. Picó, Journal of Chromatography A 1605, 2019, pp 360348

3. L. Leone, Stele daunie: semata funerari o statue votive, 2003, pp. 67–76

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Determination of Carbazole Alkaloids in Murraya Koenigii leaves by means LC-

MRM/PI/IDA/EPI analysis

E. Viteritti1, F. Eugelio1, E. Oliva1, F. Fanti1, S. Palmieri1, E. Bafile2, A. Pepe1, D.

Compagnone1, M. Sergi1

1University of Teramo, Faculty of Bioscience and Technology for Food, Agriculture and Environment, Teramo, Italy. 2Sintal Dietetics

Summary: Several biological activities of Murraya Koenigii are attributed to Carbazole alkaloids (CAs). An

LC-MS/MS method for simultaneous, quantification and putative identification of CAs was developed. The

analyses were performed by using predictive multi experiment approach, coupling multiple reaction

monitoring (MRM) and precursor product ion scan (PIS) with information-dependent acquisition (IDA) and

enhanced product ion scan (EPI).

Keywords: Carbazole alkaloids; HPLC-MS/MS; EPI.

1. Introduction Carbazole Alkaloids (CAs) represent an important

classes of natural products, as these compounds

showed various biological activities [1]. A large

number of carbazole alkaloids have been isolated

from higher plants of the Rutaceae family, and in

particular, from the genus Murraya [2]. In this

work, a sensitive method involving the use of High

Performance Liquid Chromatography (HPLC)

coupled to a hybrid Triple Quadrupole-Linear Ion

Trap (LIT-QqQ) mass spectrometer was

developed. A putative identification and

simultaneous quantification was provided for CAs

on the leaves of Murraya Koenigii. This method

was performed using a combination of predictive

multi experiment approach coupling multiple

reaction monitoring (MRM), precursor ion scan

(PI), information-dependent acquisition (IDA) and

enhanced product ion scan (EPI). An extraction and

clean-up procedure was also developed to obtain

good recoveries of all analytes (from 60% to 85%)

by solid-phase extraction (SPE).

2. Experimental The samples were analyzed by two different

acquisition modes in a single run: MRM/PI/IDA-

EPI. The MRM acquisition mode, coupled with

IDA-EPI experiment, allowed the quantitative

analysis for analytes, whose standards were

available; moreover, it allowed the putative

identification of 11 more CAs and their isomers,

whose MRM transitions were generate by means of

in silico experiments using the Competitive

Fragmentation Modeling for Metabolite

Identification (CFM-ID) [3,4] and added to create

the inclusion list. The IDA method was then used

to trigger the EPI scans by analyzing MRM signals,

so unknown compounds were confirmed by

matching dependent MS/MS spectra acquired in

EPI mode with the literature or fragment prediction

made by CFM-ID. The PI-IDA-EPI approach

allowed to find and predict, in a single acquisition,

the presence of further compounds not included in

the MRM list, but with similar structure. In this

work we applied an IDA approach with 2 survey

scans (MRM+PIS) and an EPI experiment as a

dependent scan. The use of PIS as survey scan

greatly helped to extend the number of ions to

include in the IDA criteria and therefore in EPI

experiment, so widening the number of potential

candidates for the putative identification in the

selected matrices.

3. Results The proposed analytical method allowed to

perform a qualitative and quantitative analysis of

the CAs in Murraya Koenigii leaves. Targeted

analysis using the MRM-IDA-EPI acquisition

mode provided a sensitive and robust quantitative

analysis on targeted compounds with LOQs

between (4.2 and 12.7 pg mg-1); semi-untargeted

analyses with MRM-IDA-EPI scan mode was used

to confirm the standard analytes and to investigate

their isomers and the analogs of CAs, whose peaks

have been obtained by means of the in silico MRM

transitions. The acquisition of the EPI spectra of

the following analytes was used to propose a

hypothetical identification. For example, the MRM

transitions optimized for Koenigicine standard,

showed peaks at RT 5.84 min and 6.27 during

samples acquisition. The peak at RT 6.27

corresponds to Koenigicine, identification

confirmed through the comparison with the

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analytical standard. The EPI spectrum of this peak

was acquired and matched with the spectrum of

Koenigicine reported in literature [5]. It was

possible to confirm the fragments m/z: 309.0,

293.0; 282.0; 278.0; 268.0; 238.0; 210.0; 167.0;

from the fragmentation pattern it is possible to

hypothesize that the compound is Koenigicine. The

other peak at RT 5.84 has been hypothesized to

belong to Mukonicine, an isomer of Koenigicine

which differs by the position of a methoxyl group.

From the analysis of the EPI spectrum, which was

compared with the MS/MS spectrum predicted by

CFM-ID, a fragmentation pattern similar to that of

Koenigicine is evident, and the following

fragments have been confirmed: 309.0; 278.2;

266.0; 250.1; 238.0; 210.1. For the correspondence

of the fragments it was possible to hypothesize that

this compound is probably Mukonicine.

The ions at m/z: 258.0; 264.1; 334.2; 346.2; 364.2;

376.1; 393.1 corresponding probably to the mass

6,7-Dimethoxy-1-hydroxy-3-methylcarbazole;

Girinimbine; Mahanimbinol; Murrayamine J;

Murrayanol; Murrayakonine C; Bis-2-hydroxy-3-

methylcarbazole was identified in the PI-IDA-EPI

scan m/z 210.1. For the m/z 264.1 a peak was

obtained at RT 2.84 min. The peak was associated

with Girinimbine. In fact the acquired EPI

spectrum was compared with the data reported in

the literature [6] and it was possible to compare the

following fragments: 249.0; 246.0; 236.0; 222.0;

206.4.

4. Conclusions The presented method was performed using a

combination of predictive multi experiment

approach that provides a targeted and semi-

untargeted approach to obtain both reliable

quantitative data, by MRM-IDA-EPI acquisition,

and structural information on analogues CAs using

MRM/PI-IDA-EPI acquisition mode. Accuracy,

linearity, precision, LOD and LOQ were evaluated

for validation, with good performance in each

parameter. The presented method could represent

an useful tool for studying the content of CAs in

different matrices, with good sensitivity, and also

for screening and confirming unknown analogues

included in CAs.

References 1. Y. Zang, C. Li, X. Song, J. Ma, J. Yang, and D. Zhang, J. Asian Nat. Prod. Res., vol. 6020, no. May, pp. 1–7,

2017.

2. F. F. Zhang, L. L. Gan, and C. H. Zhou, Bioorganic Med. Chem. Lett., vol. 20, no. 6, pp. 1881–1884, 2010.

3. F. Allen, R. Greiner, and D. Wishart, Metabolomics, vol. 11, no. 1, pp. 98–110, 2015.

4. Y. Djoumbou-Feunang et al.,Metabolites, vol. 9, no. 4, pp. 1–23, 2019.

5. A. A. Ochung’ et al., J. Korean Soc. Appl. Biol. Chem., vol. 58, no. 6, pp. 839–846, 2015.

6. J. L. Songue, Kouam, E. Dongo, T. N. Mpondo, and R. L. White, “Chemical constituents from stem bark and

roots of Clausena anisata,” Molecules, vol. 17, no. 11, pp. 13673–13686, 2012.

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Comprehensive two-dimensional liquid chromatography coupled to mass

spectrometry detection for characterization of bioactive compounds in food and

natural products

F. Cacciola1, K. Arena2, P. Dugo2,3, L. Mondello2,3,4

1 Department of Biomedical, Dental, Morphological and Functional Imaging Sciences, University of Messina, Messina,

Italy 2 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy

3 Chromaleont s.r.l., c/o Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University

of Messina, Messina, Italy 4 Department of Sciences and Technologies for Human and Environment, University

Campus Bio-Medico of Rome, Italy

Summary: The present contribution aims to highlight the use of comprehensive two-dimensional liquid

chromatography coupled to mass spectrometry detection for the characterization of bioactive compounds in

food and natural products. The developed approach is estimated as a valuable tool for the quali-quantitative

study of complex food and natural products.

Keywords: LC×LC; natural products; mass spectrometry.

1. Introduction Polyphenols comprise a complex class of bioactive

compounds, whose nature and high content in

commonly consumed foodstuffs, make their

analysis challenging. Comprehensive two-

dimensional liquid chromatography is a well-

established method for the unraveling of complex

food and natural products samples.

2. Experimental Combination of reversed-phase×reversed-phase

(RPLC×RP-LC) and hydrophilic interaction liquid

chromatography×reversed-phase (HILIC×RP-LC)

were investigated for unravelling polyphenol

profiles in food and natural products samples.

Moreover, the use of active modulation in the

HILIC×RP-LC approach, performed by the

addition of a make-up flow, efficiently helped to

compensate for the solvent strength mismatch

produced between the two dimensions.

3. Results The developed LC×LC methods allowed an

improved separation of bioactive compounds with

enhanced practical peak capacity and orthogonality

with respect to the current conventional available

methodologies. In addition, a quantification

approach was carried out through external

calibration curves yielding satisfactory LODs,

LOQs, intraday and interday precision and

recovery values.

4. Conclusions The developed approach can be estimated as a

valuable tool for the quali-quantitative study of

natural products, considering their great

complexity and high dimensionality. Furthermore,

their polyphenolic characterization will be of valid

aid to confirm their potential use for the human

health.

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Quantification of glyphosate in milled and brown rice in LC-ICP-MS/MS

P. Scardina1, Andrea Carcano1, Gian Maria Beone2, Maria Chiara Fontanella2,

Agnese Salvatico2

1Agilent Technologies Italia S.p.A. – Cernusco Sul Naviglio – MI – Italy

2Univ. Cattolica del Sacro Cuore – Dipartimento di Scienze e Tecnologie Alimentari – Piacenza -Italy

Summary: We developed a new method for the quantification of Gliphosate in rice by coupling an Agilent

bio-inert 1260 HPLC system with an Agilent 8900 QQQ ICP MS/MS System. The result is a very sensitive

and selective method with a minimum sample preparation; LOQ are lowed than the EU community limits

and this methos can be extended to other food matrix

Keywords: LC-ICP-MS/MS – Gliphosate - Rice

1. Introduction Gliphosate (GLY) is a potential carcinogenic

pesticide regulated by UE regulation 293/213 in

food and in particular in rice with a limit of 0.1

mg/kg considered the actual instrumental LOQ. A

new method developed coupling an Agilent bio-

inert 1260 HPLC system with an Agilent 8900

QQQ ICP MS/MS System is able to improve of

classical LC-MS method LOQ with a simplified

sample preparation.

2. Experimental ICP-MS/MS is a very selective and sensitive

detector for phosphorus, a controlled reaction with

oxygen can remove all the potential on mass

polyatomic interferents.

Rice samples prepared by extraction and after

filtration is introduced in a coupled LC-ICP-

MS/MS system, different instrumental set-up

has been tested on milled and brown rice

3. Results Method shows a very good linearity and

reproducibility with excellent LOQ both in milled

and brown rice with a good recovery even on very

low spike amount

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4. Conclusion This work isa proof that actual triple quadrupole

ICP-MS-MS system can be extended to organic

method when coupled to HPLC system, with the

advantage of a simplified sample preparation and

an improvement of LOQ.

This method can be implemented to other food

matrix and to other organic pollutant containing

elements like phosphorus and/or sulphur.

References

1. A. L. Valle · F. C. C. Mello · R. P. Alves-Balved L. P. Rodrigues· L. R. Goulart · Environmental Chemistry Letters

(2019) 17:291–317

2. B. Lajin, W. Goessler - TalantaVolume 196, 1 May 2019, Pages 357-361

3. E. M. Pimenta, F F. da Silva, É. S. Barbosa, A. P. Cacique,D. L. Cassimiro, Gevany P. de Pinhoa, F. O. Silvério - J.

Braz. Chem. Soc., Vol. 31, No. 2, 298-304, 2020

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Raw materials in food manufacturing: a complexity ascertained by high-


M.A. Acquavia1,2, R. Pascale3, A. Onzo1, C. Gaeta4, P. Iannece4, R. Ciriello1, F. Lelario1, C. Tesoro1, R. Rubino5, G. Bianco1

1Università degli Studi della Basilicata, Dipartimento di Scienze, via dell’Ateneo Lucano 10, 85100, Potenza, Italy

2THEMA INFORMATIK S.r.l Via Ressel 2/F, 39100, Bolzano, Italy 3Gnosis by Lesaffre, Pisticci, 75015 Matera, Italy 4Università Degli Studi di Salerno, Dipartimento di Chimica e Biologia, Via Giovanni Paolo II 132, Fisciano, Italy

5ANFoSC - Via San Leonardo 62/A, 84131 Salerno, Italy.

Summary: The raw materials for food manufacturing are commonly considered similar in the whole

agricultural world, due to the parameters used to evaluate their quality. Here, HRMS-based metabolomics

was used to ascertain the complexity and establish the differences among several species and varieties of

Italian agro-food products.

Keywords: agro-food products; metabolomics; HRMS

1. Introduction The market of agro-food products constitutes a

highly specialized and competitive sector.

Nevertheless, the raw materials for food

manufacturing are often considered similar in

the whole agricultural world, as the parameters

commonly employed to evaluate their quality

are poorly connected to their flavor. Indeed, they

are mainly indices of their nutritional value (i.e

proteins, fats and carbohydrates content) or of

the type of technological processing to which

they are subjected, in order to obtain the final

products. As example, for cereal-based food,

such as pasta, the aspects of major importance to

account their quality include test weight, 1000-

kernel weight, physical defects, vitreousness,

moisture content, weather damage and grain

protein percentage, as well as the milling

process of the grain [1]. Actually, the natural

conditions (terroir) and the local know-how

(savoir-faire) have proved to be important

sources of distinctiveness for the agricultural

productions and their derived food [2]. Both the

terroir and the know-how account for the

differentiation of the products, which is

reflected in their metabolic composition [3,4].

Mass spectrometry-based metabolomics

approaches could be successfully applied as tool

for the differentiation of agro-food products,

since they allow to get a chemical fingerprint of

food matrices and differentiate them on the basis

of the molecules occurring in the sample. Here,

we investigated the molecular complexity of

common Italian raw materials for food

manufacturing by high-resolution mass

spectrometry.

2. Experimental The extracts of different Italian agro-food

products, i.e. beans, peppers and aubergines as well

as of the flours obtained by wheat cultivated in

several area of the Basilicata region (South Italy),

were analyzed by electrospray ionization (ESI) FT-

ICR MS technique. High-resolution mass spectra

were acquired with a 7 T solariX XR FT-ICR MS

(Bruker Daltonik GmbH, Bremen, Germany) and

then exported to peak lists. To obtain unequivocal

formulas for each m/z signal, several constraints

were applied, such as atoms number limitations,

restrictions on atoms to carbon number ratios,

RDBE > 0, nitrogen rule, and isotopic pattern

filtering. HRMS data were processed by using Data

Analysis (v4.2, Bruker Daltonik GmbH, Bremen,

Germany) and the R software (, v3.6.3) and were

represented using van Krevelen diagrams, which

sort them onto two axes according to H/C and O/C

atomic ratios.

3. Results Direct-injection High Resolution ESI(±)-FT-ICR

MS data were used to obtain a general description

of the metabolome of different Italian agro-food

products. The processed mass spectra showed a

huge number of peaks, thus revealing the wide

diversity of metabolites occurring in the samples.

As a matter of fact, the number of identified

phytoconstituents remarkably increased from

beans (100-200 detected metabolites) to aubergines

(7000 detected metabolites), thus showing how

their content is related to the plant species (Fig. 1).

Besides, the number of metabolites, albeit to a

lesser extent, also varies within the same species,

both in the case of different varieties and for the

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same variety of a given agro-food product. As

example, for Peperoni di Senise peppers, the

secondary metabolites are about one third higher

for those protected by the PGI quality mark, thus

suggesting the influence of agricultural practices

on the chemical peculiarities of the matrix.

Moreover, the content of biomolecules is different

also in the food products obtained from the

processing of raw materials. Indeed, as

experimental evidence, the HRMS data obtained

for flours derived from the wheat cultivated in

different areas of the Basilicata region, were

different. In addition to the number of metabolites,

the differences were evident in their distribution

within the molecular maps (Van Krevelen

diagrams) too. The variability in the metabolic

fingerprint of the Carosella flours (Fig. 2), which

excel from a molecular point of view, is presumably due to the areas of wheat cultivation,

thus supporting the thesis of raw materials

differentiation for food manufacturing.

4. Conclusions High-resolution mass spectrometry proved to be a

valid tool to ascertain the complexity of agro-food

products. The high levels of resolution and

accuracy reached made possible to assess the

metabolic differences existing among vegetables

belonging to different species. More importantly,

the construction of 2D Van Krevelen plots allowed

to highlight a consistent variability in the

metabolomic fingerprints of agro-food matrices

belonging to the same variety. Such a variability

was reflected also in the products derived from

their manufacturing and could account for their

different quality and organoleptic properties.

Fig. 1. Number of metabolites identified by ESI(±)-FT-ICR MS data in agro-food products (beans, peppers, aubergines)

and flours of Basilicata region (South Italy).

Fig. 2. Van Krevelen plots of Carosella flour (262 metabolites) obtained by wheat cultivated in Episcopia (PZ) (A); Van

Krevelen plots of Carosella flour (3750 metabolites) obtained by wheat cultivated in Valsinni (PZ) (B). Types of the

formula are distinguished by colors.

References 1. Sicignano, A., Di Monaco, R., Masi, P., and Cavella, S. (2015) F. J. Sci. Food Agric., 95 (13), 2579–2587.

2. Bowen, S., and Zapata, A.V. (2009). J. Rural Stud., 25 (1), 108–119.

3. Dias, C., and Mendes, L. (2018) P. Food Res. Int., 103 (September 2017), 492–508.

4. Pascale, R., Bianco, G., Cataldi, T.R.I., Kopplin, P.S., Bosco, F., Vignola, L., Uhl, J., Lucio, M., and Milella, L.

(2018). Food Chem., 242 (June 2017), 497–504.

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Use of native-mass spectrometry to elucidate the oligomeric state and substrate-

binding affinity of FrlB, a bacterial deglycase

A. Di Capua1,2, S. Kovvali3, C. E. Bell4, V. Gopalan1, V. H. Wysocki1,2

1Department of Chemistry & Biochemistry, 2Resource for Native MS-Guided Structural Biology, 3Department of

Microbiology, 4Department of Biological Chemistry & Pharmacology, The Ohio State University, Columbus, OH

Summary: Here, we used native mass spectrometry to confirm the oligomeric state of FrlB, a bacterial

deglycase, and six of its mutant derivatives. We also elucidated the ligand binding affinity and stoichiometry

of its natural substrate, 6-phospho-fructolysine, to better identify the active site and catalytic mechanism of

FrlB.

Keywords: native mass spectrometry, ligand binding affinity, FrlB deglycase

1. Introduction Amadori compounds are stable sugar-amino acid

conjugates that are formed non-enzymatically via

the Maillard reaction. Most of the sugar-amino

acids can be metabolised through a common

pathway that involve a putative permease, a kinase,

and a deglycase, to give as final products a glucose-

6-phosphate and the respective amino acid.[1]

Fructose-lysine (F-Lys, ε-conjugated) is one of the

most abundant Amadori compounds in processed

foods and is a key intermediate in the formation of

advanced glycation end products, which in turn are

implicated in inflammation and disease. FrlB, a

deglycase, converts 6-P-F-Lys to L-lysine and

glucose 6-phosphate, with the latter feeding into

glycolysis among other routes. To better

understand the catalytic mechanism of FrlB, we

sought to identify the residues involved in its

catalytic mechanism.[2] Towards this goal, we

investigated Salmonella FrlB wild type (WT) and

six mutant derivatives.

Native Mass Spectrometry (nMS) is a powerful

analytical tool that allows study of macromolecular

complexes in the gas phase where proteins remain

folded and non-covalent interactions can be

preserved. The general workflow is based on a

protein buffer exchange with a solution of a volatile

salt (usually ammonium acetate) followed by nano-

ESI-MS analysis. The use of a volatile electrolyte

at the physiological pH and ionic strength allows

for transfer of biomolecules from solution to the

gas phase without perturbing protein-binding

interactions. [3] Here, we used native MS to confirm

the oligomeric state of WT FrlB and six individual

mutants and to investigate their affinity for the

substrate 6-P-F-Lys.

2. Methods Recombinant WT FrlB and six individual mutants

of FrlB were overexpressed in Eschericha coli and

purified by affinity chromatography. For native

mass spectrometry analysis, proteins were buffer-

exchanged into 200 mM ammonium acetate

(AmAc, pH 6.8) using 6-kDa cutoff Micro Bio-

Spin columns. For FrlB-6-P-F-Lys (enzyme-

substrate) complex experiments, 6-P-F-Lys was

diluted using 200 mM AmAc to the desired

concentration and then mixed with FrlB. Samples

were introduced into the mass spectrometer using

nano-electrospray ionization. For Online Buffer-

Exchange nMS (OBE-nMS), samples were kept in

their storage buffer and injected into the mass

spectrometer through a desalting (buffer exchange)

cartridge connected to a Vanquish liquid-

chromatography system (Thermo Scientific). [4-7]

Experiments were performed on an in-house

modified Thermo (Q) Exactive UHMR which was

modified to include an SID device after the

quadrupole.[8]

3. Results Native mass spectrometry was used to determine

the oligomeric state of FrlB WT as well as six

mutants. By using nano-ESI mass spectrometry, we

confirmed the dimeric oligomeric state for FrlB

WT and mutants. To determine the exact molecular

mass of monomers, surface-induced dissociation

(SID) was used. SID is an activation method where

proteins in the gas phase collide with a surface and

generate native-like subcomplexes.

Once we confirmed the exact mass and the

oligomeric state of our proteins, we investigated

the binding affinity of FrlB for its natural substrate

6-P-F-Lys. When FrlB was titrated with different

concentrations of 6-P-F-Lys, we observed two

copies of 6-P-F-Lys bound to the FrlB dimer (at a

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protein:ligand ratio 1:400). We then investigated

the binding competence of the six FrlB mutants,

and we observed that one of them (S46A) exhibited

a lower affinity for the natural substrate 6-P-F-Lys

and the rest were incompetent. These results are in

agreement with the kinetic studies conducted on

the FrlB mutant derivatives, where only one mutant

(S46A) showed cleavage of the substrate and the

rest are inactive. Collectively, these data provide

insights into the active site of FrlB.

Moving forward, electron capture dissociation

(ECD) coupled with high-resolution mass

spectrometry will be performed to further refine

this picture of the FrlB catalytic center.

References

1. V.M. Deppe, J. Bongaerts, T. O’Connell, et al. Applied microbiology and biotechnology (2011), pp. 399–406. 2. B. Graf von Armansperg, F. Koller, N. Gericke, et al. Molecular microbiology (2021), pp. 175–190.

3. K.R. Karch, D.T. Snyder, S.R. Harvey, V.H. Wysocki Annual review of biophysics (2022), 51.

4. Z.L. VanAernum, F. Busch, B.J. Jones, M. Jia, Z. Chen, S.E. Boyken, et al. Nature protocols (2020), pp. 1132-

1157.

5. M. Jia, S. Sen, C. Wachnowsky, I. Fidai, J.A. Cowan, V.H. Wysocki. Angewandte Chemie (International ed. in

English), (2020), pp. 6724-6728.

6. F. Busch, Z.L. VanAernum, S.M. Lai, V. Gopalan, V.H. Wysocki. Biochemistry, (2021), pp. 1876-1884.

7. B.E. Szkoda, A. Di Capua, J. Shaffer; E.J. Behrman, V.H. Wysocki, V. Gopalan. Journal of molecular biology

(2022), 167480.

8. Z.L. VanAernum, J.D. Gilbert, M.E. Belov, A.A. Makarov, S.R. Horning, V.H. Wysocki. Analytical chemistry

(2019), pp. 3611-3618.

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Assaying vegetable specimens by FT-ICR mass spectrometry, an election

technique in metabolomic studies

A. Lasalvia, A. Maccelli, S. Fornarini, M. E. Crestoni

Dipartimento di Chimica e Tecnologie del Farmaco, Università di Roma “La Sapienza”, P. le Aldo Moro 5, 00185,

Roma, Italy

Summary: Untargeted profiling studies have been performed on two different extracts of Goji berry (Lycium

barbarum L.) and Sulmona red garlic (Allium sativum L.). Direct infusion electrospray ionization (ESI) source

hyphenated with Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (MS) has allowed to

investigate and characterize, in a multimethodological approach, the phytochemical composition of food

matrices.

Keywords: FT-ICR MS, Metabolomics, foodstuffs

1. Introduction The omics sciences aim to identify, characterize,

and quantify all molecules that are involved in

biological pathways. Metabolomics, a branch of

omics sciences, investigate all chemical processes

concerning metabolites, which are the end products

of cellular pathways in cell, tissue, and organ. In

this field, mass spectrometry represents one of the

election techniques due to its analytical sensitivity

and specificity. The study of a metabolomic

fingerprint may follow either a targeted or an

untargeted approach. In this context, based on

recent work[1], an untargeted study has been

carried out by high resolution FT-ICR mass

spectrometry on two different foodstuff extracts of

Goji berry (Lycium barbarum L.) and Sulmona red

garlic (Allium sativum L.) for a comprehensive

qualitative characterization. At same time this

work winks at sustainability by shedding light on

waste material, such as goji berries leaves or garlic

tunics, because they can be renovated into a rich

source of ingredients useful to different purposes.

Goji berries (GBs), excellent source of macro- and

micronutrients, have gained increasing interest in

western world justifying the naming as

“superfood” due to the wide variety of

pharmacological functions, including antioxidant,

immunomodulatory and anticancer properties[2].

Not only berries, but also Goji leaves hold a high

amount of antioxidant and bioactive compounds,

which makes them promising in nutraceutical and

cosmetic field.

Garlic (Allium sativum L.) belonging to the

Amaryllidaceae family represents one of the most

diffuse aromatic and food herb. Garlic bulbs are

potentially involved in the prevention of chronic

and degenerative disorders[3] thanks to their

complex composition in bioactive compounds

including metabolites with antimicrobial,

antiradical and antioxidant activities, thus gaining

great interest as functional foods. ‘Rosso di

Sulmona’ is a garlic variety inserted into the list of

the officially recognized commercial varieties of

red garlic (DM 296/2009 delivered by Italian

Ministry of Agricultural, Food and Forestal

Policy). This crop presents a whole bulb covered

by a white tunica and a characteristic reddish tunica

surrounding each bulbil once the bulb is divided.

Its identification is related to morphological and

morphometric characteristic factors, while the

chemical properties have never been studied.

2. Experimental Samples have been analysed by direct infusion

ESI-MS. High resolution mass analysis of each

sample was carried out by using a Bruker BioApex

FT-ICR. All mass spectra were recorded in the m/z

100-1000 range and the measurements were based

on the “monoisotopic” ion. The extreme accuracy

of high resolution gives a univocal molecular

formula which has been assigned to several

metabolites with an uncertainty of less than 3 ppm.

Further confirmation of the identity of ionic species

was obtained by CID MS/MS experiments on

mass-selected ions giving complementary proof of

the peak assignment. Further tests by cross-

reference with online libraries were also

performed.

3. Results and Conclusions MS analyses of GBs have regarded hydroalcoholic

(HA) and organic (O) extracts of fruits and leaves

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of two different GBs cultivars: Sweet Rose and Big

Life of L. barbarum L., at red (ripe) and green

(unripe) ripening stages. In addition, a sample of

BL grown under less sun exposure (LE) was

analysed. Each sample revealed up to 1400

molecular formulas for fruits and more than 600 for

leaves. Garlic analyses were conducted on two HA

extracts of ‘Sulmona red garlic’ comparing

chemical and biological properties of the smaller

bulbs, the external white tunica and the inner red

tunica. The qualitative analyses of HA extracts

from samples, collected in two different years,

have revealed more than 400 molecular formulas

for each of them. Lastly, the analyses, for both GBs

and Sulmona red garlic extracts, have been

completed by graphical visualisation tools, such as

van Krevelen diagrams and elemental composition

histograms, and multivariate statistical analysis

(PCA). These tools allow to obtain interesting

information about density of molecular classes,

metabolic pathways linking metabolites and

correlation between samples. To conclude, the

obtained results corroborated the rich composition

in bioactive compounds (such as the Lycium

barbarum polysaccharides, carotenoids,

polyphenols, hydroxycinnamic acids) of Goji

berries and leaves imparting them beneficial

properties for health and well-being and making

them a promising source in the pharmaceutical,

food, and cosmetic fields. Likewise, Sulmona red

garlic MS analyses highlighted the great

abundance of lipids, polyketides, amino acids and

organosulfur compounds. Moreover, this work

confirmed MS as highly sensitive method and

therefore the election technique to obtain accurate

mass measurements of metabolites in complex

matrices, wherein several hundred signals can be

revealed in a single measurement. In addition,

application of the graphical visualisation tools and

statistical method to mass spectrometry analyses

allows to condense a p-dimensional information in

a two-dimensional space in order to gain an

immediate graphic visualization of the data and to

understand the different correlation between the

variables by verifying the assumptions on the data.

References 1. Maccelli, S. Cesa, F. Cairone, D. Secci, L. Menghini, B. Chiavarino, S. Fornarini, M.E. Crestoni and M. Locatelli;

Journal of Mass Spectrometry. (2020). 55:e4525.

2. Yao, R.; Heinrich, M.;Weckerle, C.S. J. Ethnopharmacol 2018, 212, 50–66.

3. V.P.Londhe, A.T.Gavasane, S.S.Nipate, D.D.Bandawane, P.D.Chaudhari. Angiogenesis. 2011, 12(13), 129–1

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ORALS

La spettrometria di massa nelle Scienze della vita

Chairs: Fulvio MAGNI & Donatella CARUSO

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Metabolomics, analytical tools for the systems-level analysis of metabolism

G. Paglia

School of Medicine and Surgery, University of Milano-Bicocca.

Summary: The use of Ion Mobility Mass Spectrometry in Metabolomics and its application in sport medicine and

transfusion medicine

Keywords: Metabolomics, Lipidomics, Ion Mobility Mass Spectrometry, Sport Medicine and Transfusion

Medicine.

Metabolomics and lipidomics are rapidly evolving

analytical approaches in life and health sciences and

they aim to the complete characterization of

metabolites and lipids in cells, tissues, and biological

fluids. Mass spectrometry (MS) is the most widely

used tool for metabolomics and lipidomics, enabling

the analysis of complex mixtures of small molecules

present with a wide dynamic range of concentrations

in biological samples. MS alone, however, is not able

to completely

resolve and characterize the thousands of metabolites

and lipids, including many isobaric and isomeric

species. One of the most attractive technological

innovations in the field of MS-based

metabolomics/lipidomics is the coupling of ion

mobility spectrometry (IMS) to MS, combination that

provides a powerful analytical platform that can

separate ions beyond mass to charge ratio (m/z).

However, metabolomics is not only measuring as

many metabolites as possible but should be considered

also a tool for the systems-level analysis of

metabolism. Considering that the metabolome and

lipidome are both impacted by genetic background

and environmental exposure, metabolomics and

lipidomics can provide the ultimate molecular

description of the phenotype of a biological system.

This report will first describe the benefits of coupling

IM-MS in metabolomics studies and then will present

applications of metabolomics to study metabolism in

cells and biological fluids, ranging from sport

medicine, transfusion medicine and population

studies.

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Proteomic profile of extracellular vesicles secreted by astrocytes using high


M. G. G. Pittalà1, L. Leggio2, G. Paternò2, A. Cucina1, V. Cunsolo1, A. Di Francesco1, B.

Marchetti2, S. Foti1, N. Iraci2, R. Saletti1

1Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Via S. Sofia 64,

95123 Catania, Italy; 2 Laboratory of Molecular Biology, Department of Biomedical and Biotechnological Sciences, University of Catania, Via S.

Sofia 97, 95123 Catania, Italy.

Summary: We characterized the proteome of astrocyte-derived extracellular vesicles (EVs) from the main brain

regions affected in Parkinson’s disease (PD), in basal and activated conditions. Our data suggest that specific

proteins are sorted towards EVs depending on the cellular origin and treatment, with potential implications for

PD diagnosis and treatment.

Keywords: Parkinson's disease, exosomes, shotgun approach

1. Introduction Parkinson's disease (PD) is a devastating

neurodegenerative disease caused by progressive

loss of dopaminergic neurons in the Substantia Nigra

pars compacta within the ventral midbrain (VMB),

and their terminals in the striatum (STR) [1]. During

PD onset and progression, extracellular vesicles

(EVs) secreted by astrocytes (AS-EVs) were

identified as potential contributors in the propagation

of molecules able to enhance neurotoxicity.

However, EVs can have also a neuroprotective role

in PD [2].

To identify the mechanisms involved in the

neuroprotective effects of AS-EVs [3], we have

investigated their proteomic profile, comparing

vesicles secreted by astrocytes from the VMB vs. the

STR, both in basal conditions (AS-EVs Ctrl) and

activated by the pre-treatment with the

neuroprotective chemokine CCL3 (AS-EVs CCL3).

2. Experimental Intact EVs were purified via differential

ultracentrifugation from primary cultures of

postnatal VMB and STR astrocytes. EVs were

incubated in ice for 15 min and then lysed using 0.1%

RapiGest SF. Proteins were purified from salts, lipids

and other contaminants, reduced with DTT, alkylated

by addition of iodoacetamide and in-solution

digested using porcine trypsin. Analysis was

conducted in triplicate by a shotgun approach and

nanoUHPLC/High-Resolution nanoESI-MS/MS [4].

Finally, we have performed Gene Ontology analysis

in order to unravel molecular processes and

biological functions potentially coordinated by AS-

EVs.

3. Results MS analysis revealed a different proteomic profile of

AS-EVs Ctrl and AS-EVs CCL3 extracted from

VMB and STR. In particular, the data shown, among

the common proteins identified, the presence of

classical EV markers, such as Cd81, Sdcbp and Tfrc

but also of astrocyte markers, such as Aldh1l1 and

GFAP. Furthermore, both cytosolic and

mitochondrial proteins (e.g. Atp5f1a and b) and

glycolytic enzymes (e.g. Eno1 and Pkm) were found.

Instead, among the proteins identified exclusively in

the AS-EVs CCL3 samples, many of them are

involved in regulation and development of nervous

system, in secretion of synaptic vesicles and in

differentiation and proliferation of neurons and glia

cells. Gene Ontology Enrichment Analysis showed

that EV proteins correlated with neurological disease

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pathways including neurodegeneration, loss of

synapse and neuroinflammation; interestingly

they are also enriched in terms related to cellular

growth, neural and glial proliferation, outgrowth

of neurites and branching of axons, in line with

the neuroprotective potential of the donor

astrocytes.

4. Conclusions

Our results suggest the existence of a molecular

machinery capable of organizing in the astrocytes

the sorting of specific proteins towards EV-

mediated secretion. Furthermore, it implies the

possibility that the secreted proteins may have

specific functions when transferred to target

cells and the possibility to reprogram AS-EVs for

brain repair. Thus, the knowledge generated

within this study, may pave the way for the

development of innovative therapeutic

approaches to tackle PD.

References

1. E. C. Hirsch, P. Jenner, S. Przedborski; Mov. Disord., 28 (2013), pp 24–30.

2. B. Marchetti, L. Leggio, F. L'Episcopo, S. Vivarelli, C. Tirolo, G. Paternò, C. Giachino, S. Caniglia, M. F.

Serapide, N. Iraci; J. Clin. Med., 9, (2020), 1941.

3. L. Leggio, F. L’Episcopo, A. Magrì, M. J. Ulloa-Navas, G. Paternò, S. Vivarelli, C. A. P. Bastos, C. Tirolo, N.

Testa, S. Caniglia, P. Risiglione, F. Pappalardo, N. Faria, L. Peruzzotti-Jametti, S. Pluchino, J. M. Garcia-

Verdugo, A. Messina, B. Marchetti, N. Iraci; BiorXiv, (2021), preprint.

4. M. G. G. Pittalà, S. Conti Nibali, S. Reina, V. Cunsolo, A. Di Francesco, V. De Pinto, A. Messina, S. Foti, R.

Saletti; Int. J. Mol. Sci., 22 (2021), 12833.

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Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS)

Profiling of Commercial Enocianina and Evaluation of Their Antioxidant and

Anti-Inflammatory Activity

L. Della Vedova1, G. Ferrario1, F. Gado1, A. Altomare1, M. Carini1, P. Morazzoni2, G.

Aldini1 and G. Baron1

1Department of Pharmaceutical Sciences (DISFARM), , Via Mangiagalli 25, 20133 Milan, Italy. 2Divisione

Nutraceutica, Distillerie Umberto Bonollo S.p.A, 35035 Mestrino, Italy.

Summary: Enocianina is an anthocyanin rich extract obtained from grape pomace used as colorant for the

food industry. To understand whether enocianina can offer potential health benefit applications, we

characterized the qualitative and semi-quantitative profile of four commercial enocianinas by LC-HRMS and

assessed their radical scavenging, enzymatic antioxidant and anti-inflammatory activity.

Keywords: Enocianina, LC-HRMS, antioxidant and anti-inflammatory activities

1. Introduction Several extracts rich in anthocyanins, isolated from

fruits and vegetables, including grape skin, currant,

elderberry, cranberry, bilberry, maize, cabbage,

and carrot, are widely used as food colorants [1].

Oenocyanin, enocyanin or enocianina is the name

of anthocyanins when extracted from grape

pomace, composed of the stalks, skin, pulp, and

seeds that are left over after the pressing of the

grapes during the winemaking process [2,3]. Hence, enocianina could be considered as an early

example of a circular economy, in fact it is used as

a colorant for the food industry (E-163, grape peel

extract). To understand whether enocianina,

besides its coloring effect, can offer potential

health benefit applications, the aim of the study is

to fully characterize the qualitative and semi-

quantitative profile (by LC-HRMS [4]) of four

commercial enocianinas and to assess their radical

scavenging (DPPH test), enzymatic anti-oxidant

(Nrf2 activation) and anti-inflammatory activity

[4].

2. Results LC-ESI-MS/MS in positive and negative ion mode

identified 90 phytochemicals, among which 41

different anthocyanins, 9 phenolic acids, 1

stilbenoid, 31 flavonols and 8 flavanols. The

relative content of each anthocyanin was assessed

by a semi-quantitative analysis, malvidin

derivatives being the most abundant, followed by

compounds containing peonidin and petunidin,

while delphinidin and cyanidin derivatives

represent a minor fraction.

Fig. 3. Venn diagram showing the number of compounds identified in positive (n=52) and negative (n=15) ion modes

and those identified by both the two ion modalities (n=23). Distribution of compounds among the flavonoid classes are

also detailed. By combining the compounds identified by the two ion modes a total number of 90 compounds were

identified.

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UV-VIS spectroscopy detected a total amount of

polyphenols and anthocyanins of 23% and 3.24%,

respectively, indicating that anthocyanins represent

a minor fraction of total polyphenols. Multiple

linear regression analysis indicates that the radical

scavenging activity is related to the total

polyphenol content and not to anthocyanins. All

four enocianins tested were found to dose-

dependently activate Nrf2 and such activity was

related to the content of catechol containing

polyphenols. Finally, all the tested enocianinas

showed a dose-dependent anti-inflammatory

activity which at the highest concentrations tested

was found to be strictly related to the total

polyphenol content and was explained by radical

scavenging, Nrf2 activation and other mechanisms

related to the polyphenolic components.

3. Conclusions

In conclusion, enocianina is a mixture of highly

valuable phytochemicals derived from wine waste

product which exert direct and indirect antioxidant

and anti-inflammatory effects mediated by

mechanisms involving both catechol (Nrf2

activation) and non-catechol containing derivatives

References

1. Khoo H.E., Azlan A., Tang S.T., Lim S.M.; Food Nutr Res, 61 (2017), pp 1361779.

2. Da Porto C., Zironi R., Celotti E., Bertolo A.; Journal International des Sciences de la Vigne et du Vin, 32 (1998),

pp 8.

3. Prodanov M.P., Domínguez J.A., Blázquez I., Salinas M.R., Alonso G.L.; Food Chemistry, 90 (2005), pp 11.

4. Baron G., Ferrario G., Marinello C., Carini M., Morazzoni P., Aldini G.; Molecules, 26 (2021), pp 5454.

.

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Emerging per- and polyfluoroalkylated substances (PFASs) in wild boar liver

S. Moretti 1, C. Barola 1, F. Paoletti 1, S. Sdogati 1, D. Giusepponi 1, G. Brambilla 2, R. Galarini 1

1Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ”Togo Rosati”, Perugia, Italy; 2Istituto

Superiore di Sanità, Dipartimento Alimenti, Nutrizione e Sanità Pubblica Veterinaria, Rome, Italy

Summary: Per- and polyfluoroalkylated substances (PFASs) are a wide group of contaminants used in several

industrial products. The presence of novel perfluoro-polyether carboxylic acids has been recently reported in

environmental matrices. In this work, substances belonging to this group have been detected for the first time in liver

of wild boars.

Keywords: Per- and polyfluoroalkylated substances (PFASs), wild boar liver, LC-Q-Orbitrap

1. Introduction

Per- and polyfluoroalkylated substances

(PFASs) are a wide group of synthetic molecules

distributed globally. The EU established

regulations that restrict some types of long-chain

PFASs and related compounds; therefore new

molecules have been synthetized as alternatives.

Several studies have proven that wild boars can

bioaccumulate PFASs [1]. The presence of

emerging PFAS molecules was investigated in a

pool of wild boar livers.

2. Experimental

Twenty-eight wild boar liver samples from male

animals shot in different Italian areas were

pooled and homogenized. Two grams of pooled

livers were then extracted and purified as

described in Barola et al. [2]. PFAS analysis was

performed by liquid-chromatography coupled to

a Q-Orbitrap mass analyser (LC-Q Exactive,

Thermofisher Scientific, San Jose, CA, USA).

Negative ionization mode was applied.

Acquisition of target compounds (with or without

reference standards) was carried out using t-SIM-

ddMS2 experiment on the basis of an inclusion

list [2-4]. Untargeted analysis was performed

combining three different experiments: full

MS/dd-MS2 (TopN), full MS/AIF/NL dd-MS2 and

full MS/DIA.

3. Results

Target analysis revealed the presence of

dodecafluoro-3H-4,8-dioxanonanoate

(ADONA), potassium 9- chlorohexadecafluoro-

3-oxanonane-1-sulfonate (9Cl-PF3ONS) and

potassium 11-chloroeicosafluoro-3-

oxaundecane-1-sulfonate (11Cl-PF3OUdS).

Moreover, congeners of perfluoroether

carboxylic acids have been detected and

tentatively identified, too. Their general structure

was hypothesized in Figure 1.

Fig. 1 -General structure of perfluoroether carboxylic acid oligomeric series (R=Cl: ClPFPECA; R = H:

HPFECA). The R group position is likely variable [3,4]

Eight major oligomeric forms of chloro-

perfluoro-polyether carboxylates (ClPFPECA)

and hydro- perfluoroether carboxylic acid

(HPFPECA) were identified with “e” from 0 to

2 and “p” from 0 to 3. These molecules have

been recently detected in environmental

samples collected in New Jersey (USA) [3,4]

and they could be traced back to the mixture of

telomers (CAS 329238-24-6) produced bySolvay with the trade name ADV 7800 [5].

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Interestingly MS2 spectra recorded for some

nearly co-eluting ClPFPECA isomers were not

compatible with the structure shown in Figure 1

due to the missing of characteristic fragment ions

such as C3ClF6O- (m/z 200.9547) [3,4]. Moreover,

by means of untargeted analysis, another group

of perfluoro polyether acids was also detected.

Based on the molecular ions, Compound

DiscovererTM software (Thermofisher Scientific)

proposed raw formulas. Later, analyzing the relevant

MS2 spectra with some characteristic fragment ions

such as C2F3O2-and C4F7O3

-, the oligomeric structure

shown in Figure 2 was hypothesized. This group of

PFASs could be traceable to CAS No. 69991-62-4

[6].

Fig. 2 – Presumed general structure of perfluoroether di-carboxylic acids series (PFPEdCA)

4. Conclusions

The results demonstrated that emerging and

novel poly- and perfluoro-polyether PFASs

bioaccumulate in liver of wild boars. Further

investigations are necessary especially to

confirm the proposed structures for PFPEdCAs

and assess their presence, persistence, mobility

and bioaccumulation in other environmental

and animal matrices.

References 1. F. Arioli, F. Ceriani, M. Nobile, R. Vigano', M. Besozzi, S. Panseri, L. Maria Chiesa. Food Additives &

Contaminants Part A. 36 (2019) 1244–1252.

2. C. Barola, S. Moretti, D. Giusepponi, F. Paoletti, G. Saluti, G. Cruciani, G. Brambilla, R. Galarini; Journal of

Chromatography A, 1628, (2020), 461442.

3. J. W. Washington, C. G. Rosal, J. P. McCord, M. J. Strynar, A. B. Lindstrom, E. L. Bergman, S. M. Goodrow, H.

K. Tadesse, A. N. Pilant, B. J. Washington, M. J. Davis, B. G. Stuart, T. M. Jenkins. Science. 2020, 368(6495):

1103– 1107.

4. J. P. McCord, M. J. Strynar, J. W. Washington, E. L. Bergman, S. M. Goodrow. Environmental Science &

Technology Letters 2020 7(12): 903–908.

5. EFSA Scientific Opinion. EFSA Journal 2010; 8(2):1519

6. J. Glüge, M. Scheringer, I. T. Cousins, J. C. DeWitt, G. Goldenman, D Herzke, R. Lohmann, C. A. Ng, X. Trieri,

Z. Wangj. Environmental Scienceç processes & Impacts 2020 22, 2345-2373

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Plasma proteome investigation of COVID-19 patients with different outcomes

through an untargeted label-free LC-MS/MS approach

L. Pagani1, C. Chinello1, A. Mahajneh1, F. Clerici1, L. Criscuolo1, A. Favalli2, P. Gruarin2, R. Grifantini2, A. Bandera3,4,5, A. Lombardi3, R. Ungaro3, A. Muscatello3, F.

Blasi3,4, A. Gori3,4,5, F. Magni1

1 Proteomics and Metabolomics Unit, Department of Medicine and Surgery, University of Milano-Bicocca, 20854

Vedano al Lambro, Italy 2 Istituto Nazionale di Genetica Molecolare (INGM), 20122 Milan, Italy 3 Fondazione

IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, Italy 4 Department of Pathophysiology and

Transplantation, University of Milano, 20122 Milan, Italy 5 Centre for Multidisciplinary Research in Health Science

(MACH), University of Milano, 20122 Milan, Italy

Summary: Mass spectrometry-based proteomics represents an attractive opportunity for the study of SARS-

CoV-2 disease. Herein, an untargeted and label-free proteomic nLC-ESI MS/MS approach have been

developed and applied to plasma samples of COVID-19 patients presenting different outcomes, with the aim

to investigate proteome changes associated to the severity of the disease.

Keywords: COVID-19, Mass spectrometry, Proteomics

1. Introduction

Coronavirus Disease 2019 (COVID-19) still

represents a life-threatening disorder, since it has

caused more than 6 million deaths, and nowadays

it presents a fatality rate of 2% [1]. In this pandemic

scenario, the omics science, as mass spectrometry-

based proteomics, represent a useful tool for

COVID-19 investigation, allowing the study of

different aspects of the disease [2] and providing a

valid platform for biomarker discovering and

sample profiling [3].

Therefore, the aim of our project was to develop

an untargeted and label-free nLC-ESI MS/MS-

based method able to analyse plasma samples of

people affected by COVID-19, in order to evaluate

our sensibility in SARS-CoV-2 proteins detection

and to highlight proteomics changes in plasma

samples of patients with different outcomes.

2. Experimental and Results At first, we established our limit of detection

(LOD) for purified SARS-CoV-2 proteins,

analysing four recombinant proteins (S1-S2-RBD

of Spike glycoprotein (S) and Nucleoprotein (N))

which were purified, de-glycosylated, digested

with trypsin and analysed in different amount with

nLC-UHRTOF. A LOD of 10pg for both S and N

proteins has been determined.

As a second step, we build a MS-compatible

method to process patient plasma and inactivate

viral particles. The protocol was tested with

healthy plasma samples spiked with purified viral

proteins. After the physical and chemical

inactivation, biofluids were subjected to a

deglycosylation step followed by an enhanced

trypsinization prior to the nLC-MS/MS analysis.

Based on this workflow we were able to detect both

S and N proteins adding at least 1 µg of each viral

protein to plasma, while about 300 human proteins

were identified [4].

Finally, the developed method was applied to

plasma samples of 48 COVID-19 patients and

permitted to identify and quantify 589 and 331

human proteins, respectively. Patients were divided

into two groups, mild and severe, based on WHO

severity classification. Considering a fold change

of 1.5, 16 proteins were down- expressed, while 12

were up-regulated in the mild group, using a

supervised approach. A significant correlation

between Fetuin-A concentration and disease

severity has been observed, in line with literature

[5-6]. In order to increase the robustness of data and

remove possible confounders, analyses were

adjusted by sex and age. Excluding this

interference, 15 proteins resulted down- expressed,

while 11 were up-expressed in mild patients.

Interestingly the trend of Fetuin-A abundance

related to patient outcome remained confirmed, as

well as the expression of almost all the proteins

already observed before the adjustment.

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3. Conclusions

An untargeted mass spectrometry-based method

able to both identify purified SARS-CoV-2

proteins and the host human proteome in plasma

samples has been developed. Applying this

workflow, a panel of proteins, including Fetuin-

A, has been recognized as significantly associated

to the severity of the disease, suggesting a

possible role as biological indicators of the

disease development and a potential involvement

in the pathophysiology of SARS-CoV-2.

References 1. Kostopanagiotou K, Schuurmans MM, Inci I, Hage R.; Ann Med. 2022;54(1):588-590.

2. Costanzo M, Caterino M, Fedele R, et al.; Int J Mol Sci. 2022;23(5):2414. Published 2022 Feb 22.

3. Park J, Kim H, Kim SY, Kim Y, Lee JS, Dan K, Seong MW, Han D.; Sci Rep. 2020 Dec 29;10(1):22418.

4. Pagani L, Chinello C, Mahajneh A, Clerici F, Criscuolo L, Favalli A, Gruarin P, Grifantini R, Bandera A, Lombardi

A, Ungaro R, Muscatello A, Blasi F, Gori A, Magni F.; BioChem. 2022; 2(1):64-82.

5. Völlmy F, van den Toorn H, Zenezini Chiozzi R, Zucchetti O, Papi A, Volta CA, Marracino L, Vieceli Dalla Sega

F, Fortini F, Demichev V, Tober-Lau P, Campo G, Contoli M, Ralser M, Kurth F, Spadaro S, Rizzo P, Heck AJ.;

Life Sci Alliance. 2021 Jul 5;4(9):e202101099.

6. Kukla M, Menżyk T, Dembiński M, Winiarski M, Garlicki A, Bociąga-Jasik M, Skonieczna M, Hudy D, Maziarz

B, Kuśnierz-Cabala B, Kapusta M, Skladany L, Grgurevic I, Mikolasevic I, Filipec-Kanizaj T, Wójcik-Bugajska

M, Grodzicki T, Rogula T, Stygar D.; Biomolecules. 2021 Sep 28;11(10):1422.

Acknowledgments:

This research was funded by FAR 2017–2020; Fondazione Gigi & Pupa Ferrari Onlus; Regione

Lombardia POR FESR 2014–2020, Call HUB Ricerca ed Innovazione: ImmunHUB; and Regione

Lombardia: Programma degli interventi per la ripresa economica: sviluppo di nuovi accordi di

collaborazione con le università per la ricerca, l’innovazione e il trasferimento tecnologico.

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A novel Spatial Multi-omics mass spectrometry imaging workflow to assist

clinical investigations

V. Denti1, G. Capitoli2, I. Piga1, G. Paglia1, F. Magni1,

A. Smith1

1 Department of Medicine and Surgery, Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al

Lambro, Italy 2 Bicocca Bioinformatics Biostatistics and Bioimaging B4 Center, School of Medicine and Surgery,

University of Milano-Bicocca, 20900 Monza, Italy

Summary: Here we present the development and the feasibility of a novel spatial multi-omics MALDI-MSI

workflow for the in-situ analysis of lipids, N-Glycans, and tryptic peptides on a single FFPE tissue section.

Keywords: multimodal, spatial omics, MALDI-MSI

1. Introduction The field of spatial omics defines the gathering of

different techniques that allow the detection of

significant alterations of biomolecules in the

context of their native tissue or cellular structures.

As such, they extend the landscape of biological

changes occurring in complex and heterogeneous

pathological tissues, such as cancer. However,

additional molecular levels, such as lipids and

glycans, must be studied to define a more

comprehensive molecular snapshot of disease and

fully understand the complexity and dynamics

beyond pathological condition [1]. Among the

spatial-omics techniques, matrix-assisted laser

desorption / ionisation (MALDI) – mass

spectrometry imaging (MSI) offers a powerful

insight into the chemical biology of pathological

tissues in a multiplexed approach where several

hundreds of biomolecules can be examined within

a single experiment [2]. Thus, MALDI-MSI has

been readily employed for spatial multi-omics

studies of proteins, peptides and N-Glycans on

clinical formalin-fixed paraffin-embedded (FFPE)

tissue samples.

2. Experimental In this work, we describe a spatial multi-omics

MALDI-MSI workflow which enables the

sequential analysis of lipids, N-glycans, and tryptic

peptides on a single FFPE tissue section (Figure1).

In doing so, we first highlight the feasibility using

technical replicates of murine brain tissue.

Following, as a proof-of-concept, the approach was

applied to four clear cell renal cell carcinoma

(ccRCC) specimens to assess the ability of this

multiplexed MALDI-

MSI approach to more comprehensively characterise

the tumour tissue when combining the multi-level

molecular information.

Fig. 1. Multimodal MALDI-MSI workflow. Sample

preparation for lipids (A), N-Glycans (B), and tryptic

peptides (C) on the same FFPE tissue section

3. Results First, comparing the average spectra obtained from

lipid, N-Glycan, and tryptic peptide imaging of the

three technical replicates which were analysed on

three separate days, respectively, a high degree of

similarity can be observed. Accordingly, the mean

CV% obtained from each of the sequential

MALDI- MSI analysis lay within a range

comparable to the CV threshold of 20%

recommended by the European Medicine Agency

(EMA) for analytical techniques.

When the spatial distribution of the detected lipid,

N-Glycan, and tryptic peptides was evaluated, it

soon became clear that each

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molecular class was able to better underline

different regions of the brain tissue, indicating their

complementary nature. This was also consistent

among the three technical replicates. Additionally,

the potentiality of this spatial multi-omics MALDI-

MSI workflow in ccRCC pathological tissue was

assessed and, as observed in murine brain tissue,

each of the molecular levels showed tendencies to

better underline different histopathological regions.

To confirm the capability of each molecular class to

distinguish among the different histopathological

regions of ccRCC tissue, qualitative statistical

analyses were performed. In support of what was

observed in the MALDI-MS images themselves,

each dataset led to a complementary separation of

the histopathological regions.

When these individual datasets were then combined

into one large multi-omics dataset, the

histopathological regions were separated with

greater power and, in fact, could all easily be

distinguished from one another. This was

not possible using the lipidomics, N-Glycan, or

tryptic peptide dataset in isolation.

4. Conclusions Taken together, the spatial multi-omics MALDI-

MSI workflow presented here provides the ability

to map the distribution of lipids, N-Glycans, and

tryptic peptides on a single FFPE tissue section.

Whilst this study focuses on known

histopathological regions as proof of concept, it

underlines the increased molecular coverage that is

obtained by using a multiplexed MALDI-MSI

approach and can lead to a more comprehensive

characterisation of diseased tissue [3]. Finally,

these findings also pave the way for further

development of more powerful bioinformatics

tools which can be used to mine these spatial multi-

omic datasets and uncover hidden molecular

patterns which arise as a result of the relationship

between these multiple molecular levels.

References 1. G. C. Bingham, F. Lee, A. Naba, and T. H. Barker, Matrix Biol., vol. 91–92, pp. 152–166, 2020

2. A. Smith, I.Piga, V. Denti, C. Chinello and F. Magni, Methods in molecular biology (Clifton, N.J.), 129–142, 2021

3. Subramanian, I., Verma, S., Kumar, S., Jere, A. & Anamika, K., Bioinformatics and Biology Insights, 14, 2020.

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The detection of new products by HPLC-HRMS validates a novel stoichio-

kinetic model for the reaction between the 2,2-diphenyl-1-picrylhydrazyl radical

(DPPH•) and common antioxidants

L. Angeli, S. Imperiale, Y. Ding, M. Scampicchio, K. Morozova

Faculty of Science and Technology, Free University of Bozen-Bolzano, Piazza Università 1, 39100 Bolzano, Italy

Summary: A kinetic model for the DPPH assay was developed to describe the antioxidant activity of single

molecule and natural extracts. The mechanism involved the main reaction and the side reaction, which leads

to the generation of unknown products. The products were putatively identified with HPLC-HRMS validating

the kinetic model.

Keywords: antioxidant activity; kinetic model; products characterization

1. Introduction Food antioxidants play an important role in

improving public health since they help to fight

oxidative stress in cells. In vitro assays are usually

applied to evaluate such activity in food matrices.

The 2,2-diphenyl-1-picrylhydrazil (DPPH•) is a

stable radical used to assess the antioxidant

activity of food products. However, the common

DPPH• protocol uses a single-point measurement

and does not give information about the kinetics

of the reaction. Kinetic approaches involve the

reaction between the antioxidant and DPPH•

giving the antioxidant oxidized and the reduced

form for DPPH• [1]. The mechanism of the

reaction between DPPH and antioxidants (AOH)

is described in Eq.1.

(1)

However, the possible secondary reactions

between the transient radicals of the

antioxidant generated in the first reaction have

not been fully considered yet (Eq.2).

(2)

The radical form of the antioxidant is generally

able to quench another DPPH. Moreover,

secondary reactions lead to unknown products.

This reaction is underestimated in literature and

could explain the variability of results of the

common DPPH assay. The aim of the work was

to develop a kinetic model able to fit

experimental data using both the equation and to

calculate the stoichiometry of the reaction.

Thus, the reaction of DPPH and different

concentration of ascorbic, chlorogenic, sinapic,

ferulic, caffeic, gallic acid and Trolox was

studied. The method was then validated with

HPLC-HRMS and applied to eight herbal

extracts.

2. Experimental The absorption decay of DPPH• at 515 nm during

the reaction with antioxidants was monitored

spectrophotometrically. 100 µM of DPPH• reacted

with different concentrations (10-100 µM) of

gallic, ferulic, caffeic, sinapic, ascorbic acid and

Trolox, separately. High-resolution mass

spectrometry with Q-Exactive Orbitrap was used

to validate the method and find the products of the

side reactions. A reaction mixture with an excess

of DPPH• was injected in the HPLC after 1 h in

the dark [2]. The MS operated in negative

ionization mode with a capillary voltage of 4.5 kV

at 350 °C. Full-MS analysis was performed at a

resolution of 70,000 with AGC Target at 2e5. For

data-dependent analysis (dd-MS2) the instrument

operated at a resolution of 17,500. Eight herbal

extracts were analyzed for their total phenolic

content (TPC) and subjected to the DPPH kinetic

analysis. The fitting of the data was achieved

using the software COPASI.

3.Results

A kinetic model of second order describes the

reaction between DPPH• and common

antioxidants. Such reaction can follow a simple

model or a more complex model employing a

secondary reaction, related to the development of

reaction products, called side reaction. The fitting

of such decay yields the rate constant k1, which

describes the main reaction between antioxidants

and DPPH• and it is responsible for the velocity,

and the rate constant k2, which is attributed to a

slower side reaction considering the products

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generated between the transient radicals (AO•) and

another molecule of DPPH•. This side reaction is

involved in the ability of the antioxidant to

continue the scavenging mechanism over time.

Moreover, the stoichiometric factor of the reaction

could be easily assessed. The model was first

applied to antioxidant standards. Sinapic acid,

Trolox and ascorbic and chlorogenic acids did not

show any side reaction. Instead gallic, ferulic and

caffeic acids achieved the best fitting with k2.

High-resolution mass spectrometry (HPLC-

HRMS) was used to characterize the reaction

products of those antioxidants with the DPPH

radical. Some antioxidants, such as gallic, ferulic

and caffeic acid, showed complex reaction

products that are justified by the presence of

a side reaction in the kinetic model, as shown in

Fig. 1.

Fig. 1. Products and fragmentation spectra for the reaction between gallic acid and DPPH

For other antioxidants, such as ascorbic acid,

Trolox and sinapic acid the oxidized form or a

dimer were found. Indeed, they did not show any

side reaction in the kinetic model. Finally, the

kinetic model was applied to evaluate the

antioxidant activity of more complex matrices.

Eight herbal extracts were employed and could be

described by the kinetic model involving the side

reaction with a very good fitting (R2 > 0.99) [3].

4. Conclusion In summary, this study describes a promising

stoichiometric and kinetic approach coupled to

high-resolution mass spectrometry to monitor the

antioxidant activity of food antioxidants. These

achievements help to elucidate why a kinetic model

is superior to single-point measurement methods.

References 1. M. C. Foti et al; Journal of Organic Chemistry, 69 (2004), 2309-2314.

2. S. B.R. Berton et al.; Industrial Crops & Products, 154 (2020), 112701.

3. L. Angeli et al.; Antioxidants, 10 (2021), 1019.

ì

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MS-based approaches in drug development: elucidation of RNA-ligand-protein

interactions for the investigation of bis-3-chloropiperidines targeting TAR

A. Sosic1, R. Goettlich2, D. Fabris3, B. Gatto1

1Dipartimento di Scienze del Farmaco, Università di Padova, Padova, Italy 2Institute of Organic Chemistry, Justus Liebig University Giessen,Germany

3Department of Chemistry, University at Albany-SUNY, Albany, NY, USA

Summary: RNA has gained its credibility as a druggable target. We have recently probed the TAR RNA of

the HIV-1 genome with bis-3-chloropiperidines (B-CePs). To thoroughly characterize B-CePs effects on TAR

and its recognition by the nucleocapsid (NC) protein, we developed quantitative MS-based approaches for

the study of multicomponent RNA-based interactions.

Keywords: RNA-based interactions, STHEM-ESI, native MS

1. Introduction The Trans-Activation Response element (TAR) is

an RNA stem–bulge–loop structure involved in

several steps of HIV-1 replication.[1] We recently

showed how RNA targeting can inhibit HIV-1

nucleocapsid (NC), a highly conserved protein

known to catalyze nucleic acid melting and strand

transfers during reverse transcription. Our RNA

targeting strategy consists of the employment of

bis-3-chloropiperidines (B-CePs) to impair RNA

melting through bifunctional alkylation.[2]

Bis-3-chloropiperidines (B-CePs) are recently

developed alkylating agents, thoroughly

investigated for their mechanism of action toward

DNA.[3] Interestingly, the analysis of a large

library of B-CePs revealed that high in vitro

reactivity toward DNA mirrors a poor cytotoxicity

despite a good cell permeability.[4] The most

potent B-CePs react fast with available

nucleophiles before reaching the cell nucleus,

resulting in lower genomic DNA damage, and

higher susceptibility of competing reactions in the

cytoplasmic environment. Given this observation,

we reasoned that fast-reacting B-CePs may hide

an unveiled RNA-targeting potential and could

possibly disclose new candidates for alternative

therapeutic applications.

We therefore thoroughly characterized binding

modes by electrospray ionization mass

spectrometry (ESI-MS) analysis and we

unambiguously elucidated at the molecular level

the reaction of B-CePs with RNA.

2. Experimental All RNA substrates were purchased from IDT

(Coralville, IO). RNA constructs were prepared

by heat-refolding in order to assume their desired

secondary structure. Bis-3-chloropiperidines were

synthesized in house. Typical probing reactions

involved different B-CePs to RNA ratio in phosphate

buffer (pH 7.4). After 2h incubation at 37°C,

reactions were quenched by ethanol precipitation and

buffer-exchanged in 150 mM ammonium acetate (pH

7.0). Samples were analyzed by direct infusion

nanospray ionization either on a Thermo Fisher

Scientific (West Palm Beach, CA) LTQ-Orbitrap

Velos mass spectrometer or on a Synapt G2 HDMS

traveling- wave ion mobility spectrometry (IMS)

mass spectrometer (Waters, Manchester, UK).

Duplex dissociation was carried out in solution by

using a home-built heating block to finely adjust the

emitter temperature between 4 and 100°C.

3.Results We explored the previously uncharted reactivity

of bis-chloropiperidines (B-CePs) towards RNA.

We characterized at the molecular level the

different adducts induced by the fast reacting B-

CeP with RNA. Following an approach based on

solution thermal melting coupled with ESI mass

spectrometry (STHEM-ESI), we proved the

ability of B-CePs to induce inter-molecular cross-

links between guanines in double stranded RNA

(Figure 1), which had not been observed for any

DNA construct previously examined.[5] We

leveraged this unique feature to study specific

interactions between B-CePs and TAR RNA,

which were analytically analysed by mass

spectrometry, allowing the elucidation of B-CePs’

recognition of TAR, and highlighting an RNA-

directed mechanism of protein inhibition.

We further performed a systematic evaluation of B-

CePs-induced adducts onto TAR-NC binding to

better understand their putative inhibitory

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properties. This MS-based evaluation involved

accomplishing: (i) the qualitative and quantitative

analysis of conditions, but significant differences

were immediately evident, which could be

ascribed B-CePs-TAR adducts for the selected

test compounds; (ii) the quantification of the

crosslinks induced within the TAR RNA hairpin

structure; (iii) the quantification of B-CePs

inhibition of TAR-NC binding (Figure 2); and

(iv) analogous analyses on samples in which TAR

RNA was replaced by its complementary cTAR

DNA construct.[6] The results afforded new

valuable insights into the mechanisms of B-CePs

at the molecular level.

Figure 1. STHEM-ESI analysis of a sample obtained by mixing 2 M of double-stranded oligoribonucleotide dsRNA

with 5 M bis-3-chloropiperidine 1 and incubating for 2 h at 37◦C. (A) Representative ESI-MS spectrum obtained at

25◦C. (B) Representative ESI-MS spectrum obtained at 95◦C.

Figure 2. Representative ESI-MS spectrum of samples obtained by adding NC protein to TAR pre-treated with B-CeP 1

(50:1 compound:RNA ratio) for 2h at 37 °C. NC protein and TAR are 1 μM each. The spectra were recorded in 150 mM ammonium acetate. The inset shows an enlarged view of the detected ternary complexes.

4. Conclusions The employment of agents able to react with the RNA partners of NC blocking structure remodeling represents an original strategy that draws the spotlight on the RNA-mediated processes. At the same time,

our studies provided the opportunity to assess the merits of our MS- based approaches as a successful analytical strategy for the in vitro investigation of multicomponent complexes involving RNA.

References 1. M. Mori; L. Kovalenko; S. Lyonnais; D. Antaki; B.E. Torbett; M. Botta; G. Mirambeau; Y. Mely; Curr. Top.

Microbiol Immunol. 389 (2015), 53-92. 2. A. Sosic; G. Olivato; C. Carraro; R. Goettlich; D. Fabris; B. Gatto; Molecules, 26 (2021), 1874. 3. A. Sosic; I. Zuravka; N.K. Schmitt; A. Miola; R. Goettlich; D. Fabris; B. Gatto; ChemMedChem, 12 (2017), 1471-

1479. 4. C. Carraro; T. Helbing; A. Francke; I. Zuravka; A. Sosic; M. De Franco; V. Gandin; B. Gatto; R. Goettlich;

ChemMedChem, 16 (2021), 860-868. 5. A. Sosic; R. Goettlich; D. Fabris; B. Gatto;Nucleic Acids Res., 49 (2021), 6660-6672. 6. A. Sosic G. Olivato; C. Carraro; R. Goettlich; D. Fabris; B. Gatto; Int. J. Mol. Sci., 23 (2022), 582.

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Discovery and characterization of a novel lipid class via

LC-ESI-MS & MSn analysis: the case of Acyl-Glucuronosylglycerols

G. Ventura1, D. Coniglio1, C. D. Calvano1,2, I. Losito1,2,

T. R. I. Cataldi1,2

1 Department of Chemistry, University of Bari Aldo Moro, 70126 Bari, Italy 2Interdepartmental Research Center SMART, University of Bari Aldo Moro, 70126 Bari, Italy

Summary: A lipid class named acyl-glucuronosylglycerols, was discovered and characterized in red ripe

tomatoes (Solanum lycopersicum L.) by an RPLC-ESI-MS and MSn investigation. Fragmentation rules were

established, thus allowing a deep chemical characterization and regiochemical assignment.

Keywords: Lipidomics, tandem MS, chromatography

1. Introduction Glucuronopyranosyl-diacylglycerols (GlcA-DAG

R1/R2, Figure 1), are unusual glycolipids found in

several organisms, like bacteria [1,2] and fungi [3],

whose occurrence was recently reported even in

higher plants [4,5]. GlcA-DAG formation has

received much attention and it seems to increase

under stressful circumstances, such as phosphate-

depletion conditions [4]. Extensive research for

such a lipid class in red ripe tomato (Solanum

lycopersicum L.) lipid extracts led us to a not-yet

described lipid class, i.e. acylated R3 GlcA-DAG

R1/R2 (Acyl-GlcA-DAG), together with their

derived lyso-forms (acylated R3 GlcA-DAG R1/0:0

or 0:0/R2, Acyl-GlcA-MAG, see Figure 1) [6].

High-performance liquid chromatography and MSn

analyses were employed to characterize GlcA-

DAG, Acyl-GlcA-DAG, and Acyl-GlcA-MAG.

Simple fragmentation rules by collision-induced

dissociation tandem MS (CID-MS/MS) spectra

were established, both in negative and positive ion

modes. As a result, the proper regiochemical

assignment of Acyl-GlcA-DAG was addressed and

isobaric Acyl-GlcA-MAG and GlcA-DAG species

were distinguished.

Fig. 4. Simplified Structures of Acylated 3-(O-α-D-glucuronosyl)-1,2-diacyl-sn-glycerols

(Acylated R3 GlcA-DAG R1/R2), GlcA-DAG R1/R2, and Acylated R3 GlcA-MAG (R1/0:0) or (0:0/R2).

2. Experimental The analytical approach based on reversed-phase

liquid chromatography (RPLC) coupled with

electrospray ionization (ESI) and tandem mass

spectrometry (MS/MS) using a linear ion trap is

described in this communication. The development

of an appropriate chromatographic set-up was the

key to separate isomeric Acyl-GlcA-MAG and/or

GlcA-DAG having the same sum composition.

Since acidic extraction conditions may trigger an

artifact formation of plant acyl lipids by activating

acyltransferase enzymes, a slightly modified hot 2-

propanol extraction method [7] was devised to

demonstrate that these species might be

endogenous.

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3. Results 14 GlcA-DAG species, including four

regioisomers, containing three predominant fatty

acyl chains (viz., 16:0, 18:2, and 18:3), were

identified in tomatoes lipid extracts. Moreover, 29

Acyl-GlcA-DAG were discovered, alongside 15

acylated lyso-forms, i.e., acyl-R3 GlcA-MAG.

Furthermore, MS/MS spectra of acyl-GlcA-DAG

and acyl-GlcA-MAG as sodiated adducts,

[M+Na]+, allowed us to identify the hydroxyl

linked to C2′ of the glucuronosyl ring as the

preferential acylation position. Finally, those lipid

species were also detected and characterized in

tomato plant leaves, roots, and steams. Although

further investigation is necessary to clarify the

biological importance, such glycolipids can be

significant for the recovery of compounds with

high added value from production waste.

4. Conclusion The proposed RPLC-ESI-MS/MS approach paved

the way for the characterization of GlcA-DAG and

GlcA-MAG. The presence of acylated GlcA-DAG

species, containing fatty acyl groups linked at the

C2′, was also demonstrated both in red ripe

tomatoes and tomato plant wastes.

Fragmentation rules were established to recognize

the occurrence of these acylated glycolipids,

confirming that LC-MS techniques are a very

powerful tool in the field of lipidomics.

Acknowledgements This work was supported by the project PONa3_00395/1“BIOSCIENZE & SALUTE (B&H)” of Italian

Ministero per l’Istruzione, l’Università e la Ricerca (MIUR) and by PON “Ricerca e Innovazione” 2014-2020

CCI2014IT16M2OP005 “Caratterizzazione e valorizzazione di biomasse da scarti agroalimentari per una

efficiente produzione energetica nelle bioraffinerie”, financed by the Ministero Italiano per l’Istruzione,

l’Università e la Ricerca (MIUR).

References 1 H. Y. J. Wang, R. V. V. Tatituri, N. K. Goldner, G. Dantas and F. F. Hsu, Biochimie, 178 (2020), pp 158–169.

2 G. Hölzl and P. Dörmann, Prog. Lipid Res., 46 (2007), pp 225–243.

3 H. Diercks, A. Semeniuk, N. Gisch, H. Moll, K. A. Duda and G. Hölzl, J. Bacteriol., 197 (2015), pp 497–509.

4 Y. Okazaki, H. Otsuki, T. Narisawa, M. Kobayashi, S. Sawai, Y. Kamide, M. Kusano, T. Aoki, M. Y. Hirai and

K. Saito, Nat. Commun., 4 (2013), 1510.

5 D. Yu, T. W. T. Rupasinghe, B. A. Boughton, S. H. A. Natera, C. B. Hill, P. Tarazona, I. Feussner and U.

Roessner, Anal. Chim. Acta, 1026 (2018), pp 87–100.

6 G. Ventura, C. D. Calvano, V. Cinquepalmi, I. Losito and T. R. I. Cataldi, J. Am. Soc. Mass Spectrom., 32

(2021), pp 2227–2240.

7 I. A. de la Roche, C. J. Andrews and M. Kates, Plant Physiol., 51 (1973), pp 468–473.

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Quali-quantitative determination of toxic carbonyl compounds in the

emissions of both heated tobacco products (HTP) and conventional

cigarettes

S. Ronsisvalle1,3, F. Sipala1, N. Santuccio1, R. Rapisarda1, M. Nicolosi1, M.

Caruso2,3, G. Li Volti2,3

1 Department of Drug and Health Sciences 2 Department of Biomedical and Biotechnological Sciences3 Center of

Excellence for the Acceleration of Harm Reduction (CoEHAR), University of Catania, Via S. Sofia, 89, 95123, Catania

(Italy)

Summary:Tobacco users continually come into contact with toxicologically relevant substances responsible for multiple adverse health effects. The aim of this work was to perform a quali-quantitative

analysis of carbonyl compounds such as acetaldehyde, acrolein and formaldehyde, in the emissions of

conventional cigarettes and heated tobacco products using UPLC-Ms/Ms techniques.

Keywords:Cigarettes, UPLC-Ms/Ms analysis

1. Introduction Tobacco users continually come into contact

with various toxicologically relevant

substances that are responsible for multiple

adverse health effects [1, 2, 3]. As a result, the

focus today is on developing alternative

products that are less dangerous to health.

Several scientific evidences show that,

compared to traditional tobacco cigarettes,

new heated tobacco devices and/or electronic

cigarettes are able to contain lower levels of

compounds that are harmful to the health not

only of smokers, but also of nonsmokers who

are exposed to secondhand smoke [4, 5, 6].

2. Aim The aim of this work is to verify the hypothesis

that in the emissions of heated tobacco

products (HTP) there are lower harmful

chemicals than in conventional cigarette (CC),

given the lack of direct tobacco combustion.

The goal was achieved quantifying and

comparing the levels of carbonyl compounds

(acetaldehyde, acrolein and formaldehyde), in

the aerosols of these two products, using

UPLC-MS/MS methods for quali-quantitative

analysis. The conventional cigarette used is a

1R6F Certified Reference Cigarette provided

by the Center for Tobacco Reference Products

(CTRP) at the University of Kentucky, which

is widely used as a standard for tobacco and

tobacco product analysis [7]. The heated

tobacco product (HTP) used for the study is the

IQOS 3 device, developed by "Philip Morris

International" based in Switzerland, which was

launched on the market in Italy, Switzerland

and Japan in 2014, and is widely used today

[8].

3. Results and conclusion At the end of the UPLC-MS/MS analyses, the

levels of acetaldehyde, acrolein and

formaldehyde were compared with the

certified reference values, established by the

Health Canada Intense (HCI) Smoking

Regime for the certified reference cigarette

1R6F and for the HTP IQOS 3. The result

showed that the levels of aldehydes, including

acetaldehyde, acrolein, and formaldehyde

emitted by HTP are lower than the levels

emitted by conventional cigarette. The results

suggest that, the use of heated tobacco

products as a substitute for combustible

cigarettes can play an important role in

reducing smoking-related harm.

References 1. Lopez, A. D., Collishaw, N. E. & Piha, T. Tobacco Control 3, (1994).

2. U.S. Department of Health and Human Services. National Library of Medicine 2012, (2004).

3. Athyros, V. G., Katsiki, N., Doumas, M., Karagiannis, A. & Mikhailidis, D. P. Current Medical Research

and Opinion vol. 29 (2013).

4. Caponnetto, P., Keller, E., Bruno, C. M. & Polosa, R. Internal and Emergency Medicine vol. 8 (2013).

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5. Polosa, R., Rodu, B., Caponnetto, P., Maglia, M. & Raciti, C. Harm Reduction Journal vol. 10 (2013).

6. Caruso, M. et al. Scientific Reports 11, 24182 (2021).

7. Ortiz, A. & Grando, S. A. International Journal of Dermatology vol. 51 (2012).

8. Barreiro E, Peinado VI, Galdiz JB, Ferrer E, Marin-Corral J, Sánchez F, Gea J, Barberà JA; Am J Respir Crit

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ORALS

50 anni della DSM

Chair: Gianluca GIORGI

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The first 50 years of GSM – DSM

G. G. Mellerio

Dipartimento di Chimica, Università di Pavia

Summary: The dates of the foundation and the first activity of the mass spectrometry group (GSM), now

division (DSM), of the Italian Chemical Society (SCI), are remembered.

Keywords: Mass Spectrometry, history, Italy

The first “Italian Mass Spectrometrists’

Congress” [Convegno degli spettrometristi di

massa italiani] was organized in 1968 (April 29-

30) at the Joint Research Centre EURATOM in

Ispra (VA), thanks to efforts of Dr. Sergio

Facchetti [1]. More than 50 scientists attended

and the main topics included ionisation

phenomena, gas chromatography – mass

spectrometry coupling and inorganic solids.

Three years later, again at the Joint Research

Centre EURATOM in Ispra (VA), the second

edition of this congress took place (September

1-3, 1971) [2]. After the congress (September,

22) a promoting committee (Pompeo Capella,

Sergio Facchetti, Alberto Frigerio, Giovanni

Galli, Gian Gualberto Gallo, Salvatore

Pignataro, Antonio Selva, Luigi Zerilli) met in

Milan and founded the Group of Mass

Spectrometry. Its interests are directed to

scientific, industrial and didactic problems. In

1972, May 13, the “Group of Mass

Spectrometry”(GSM) was set up as an

interdisciplinary working group within the

Italian Chemical Society (SCI). The number of

its members was 58 in 1974 [3] and in 1977

about seventy, equally distributed between

laboratories of universities and industries, with

an availability of about one hundred instruments

of different types. An executive committee of

seven members was active, whose president for

the three-year period 1974-76 was Dr. S.

Facchetti. Immediately the GSM has sponsored

two Schools of Mass Spectroscopy, in April

1973 in Rome and in June 1975 in Milan, with

58 and 35 participants respectively from

universities and industry. The schools were both

four days long, starting from a basic level up to

somewhat specialist topics, and they included an

experimental section. The GSM also organized

a specialized meeting in Naples in December

1973 and, apart from this, the national meeting.

The third edition of the Mass Spectrometry

Congress, named for the first time “National”,

moved to Central Italy and it was organized in

1974 (April, 22-24) at the Italian National

Institute of Health [Istituto Superiore di Sanità]

in Rome [4]. In addition, mass-spectroscopic

sections have been sponsored by GSM in the

national meetings of the Italian Chemical

Society (Cagliari, 30 September 1975) and of

the Italian Vacuum Association (Perugia, 29

September-2 October 1975); these sections

concentrated on instrumental topics. The main

task of GSM for 1976 was the organization of

the 7th International Mass Spectrometry

Conference in Florence, 30 August-3 September

1976. The conference was expertly organized by

S. Facchetti and all appeared to go smoothly [5].

Over 700 registered participants arrived from

the 5 continents and 35 countries, 296 papers

and posters were presented. The 10 plenary

lectures embraced many of the topics to be

further discussed in the lecture sessions each

day (2 sessions, running 3 at a time), the round

tables (7 during the week) and the extensive

poster sessions. Salvatore Pignataro from

Catania delivered the plenary lecture: Ionisation

Techniques.

The series of the “Mass Spectrometry

Congresses” continues in 1977 (4th Edition,

September 12-14, 1977, Catania) [6], 1980 (5th

Edition, September 14-18, 1980, Rende (CS)

[7], 1983 (6th Edition, August 28-September 2,

1983, Sorrento (NA) [8], 1986 (7th Edition,

September 2-5, 1986, Torino). The ”Congress of

Mass Spectrometry” changed its name and from

1991 (L’Aquila, June, 2-5) on it became

“MASSA” followed the name of the year. These

meetings, which are held every three years, gave

an opportunity for Italian mass spectroscopists

to meet each other and discuss their problems.

The programme [9] included plenary lectures,

usually taught by experts in the field: such as in

Catania, 1977: E. Jellum, “GC-MS computer

methods applied to biomedical problems”, H.

Knöppel, “MS in environmental organic

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analysis”, and J. Seibl, “Trends in organic MS”.

Social offices were renewed during the

congress: in 1977 the new Council, which would

direct activities until 1980, was elected: A.

Selva (President and Treasurer), P. Traldi

(Secretary), A. Casalini, N. Uccella, L. Ceraulo,

G. Marino and S. Pignataro. In 1981 two

important meetings of the Italian Chemical

Society's Mass Spectrometry Group took place

in the Residential Congress Centre of the

University of Calabria at Rende (Cosenza). The

first meeting, from 8 - 12 September, was the

Scuola di Spettrometria di Massa Organica, an

advanced mass spectrometry school devoted to

organic chemists, organized in conjunction with

the Organic Chemistry Division of the Italian

Chemical Society. The programme included

lessons in theory, practical work on instruments

and open sessions for discussion, for a total of

eight hours a day. From 14--18 September was

held the 5th National Congress of Mass

Spectrometry. The programme did not consist of

the usual type of communication, but general

reports from research groups operating in Italy

about their research topics, to promote a better

knowledge of the state of art of mass

spectrometry in Italy.

In October, 1982 the Mass Spectrometry Group

(GSM) organized two important meetings in the

Area della Ricerca del Consiglio Nazionale

delle Ricerche (Research Area of the National

Research Council) in Padova (Padua). The first

meeting, from October, 6-9 was an

“Introduzione alla Spettrometria di Massa”,

organized in connection with CNR. The

program included theoretical lessons and open

sessions for discussion on the school's lesson

themes, for a total of eight hours a day. From 12-

13 October a meeting on “ La Chimica degli Ioni

Organici” (organic ion chemistry) was held.

This meeting was organized together with the

Organic Chemistry Division of SCI.

In 1985, with the president Antonio Malorni

(Naples) and the secretary Gloriano Moneti

(Florence), the “Group of Mass Spectrometry”

became the “Division of Mass Spectrometry”

(DSM) of the Italian Chemical Society with the

statement: to favour the studies in mass

spectrometry in fundamental and applied

research; to increase in Italy the knowledge of

the capabilities of mass spectrometry and to

maintain relations with other groups in foreign

countries. Members: 181, meetings per year: 5,

attendance (average participation): 40 [10].

To be continued! [11-13]

References 1. Chim. Ind., 50, 707 (1968).

2. Chim. Ind., 53, 309 (1971).

3. Bollettino del Gruppo di Spettrometria di Massa della Società Chimica Italiana – Numero I, s.l., 1974.

4. Chim. Ind., 57, 149; 59, 725 (1974).

5. Eur. Spectroscopy News, 9, 21 (1976).

6. Chim. Ind., 59, 726 (1977).

7. Chim. Ind., 63, 378 (1980).

8. Chim. Ind., 66, 290-291 (1983).



11. cfr: F. Turco, Archi e tracce. La spettrometria di massa tra fisica e chimica, La Goliardica Pavese, Pavia, 2005.

In particolare: Capitolo 7 “La spettrometria di massa in Italia” pp. 117-148.

12. 12. K. R. Jennings (ed), A History of European Mass Spectrometry, IM publications, Chichester, 2012. pp. 255-

258.

13. G. G. Mellerio, “Spettrometria di massa in Italia “, Chim. Ind., 2013 (5), 111- 115

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Mass Spectrometry in Sicily: a Historical Overview

L. Ceraulo

Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Università di Palermo, via

Archirafi N° 32,I 90123 Palermo

Summary: This short communication aims to bring back the origin and history of MS in Sicily. The first MS

laboratories and the first researchers, their scientific achievements and their overall activity in this field will

be remembered.

Keywords: Mass-spectrometry, Sicily, History

1. Introduction I gladly accepted the invitation to present this short

report on the origin of mass Spectrometry (MS) in

Sicily and on the contribution that Sicilian

scientists have made in this field and their

activities within the MS Division of the Italian

Chemistry Society (DSM-SCI).

2. The Pioneers in Sicily The first Sicilian (by birth, university education

and affiliation) who conducted researches in MS is

Salvatore Pignataro [1], graduated in chemistry

(1962) at the University of Catania.

We also find S. Pignataro in the meeting of the

promoting committee of the MS Group of the

Italian Chemical Society (Milan, September 1971)

together with P. Capella, S. Facchetti, A. Frigerio,

G. Galli, A. Selva and L. Zerilli. After a period

spent at the University of Perugia and at the

University of Bologna and one year at the MS

Laboratory of Lossing (Canadian N.R.C- Ottawa)

Pignataro came back to Catania as Full Professor

of Physical Chemistry (1975). He formed a

research group, with Orazio Puglisi, Nino

Licciardello and Giovanni Marletta, operating in

several scientific fields (MS, Photoelectron

Spettroscopy, Materials and Surfaces). In 2004 S.

Pignataro received the DSM-SCI Bruner medal

Also at the University of Catania Prof. Giorgio

Montaudo (currently Professor Emeritus of

Industrial Chemistry), leading in the early 70s the

research group on polymers, began to deal with MS

in collaboration with Prof. Helmut Ringsdorf

(University of Mainz) which had a double focusing

mass spectrometer MAT 711. These researches

were carried out together with the very young

Salvatore Foti (now Full Professor of Organic

Chemistry). Before the acquisition of the LKB

mass spectrometer by the University of Catania S.

Foti used the instrumentation of University of

Mainz (M. Prsybylski), University of Palermo (L.

Ceraulo) and University of Calabria (G. Sindona).

Later, when Prof. Montaudo became the director of

the CNR Institute for Polymers (1985), a Kratos

mass spectrometer with a FAB source was

purchased. To gain experience with the new ion

source S. Foti spent one year at the University of

Cambridge where Prof. Dudley Williams was

using an identical instrument. In the meantime,

Prof. Montaudo formed also another research

group, involving Dr. Domenico Garozzo, while

Prof. Salvatore Foti (now Associate Professor)

constituted its own group with expertise in MS of

proteins, at the Department of Chemistry. This

group is still operating and comprise Prof. Rosaria

Saletti, Prof. Vincenzo Cunsolo, Prof. Vera

Muccilli (DSM-SCI Young Researchers Award in

2006). In 2003 Prof. Montaudo received the DSM-

SCI prize for scientific research,

At the University of Messina, the first interest in

MS was shown in the second half of the 1960s by

Giampietro Cum, Professor of Industrial

Chemistry, who also had scientific contacts with

the University of Cambridge. The young Nicola

Uccella was sent to Cambridge to undertake

research in this field. Shortly after (1974) Nicola

Uccella together with the even younger Giovanni

Sindona moved to the University of Calabria,

where they conducted important research on

fundamental and applicative aspects of MS.

Subsequently both were Presidents of DSM-SCI.

In the same academic environment, Giovanni Dugo

(today Professor Emeritus of Food Chemistry) and

Giacomo Dugo founded a research group with an

excellent expertise in separative techniques also

coupled with MS. The group widened with Paola

Dugo and Luigi Mondello (currently President of

the Division of Analytical Chemistry of SCI).

As far as Palermo is concerned, the history of MS

begins in 1972 with the decision of Prof. Vincenzo

Sprio (director of the Institute of Organic

Chemistry of the Faculty of Pharmacy) to buy the

first mass spectrometer of the Athenaeum. To

achieve rapidly expertise on the field, he sent me,

young researcher with a fellowship, to the MS

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laboratory of the Institute of Chemistry of the

Milan Polytechnic. The laboratory was directed by

Prof. Antonio Selva and at the time it was one of

the most important in Italy, with more than five

years of experience! I attended the laboratory for

two years. In addition to meeting Antonio Selva,

Piero Traldi, Umberto Vettori and Roberto Tonani

who also worked there, I had the opportunity to

meet Sergio Facchetti (our first President), Luigi

Zerilli, Giovanni Galli, Alberto Frigerio, Giorgio

Mellerio, Luca Simonotti, Ermes Pella and Bruno

Gioa. Back in Palermo in 1974 (at that time as

Assistant Professor) I joined with the young

Pasquale Agozzino, Mirella Ferrugia and Liliana

Lamartina to create a peers’ research group. We

were able to purchase (in 1976) a mass

spectrometer with a resolving power of 10,000. At

the beginning our interest were focused on the

structural determination of synthetic and natural

organic compounds and the study of fragmentation

mechanisms. From this first group the expertise

was transmitted to the researchers who currently

deal with MS for environmental and agri-food

analyses at the University of Palermo. They are

Prof. Giuseppe Avellone (currently in charge of the

MS Laboratory of AtenCenter), David Bongiorno

(DSM-SCI Young Researchers Award in 2000),

Prof. Vita Di Stefano and Prof. Serena Indelicato

(winner of a DSM-SCI 2008 mobility fellowship

for young researchers). With the foundation of the

network of University Laboratories (UniNetLab

now AtenCenter) that I directed from 2008 to 2014,

the instrumentation was enhanced and in addition

to several analytical investigation, some innovative

research on the aggregation of surfactants in the gas

phase were carried out. Later I had the honor of

being the President of the DSM-SCI (2005-2007)

and in 2013 to receive the DSM-SCI award for the

activity carried out in the field of MS. Since 2010

the University laboratories were joined, in

Palermo, by the important MS laboratory of the

Regional Laboratory Quality Center (CRQ)

directed by Prof. Francesca Di Gaudio that operates

as clinical service for the AOOR Villa Sofia

Cervello.

In Palermo, another important activity linked to

MS concerns the determination of stable isotopes.

The first laboratory (equipped with a Varian MAT

250 mass spectrometer) was founded by Antonio

Longinelli (Full Professor of Isotopic

Geochemistry) and by the young Sergio Hauser

(later he will become Full Professor of Isotopic

Geochemistry). Currently, isotopic MS in Palermo

involves the University and the section of Palermo

of the National Institute of Geophysics and

Volcanology (INGV-PA). A large number of

researchers have been involved and many more are

actually involved in MS for stable isotopes of noble

gases (He, Ne, Ar, and Xe) and of (C-O-H-N) in

water and natural gas samples and in rock samples

(University of Palermo: Prof. Sergio Hauser, Prof.

Paolo Censi, Prof. Mario Nuccio, Prof. Salvatrice

Vizzini, Dr. Giovanna Scopelliti; INGV-PA: Dr.

Francesco Italiano, Dr. Andrea Rizzo, Dr. Antonio

Caracausi, Dr Rocco Favara, Dr. Giorgio Gapasso,

Dr. Fausto Grassa).

3. Meetings, Schools and Masters Numerous meetings and schools of MS have been

organized in Sicily, the most with the collaboration

of my group and that of Prof. Foti and often with

the involvement of Prof. Sindona. In Sicily both the

40th and 50th anniversaries of DSM-SCI were

celebrated. The 30th anniversary was celebrated in

the conference in Cetraro (co-chairs: G. Sindona,

L.Ceraulo and S. Foti). Finally another important

initiative has been the activation of the first

university master's degree in MS Methodologies

and Applications (AY 2007/08, 2010/11, 2011/12).

(Director L. Ceraulo; Scientific Committee: P.

Agozzino, G. Capasso, L. Ceraulo, S. Foti, G.

Sindona) at the University of Palermo, in

partnership with the University of Catania, the

University of Calabria and INGV-PA.

4. Managing Board of the DSM-SCI Sicilian scientists (S. Pignataro, L. Ceraulo, N.

Uccella, G. Innorta, S. Foti, G. Sindona, D.

Bongiorno, V. Cunsolo, G. Avellone) have been

members of the Managing Board of DSM-SCI in

different periods, since the proposing committee in

1971. They have been present to all the Managing

Boards, with the exception of the last two. Further

three Sicilian scientists have been Presidents:

Nicola Uccella (1981-1983) and Giovanni Sindona

(1996-1998) both Sicilian by birth and university

education, not as affiliation at the time of their

presidencies, and myself Leopoldo Ceraulo (2005-

2007), Sicilian by birth, university education and

affiliation.

5. Conclusions In conclusion, the activity of the Sicilian mass

spectrometrists, who also contributed to the

foundation and management of the DSM-SCI, has

been relevant. I hope that this meeting in Sicily will

further stimulate the activities of our researchers

within the Division.

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Acknowledgments: I thank Salvatore Foti (UNICT) and Giorgio Capasso (INGV-PA) for their valuable

help.

References: Foffani, S. Pignataro, B. Cantone, F. Grasso; Nuovo Cimento, 29 (1963), pp 918-929.

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ORALS

Validazione, qualità del dato e miscellanea

Chairs: Cecilia BERGAMINI &Roberta GALARINI

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Current state and future perspectives on validation of Mass Spectrometry

Methods

M. Kostakis

Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens,

Panepistimiopolis Zografou, 157 71 Athens, email: [email protected]

Summary: An overview of performance criteria for mass spectrometry that referred to validation guidelines

will be presented in this lecture. Furthermore, advantages, disadvantages, challenges and problems on

validation of promising and eventually developing non-targeted methods will be discussed.

Keywords: validation, mass spectrometry, non-targeted methods

1. Introduction In the last 20 years, mass spectrometry has become

the dominant technique in the field of research as

well as routine analysis. Mass spectrometry

minimizes the cost and time of analysis, makes

affordable lower limits of detection, and gives the

opportunity for detection of unknown compounds

or emerging pollutants. The challenges for methods

that they fitted for intended use, are becoming more

and more necessary, especially in the non-targeted

methods that guidelines are limited until now.

2. Validation of targeted methods In the first part of presentation, validation

guidelines and performance characteristics that

they related to mass spectrometry methods will be

discussed. Generic performance characteristics,

such as linearity and accuracy, mass spectrometry-

specific performance characteristics, such as

matrix effect, will be presented and its related

performance criteria.

3. Challenges in validation of non-targeted methods The following questions about non-targeted

technique validation will be addressed in the

second part of presentation [1,2]:

1. What are the differences between targeted and

non-targeted methods?

2. How is a non-targeted method validated?

3. Are all the non-targeted methods the same?

4. Are the current validation guidelines sufficient

for non-targeted methods?

References

1. J. M. Bastos da Silva, J. Chaker, A. Martail. J.C. Moreira, A David, B. Le Bot; Journal of Xenobiotics, 11 (2021),

pp 1-15.

2. K. Nachani, S. Uhlig, M. Stoyke, S. Kammlein, F. Ulberth, I. Haase, M. Doring, S. G. Walch and P. Gowik;

Preprints, (2021), 2021120420.

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Development and validation of a rapid and simple analytical method for the

simultaneous analysis of pyrrolizidine alkaloids and related N-oxides in beehive

products by salting out assisted liquid liquid combined with liquid

chromatography-tandem mass spectrometry

S. Rizzo1, R. Celano1, L. Campone2, L. Rastrelli1, A. L. Piccinelli1

1Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Salerno, Italy 2Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza Della Scienza 2, Milan, I-20126,

Italy

Summary: A simply, fast and cheap analytical procedure for the simultaneous analysis of PAs and PANOs in

beehive products was developed. The developed method, based on SALLE technique before the instrumental

analysis by UHPLC-MS/MS, provided a sensitive and accurate determination of the target analytes.

Keywords: pyrrolizidine alkaloids, beehive products, UHPLC-MS/MS

1. Introduction Pyrrolizidine alkaloids (PAs) are phytotoxins

widely distributed in about 3% of flowering plants.

They are specialized metabolites synthesized

by different plant species (Boraginaceae,

Asteraceae and Fabaceae) as defence against

phytophagous insects and herbivorous

animals. [1]. 1,2-Unsaturated PAs are defined as

hepatotoxic, genotoxic, and carcinogenic agents

and they are one of the most significant hepatic

phytotoxins class. The possible routes of exposure

of the human diet to PA occur, therefore, through

accidental ingestion of plants, herbal products

(drug, teas, food supplements), besides to products

of vegetal-animal origin, such as honey and pollen

[1, 2]. Considering the potential risk that PA

represents to human health, there is growing

interest from the scientific community in relation

to these compounds and beehive products, mainly

honey and pollen.

2. Experimental SALLE was evaluated as extraction and clean-up

technique for the analysis of PAs and PANOs in

honey and pollen. UHPC-MS/MS was selected as

highly sensitive and selective multiresidue method.

The critical parameters (pH of the aqueous

solution, extraction solvent, type and amount of

salting-out agents) affecting SALLE performance

were carefully studied and optimized using a

chemometric approach. The proposed method

was validated and applied to honey and pollen

samples.

3. Results The study of SALLE conditions allowed to select

and optimize the factors affecting on the extraction

efficiency and to provide acceptable extraction

efficiencies for PAs and PANOs. Under the

optimal experimental conditions the developed

procedure showed excellent extraction efficiency

(86-104 %) and recoveries (78-113 %) for all target

analytes, in both studied matrices. The proposed

method showed negligible matrix effect for both

studied matrices and satisfactory intra-day (< 10)

and inter-day (< 13) precisions. Method detection

and quantification limits were at very low ppb

levels for all target analytes. The proposed method

was successfully applied to the analysis of honey

(n=71) and pollen (n=6) samples.

4. Conclusions This study describes the first application of SALLE

to the determination of PAs in food matrices.

Compared with the most widely used method in PA

analysis, the proposed method provides a

comparable accuracy and higher sensitivity, with

lower costs and analysis times, and reduced use of

organic solvents. Its represents a simple and low-

cost alternative for screening and quality control

programs and occurrence studies.

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References

1. EFSA. (2011). European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (CONTAM).

Scientific Opinion on Pyrrolizidine alkaloids in food and feed. EFSA Journal 2011, 9(11), 2406; 1–134.

2. EFSA, European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (CONTAM), F. C.,

Knutsen, H. K., Alexander, J., Barregard, L., Bignami, M., Binaglia, M. (2017).. EFSA Journal, 15(7).

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Perfluoroalkyl and polyfluoroalkyl substances (PFASs) in honey by LC-Q-

Orbitrap: method development and validation study

C. Barola 1, S. Moretti 1, A. Piersanti 2, T. Tavoloni 2, A. Stramenga 2, D. Giusepponi 1, F. Paoletti 1, S. Sdogati 1, R. Galarini 1

1 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ”Togo Rosati”- Perugia 2 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ”Togo Rosati”- Ancona

Summary: A method was developed for the determination of forty PFASs at ng/kg levels in honey by means of

LC-Q-Orbitrap technique. Validation experiments showed satisfactory performances for most of the analytes.

The procedure was then applied to forty Italian honeys of different geographical and floral origins.

Keywords: Perfluoroalkyl substances (PFASs), honey, LC-Q-Orbitrap

1. Introduction Per- and polyfluoroalkylated substances

(PFASs) have been produced since the late

1940s. They are a family of more than 4000

fluorinated aliphatic synthetic compounds

used in hundreds of industrial products such

as fire-fighting foams, coatings and paper

products for food packaging [1]. Over the

past decade, PFASs have proven to be

ubiquitous in a variety of globally distributed

environmental and biological matrices

thanks to their high resistance to degradation

processes. Bee products are generated in

environments, which may be polluted by

different contamination sources and

contaminants may reach nectar, pollen, plant

exudates through air, plants, water and soil

and then be transported into the bee hives by

bees. Therefore, honey is not only a food

product with world-wide consumption, but it

can be also a good indicator of

environmental pollution. To date few

methods have been developed for PFAS

determination in honey and, in addition, they

include only a restricted number of target

analytes [2, 3]. The aim of this work was the

development and validation of an analytical

procedure for the determination of forty

PFASs in honey of different floral and

geographical origin.

2. Experimental

Honey samples were treated and analysed by

liquid-chromatography Q-Orbitrap mass

spectrometry (LC-Q Exactive Plus,

Thermoscientific, San Jose, CA, USA) under

the conditions reported in Barola et al. [4]

with modifications. The forty PFASs were

quantified applying isotopic dilution with the

addition of twenty-five labelled internal

standards. The validation study was carried

out through spiking experiments at eight

different concentrations (from 2 ng/kg to 500

ng/kg) applying a nested design [5]. The

method performance characteristics

(linearity, precision, trueness, limits of

detection and quantification) as well as the

measurement uncertainty were estimated by

means of an in-house software developed in

R environment [6,7]. Finally, forty Italian

honey samples were collected in local

markets and analysed by the validated

procedure.

3. Results At the beginning, the sample treatment

protocol already optimized for tissues was

applied without any modification [4].

However, for honey, the extraction step had

to be drastically changed dissolving the

sample directly in 20 mL of a mixture of

MeOH and a solution of 100 mM ammonium

acetate (50:50, v/v). The solubilized sample

was then purified through a double clean-up

step [4]. The validation study results were

satisfactory for the most of the forty tested

PFASs with intra-laboratory reproducibility

coefficients of variation lower than 20% and

trueness from 80 to 110%. Limits of

quantification were lower than or equal to 10

ng/kg for the majority of the analytes.

Finally, the validated method was applied to

forty honey samples of different botanical

origin (acacia, chestnut, multiflower and

orange) produced in Italy. Five PFASs

(PFPeA, PFHxA, PFHpA, PFOA, PFDA)

were detected in thirty-five honey samples.

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The most polluted honey type was chestnut

honey with concentrations of PFPeA and

PFHxA from 2 ng/kg to 25 ng/kg. In Figure

1, the LC-Q-Orbitrap chromatograms

recorded for a multiflower honey are

compared with the chromatograms of same

honeyspiked at 100 ng/kg. As shown, some

PFASs were detected also in the unspiked

honey and, although their peaks had a

negligible area (< 1 ng/kg), this observation

confirmed the ubiquity of per- and

polyfluoroalkylated substances and the need

to prevent laboratory contamination [4].

Fig.1- LC-Q-Orbitrap chromatograms of a multiflower honey sample (left) and of the same honey fortified at 100 ng/kg (right)

4. Conclusions The developed procedure demonstrated

good performance characteristics in terms of

selectivity, linearity, accuracy, detection and

quantification limits. These results were

finally confirmed by the participation in a

Proficiency Test (EURL-PT-PF_2102-HO)

organised by the European Union Reference

Laboratory of Friburg (Germany) obtaining

z-scores between -2 and +2. Analysis of

honey samples purchased in local markets

showed low contamination. The PFASs

found most frequently were the medium-

chain perfluoroalkyl carboxylates (C5-C8

PFCAs).

Acknowledgments The authors gratefully acknowledge financial support from the Italian Health Ministry (Ricerca

Corrente “Determination of PFASs in food by liquid chromatography coupled to mass spectrometry

detection” IZSUM 06/2020 and Ricerca Corrente “Multiclass methods for the determination of

antibiotics in food. Squaring the circle: eggs and honey” IZSUM RC0032019).

References

1. E. M. Sunderland, X. C. Hu, C. Dassuncao, A. K. Tokranov, C. C. Wagner, J. G. Allen. Journal of Exposure

Science & Environmental Epidemiology 29, (2019) 131–147

2. M. Surma, W. Wiczkowski, E. Cieślik, H. Zieliński, Microchemical Journal 121, (2015) 150–156

3. M. Surma, H. Zieliński, M. Piskuła. Bullettin of Environmental Contamination and Toxicology 97, (2016) 112–

118

4. C. Barola, S. Moretti, D. Giusepponi, F. Paoletti, G. Saluti, G. Cruciani, G. Brambilla, R. Galarini;

Journal of Chromatography A, 1628, (2020), 461442.

5. Eurachem Guide. Planning and Reporting Method Validation studies. First edition 2019

https://www.eurachem.org/images/stories/Guides/pdf/MV_Guide_planning_supplement_EN.pdf. 6. The Comprehensive R Archive Network. https://cran.r-project.org

7. R. Galarini. XXII International Mass Spectrometry Conference, Florence (Italy) ThP-199, 1211-1212,

(2018) https://www.imsc2018.it/images/IMSC2018_Abstract_Book.pdf

http://www.eurachem.org/images/stories/Guides/pdf/MV_Guide_planning_supplement_EN.pdf

http://www.imsc2018.it/images/IMSC2018_Abstract_Book.pdf

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Material screening of electronic devices by ICP-MS and HR-ICP-MS: towards a

low background for astroparticle physics experiments

F. Ferella1,2, S. Nisi1

1 INFN, Gran Sasso National Laboratories, via G. Acitelli 22, I-67100, Assergi (L’Aquila), Italy

2Department of Physical and Chemical Sciences, University of L’Aquila, via Vetoio, I-67100, L’Aquila, Italy

Summary: Low background is a fundamental requirement to astroparticle physics experiments to maximize

sensitivity. Gran Sasso National Laboratories are an ideal place to reduce background through rock

thickness of above mountain. Cosmic rays are reduced about 106 factor, furthermore radiopure material are

also used in experiments.

Keywords: trace analysis, low background

1. Introduction Material screening is a fundamental step to follow

to evaluate different type of materials used in setup

of astroparticle physics experiments that requires a

low radioactive background [1]. Electronic

components play a very important role during

amplification and transfer of signals obtained by

detectors, for this reason use of high purity

materials is necessary. Cables, circuits, electronic

boards, pins, supports, connections, and other

components required for set-up are subjected to a

preliminary screening to determine their purity and

therefore possible application and use. Inductively

Coupled Plasma Mass Spectrometry (ICP-MS) has

been used to characterize all materials adopted for

this procedure, to search for impurities inside

samples and to monitor contamination level]. High-

Resolution ICP-MS was used to determine amount

of impurities regard to natural radioactive elements

(Th, U, K), lower detection limits were reached

improving our analysis. Search of materials with

low level of contamination, as optical fibers, TPB,

nylon foils, circuits will be discussed to make a

preliminary screening related to materials to be

used for detectors assembly.

2. Materials and methods Chemicals used in sample preparations were HNO3

ultra-pure grade obtained from Hyper pure grade

nitric acid (VWR Chemicals, Canada) through sub-

boiling system (DuoPur, Milestone, Italy), HF

from Panreac, H2O UP (resistivity 18.2 MΩ/cm at

25°C) obtained from a Milli-Q (Millipore

Corporation, Billerica, Ma, USA). All

measurements were performed by mean Sector

Field High Resolution Inductively Coupled

Plasma-Mass Spectrometer (Element II, Thermo

Fisher Scientific, USA) equipped with an ASX520

autosampler from Cetac (Omaha, NE, USA).

Standard solution of 25 ppt Th and U in oxalate was

used as a reference material to calibrate instrument.

Instrumental calibration with certified standard

solution and recovery test were carried out to

perform measurements. ISO 6 clean room located

at LNGS was used to preparative analysis and

characterizations to avoid risk of environmental

contamination.

3. Results and discussion A preliminary step to take into consideration

information was to evaluate which radionuclides

ware sought in materials of our interest, in order to

be able to focus characterization on specific

masses; presence of radionuclides with natural

radioactivity give a contribute that get worse

background, so sensitivity could be decrease.

Instrument calibration was performed by standard

solution, recovery test was performed by adding a

known amount of solution (spike solution) in order

to evaluate total efficiency of our system.

Mineralization of samples was different according

to type of materials, in specify case ashing

treatment, acid dissolution, microwave digestion.

Regards to some types of material, it was important

to determine radioactive contribution of various

components by treating them separately, different

mineralizations were performed to discrimine

contaminations and subsequently evaluate total

contribution. HR-ICP-MS was adopted to perform

quantitative analysis, focusing on element of our

interest, and to reduce interferences; working in an

enrivoment with a very high presence of argon,

polyatomic compounds that are formed generate

various interferences especially in low resolution,

therefore with medium and high resolution it’s

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possible to reduce them even if there’s a loss of

sensitivity.

4. Conclusions ICP-MS has proved to be a highly sensitive and

versatile technique for carrying out

characterizations of materials involved in physics

experiments. Fast time of analysis, high sensitivity,

use of very small sample amounts, are great

advantages that are widely exploited in this type of

work. Over the years, rare event searches like those

hosted in LNGS laboratories investigating neutrino

and dark matter have screened different materials

through ICP-MS characterization. Also, other

techniques are used (e.g. gamma-ray spectrometry)

[2] to have a general overview of contamination

inside materials

References 1. F. Ferella, S. Nisi, F. Marchegiani, Physics, 3 (2021), pp 71-84

2. S. Nisi, M. Laubenstein, et al., Appl. Radiat. Isot., 67 (2009), pp 828-832

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Different applications of the Liquid Chromatography coupled to Isotope Ratio

Mass Spectrometry (LC-IRMS)

M. Perini1, M. Pellegrini2, S. Pianezze1

1Centro Trasferimento Tecnologico, Fondazione Edmund Mach, San Michele all’Adige (TN), Italy

2Thermo Fisher Scientific S.p.A, Rodano (MI) -Italy

Summary: The liquid chromatography interface coupled with IRMS permits the compound-specific δ13C

analysis of non-volatile and water-soluble compounds in complex mixtures without resorting to derivatization.

Applications in paleoarchaeology, nutrition and trophic, pediatrics, soil science and food genuineness are

examined here by reporting the progress and the technical constraints.

Keywords: stable isotope ratio analysis, liquid chromatography

1. Introduction Isotope Ratio Mass Spectrometry (IRMS) is a mass

spectrometry technique aiming to measure the

relative stable isotopic abundances of the elements

that comprise a specific material. The stable

isotope ratios of carbon (13C/12C), hydrogen

(2H/1H), oxygen (18O/16O), sulfur (34S/32S) and

nitrogen (15N/14N) are usually measured to provide

information about the geographical, chemical and

biological origin of substances. The IRMS can be coupled with different types of

devices, in particular liquid (LC) or gas

chromatographs (GC), so that the separation of the

specific compounds in the studied matrices is

performed before measuring the isotope ratio. The

association between the liquid chromatography and

the isotope ratio mass spectrometry (LC-IRMS),

although more recent than the GC-IRMS, has an

undisputed potential that will be here illustrated.

2. Results and Discussion The three most commonly used liquid

chromatographic techniques coupled with the

IRMS are thereafter described. Ion exchange

chromatography (technique 1) was used for the

determination of the carbon isotope ratios of

carbohydrates, amino sugars and amino acids.

However, it is difficult to completely resolve the

proteinogenic amino acids when adopting ion

exchange as the only mechanism of separation. The

resolution of many amino acids can be consistently

improved by using a reversed phase (RP) column

(technique 2). Almost all amino acids can be

resolved by mixed mode chromatography

(technique 3), i.e., the combination of RP and ion

exchange interaction sites in the same stationary

phase, which is today the most published method

for LC-IRMS analysis of non-derivatized amino

acids. The compounds that can be studied by

applying this technique range from carbohydrates

(in honey and sweet wine for the identification of

sugar adulterations or for the study of human

glucose metabolism and in the environmental

field), to organic acids (such as citric acid

fraudulently added in juices), to amino acids and

polypeptides (for the separation and the measure of

the 13C isotope enrichment of non-derivatized

amino acids in ecological, nutritional, trophic and

pediatric studies) and to herbicides.

Recently, the application of high temperatures to

the mobile and stationary phases (HT-LC-IRMS)

was proposed as an alternative approach to increase

the elution strength of aqueous eluents in reversed

phase LC. This specific variant of the method has

so far been applied to two matrices only: caffeine

and pharmaceutical products.

3. Conclusions The LC-IRMS is a powerful tool for the analysis of

a wide variety of different matrices and can be

applied to different research fields, as shown by

several studies. Compound-specific isotopic

analysis can be much more informative than the

bulk one, and the technological advances that make

it possible to isolate a specific compound before the

isotopic analysis by an LC interface lead to the

opening up of broad perspectives.

The LC technique proves to be an effective

alternative approach to the GC technique, offering

the advantage to avoid the derivatization of the

sample, which can lead to unwanted fractionations.

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References

1. M. Perini, L. Bontempo; Liquid Chromatography coupled to Isotope Ratio Mass Spectrometry (LC-IRMS): a

review, TrAC Trends in Analytical Chemistry, 147 (2022), pp 1–11.

,

.

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Validation of analytical method for the determination of cortisol in hair by

UHPLC-MS/MS

C. Maccari1, G. Magnani3, G. Niccoli3, M. Corradi1,2, R. Andreoli1,2

1Laboratory of Industrial Toxicology, Department of Medicine and Surgery, University of Parma, Italy 2Centre for Research in Toxicology (CERT), University of Parma, Italy.

3 Department of Medicine, University of Parma, Parma.

Keywords: Cortisol, chronic stress, LC-MS/MS

1. Introduction Cortisol is a steroid hormone commonly known as

the "stress hormone". Like all steroid hormones, it is

produced from cholesterol. The stages leading to its

synthesis take place at the level of the adrenal glands

under the control of the hypothalamic-pituitary-

adrenal axis. The main factors involved in controlling

cortisol secretion are: negative feedback, stress-

induced stimuli and circadian rhythm. The rate of

cortisol production shows high intra- and inter-

individual variability and is related to a physiological

circadian rhythm. Usually, cortisol levels were

quantified in saliva, urine, or blood, to describe an

extemporary, non-specific oxidative status.

Chronic hyper- or hyposecretion of cortisol is

influenced by environmental factors, psycho-

physical stress and involves long-term physiological

changes.Chronic stress can increase the susceptibility

of the individual to poor health and expose him to

different diseases: in particular, several studies have

shown that acute and chronic stress factors, can

increase the probability to have heart events, such as

heart attack.

Hence the interest in the use of cortisol as a biomarker

for chronic stress, usable as a prognostic marker for

many diseases but also as a predictor and therefore

applied in the prevention field.

In literature, the cardiovascular effects of chronic

cortisol exposure are described but there are not

enough evidence that its levels, measured in

biological fluids such as urine, blood or saliva, could

be used as a prognostic marker.

Determination of cortisol levels in hair can be a

reliable biomarker of long-term HPA axis activity

since it reflects total cortisol production for several

weeks or months and it is thought that the amount of

cortisol deposited in the hair is proportional to the

concentration of systemic cortisol.

Since the hair has a growth rate that generally stands

around 1 cm/ month; the concentration of a

compound in a specific hair segment is closely

related to the exposure suffered by the subject in the

period corresponding to the hair growth under

examination. For this reason, hair analyses allow a

retrospectively investigation of the production of

cortisol and can prove valuable to use cortisol as an

indicator of chronic stress.

2.Experimental We studied cortisol and 6β-hydroxy-cortisol (6β-

OHF) in 140 spot urine samples of healthy

volunteers, collected in the time window 8 a.m-10

a.m. to reduce the individual variability linked to the

circadian rhythm and in 14 strands of hair of healthy

volunteers. All the subjects were recruited on a

voluntary basis, were over the age of 18, and with no

chronical stress exposure history.

LC-MS/MS determinations were carried out on a

6500+ triple quadrupole mass spectrometer (Sciex),

equipped with a TurboIonSprayTM interface.

Chromatography was performed on an Atlantis dC18

column using variable proportions of 10 mM aqueous

formic acid and methanol at a flow-rate of 0.35

ml/min (Figure 1).

Fig. 2. Chromatograph with peak of two standard

Cortisol and 6β-OHF were ionized in negative ion

mode and detection was obtained in selected-reaction

(SRM) mode. For all analytes, the [M+HCOOH-H]-

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was selected by the first mass filter, to increase the

sensitivity of the analytical method (Table 1).

Table 1. working condition of mass spectrometry

Q1 Q3 compound DP EP CE CXP

407 331 cortisol -20 -10 -25 -18

407 297 cortisol -20 -10 -40 -15

407 282 cortisol -20 -10 -50 -15

423 423 6β-OHF -40 -10 -10 -15

423 347.3 6β-OHF -40 -10 -25 -18

Urine samples were analysed after thawing,

centrifugation and dilution with a solution of water

and methanol.

Working on a pool of hair, the better sample

treatment was investigated comparing three different

extraction procedures and different sample

quantity/extraction volume ratio. Particularly, the

tested extraction conditions were: (i) heating at 50 ºC

for 12 hours; (ii) ultrasonic for 20 minutes; (iii)

overnight agitation at room temperature in MeOH;

and the sample quantity/extraction volume ratios

were: (i) 50 mg of hair in 1 ml of MeOH in eppendorf;

(ii) 50 mg of hair in 1 ml of MeOH in Falcon; (iii)

100 mg of hair in 2 ml of MeOH in Falcon.

3. Results The limits of quantification, defined as signal to noise

ratios equal to ten (S/N=10) were calculated in

solvent and were 0.5 ng/L for cortisol and 0.12 ng/L

for 6β-OHF. Working calibrations were performed in

matrix and calibrating standards were treated as

samples. The imprecision of the method, calculated

as %RSD at three different concentration levels (n=6)

in all matrices, was within 1-4% for intra-day and 3-

9% for inter-day determinations.

LC-MS/MS method was validated on urine samples

because it is a biological matrix easier to collect then

hair, more accessible and it is known to have a natural

physiological content of cortisol and 6β-OHF. The

concentration levels of urinary cortisol and 6β-OHF

were found to be of the same order of magnitude as

the reference values.

Fig. 2. Analysis of extraction yields

Looking at the hair, the sample treatment that allowed

the higher recovery of cortisol was 100 mg of hair in

Falcon with 2 ml of methanol stirring at room

temperature overnight; while in none of tested

conditions, 6β-OHF was found to be in

concentrations above the limit of quantification (0.12

ng/L).

Then we obtained preliminary results related to the

natural cortisol content in hair. We divided the

strands of hair into segments of 1 cm each, starting

from the most proximal to the most distal segments

from the scalp of four healthy individuals and we

analyzed each segment.

We observed a progressively decrease of cortisol

levels starting to 3.9 pg/mg near the neck from 0.3

pg/mg corresponding to the final part of the hair; the

results were in accordance with the literature [2] and

the founded phenomenon is known as “wash-out”

effect [3].

4. Conclusions We developed a new analytical method that allowed

the quantification of cortisol in the hair samples.

Although some more tests are needed, cortisol

determination in the hair is a promising tool for a

chronic stress assessment.

References

1. Gatti, R., Codemo, C., De Palo, E.F. Biochim. Clin., 34(20109, pp 591-599.

2. Gao, W., Stalder T., Foley, P., Rauh, M., Deng, H., Kirschbaum, C. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci,

928(2013), pp 1-8.

3. Mayer, S.E., Lopez-Duran, N.L., Sen, S., Abelson, J.L. Psychoneuroendocrinology; 92 (2018), pp 57–65.

4. Iob E., Steptoe A., Current Cardiology Reports 21 (2019), pp 116.

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Fast and sensitive high-throughput antineoplastic drugs surface contamination

monitoring by ultra-high performance liquid chromatography coupled with

tandem mass spectrometry

S. Dugheri1, N. Mucci2, D. Squillaci2, G. Marrubini3, G. Bartolucci4, C. Melzi5, E. Bucaletti2, G. Cappelli2, L. Trevisani2, C. Valsecchi6, V.Consonni6, F. Gosetti6, D.

Ballabio6, G. Arcangeli2

1 Industrial Hygiene and Toxicology Laboratory, Occupational Medicine Unit, Careggi University Hospital,

50134 Florence, Italy 2 Department of Experimental and Clinical Medicine, University of Florence, 50134

Florence, Italy; 3Department of Drug Sciences, University of Pavia, Via Taramelli 12, 27100 Pavia, 4 Department

of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, 50019 Sesto

Fiorentino, Italy; 5 Metanalisi, Corso di Porta Venezia 40, 20121 Milano, Italy; 6 Milano Chemometrics and QSAR Research Group,

Department of Earth and Environmental Sciences, University of Milano-Bicocca, 20126 Milan, Italy;

Summary: Antineoplastic drugs are an heterogeneous group of substances that can cause severe toxic

effects. Hospital surfaces contaminations are a growing issue and therefore require the development and

implementation of sensible and fast monitoring methods. The objective of this work is to create a high-

throughput wipe-test sampling procedure coupled with UHPLC-MS/MS analysis.

Keywords: Antineoplastic drugs; LC-MS/MS; Wipe test;

1. Introduction

Antineoplastic drugs (ADs) are a wide and

heterogeneous group of substances that can

cause highly severe toxic effects to whoever is

exposed. From an occupational safety point of

view, surface contaminations inside preparation

and administration units are a growing issue and

therefore require the development and

implementation of sensible and fast monitoring

methods. Wipe-test sampling procedure coupled

with (U)HPLC-MS/MS analysis is at the time

being the most used method to evaluate surface

contamination. However, it still presents a

challenging environment for mass spectrometry

analysis, due to the presence of organo-metallic

compounds, such platinum-based ADs, isobaric

compounds and isomers couples. At the same

time, the variety of molecules required the use

of two separated chromatographic methods,

both in reverse and normal phase.


The methods were developed on a Shimadzu

LCMS 8050 triple quadrupole, equipped with

an electrospray source (ESI) and coupled with

a Nexera X2 UHPLC system. Each of the 34

ADs underwent a scan spectrum evaluation,

collision induced dissociation of the selected

precursor ion and optimization of the

quantitative and qualitative fragment ions

selected. Seven levels of calibration solutions

were prepared using drug products in a

concentration range from 5 to 50 ng/mL, with

the exception of cisplatin which needed

concentrations from 40 to 400 ng/mL. Internal

quality control samples were prepared to

evaluate accuracy and precision of the method.

For what concerns the normal phase method, a

co- injection program has been used to inject

bigger volumes of sample without altering the

peak shape. The optimization of the mobile

phases composition has been carried out by the

use design of experiments (DoE). Wipe

samples have been desorbed with a 50:50

water-methanol solution, filtered and injected

without further purification.

3. Results and discussion

The major difficulties have been encountered

during the development of the mass spectrometry

method, particularly for what concerns the

selection of the precursor ion. The wipes’ matrix,

even if relatively clean, might contain many salt

residues, deriving from the drugs excipients and

from the monitored surfaces, and thus create an

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abundant amount of adduct ions in ESI. This

issue has been evident in the analysis of

platinum-based ADs (PtADs), whose metal

complexes nature render particularly exposed to

the formation of collateral adducts and

modification of the very composition of the

drugs, especially considering that platinum itself

brings a very complex isotopic cluster. Cisplatin,

whose structure is the most labile among PtADs,

is divided in many different signals in the

positive ion scan, deriving not only from adducts

with the salts present in the matrix, but also from

the formation of complexes with the solvents’

molecules. For the same reason a few of the

precursor ions had to be selected between

ammonium and sodium adducts. However, the

obtained results were fitting the needed

performances, showing a precision and

accuracy error lower than 15%. The fast

sample preparation and analysis time allow the

monitoring, in the same sampling campaign, of

more than 100 points, making possible a wide

and punctual search of the ADs contamination in

a reasonable instrument work time. This way, not

only the average contamination of a preparation

or treatment unit can be checked, but also an

evaluation of contamination patterns which lead

to that result.

4. Conclusions

The developed methods gave the possibility to

monitor 34 ADs surface contaminations in the

order of pg/cm2 (ng/mL injected) with a single

sampling and two LC-MS/MS runs, with a total

time of analysis lower than 30 min/sample.

They proved to provide a useful tool not only

for the contamination monitoring but also to the

comprehension of how the contamination

might be generated.

References

1. Dugheri, S.; Mucci, N.; Squillaci, D.; Marrubini, G.; Bartolucci, G.; Melzi, C.; Bucaletti, E.; Cappelli, G.;

Trevisani, L.; Arcangeli, G. Separations 2021, 8(9), 150.

2. Dugheri, S.; Mucci, N.; Mini, E.; Squillaci, D.; Marrubini, G.; Bartolucci, G.; Bucaletti, E.; Cappelli, G.;

Trevisani, L.; Arcangeli, G. Separations 2021, 8(5), 69.

3. Dugheri, S.; Mucci, N.; Squillaci, D.; Bucaletti, E.; Cappelli, G.; Trevisani, L.;Valsecchi, C.; Consonni,V. ;

Gosetti, F.;Ballabio,D.; Arcangeli, G. Separations 2022, 9(2), 34.

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Rapid analysis and interpretation of metabolomics SWATH acquisition

A. Antonoplis, J. Causon, C. Hunter

SCIEX

SWATH acquisition is a data independent

acquisition (DIA) workflow that has been

demonstrated to improve metabolite coverage over

traditional data dependent techniques for

untargeted metabolomics. The workflow enables

creation of a digitized record of the metabolome

present in a sample, with full (MS1) and MS/MS

scans capturing every detectable analyte in a single

injection. The richness of SWATH acquisition data

offers numerous data analysis opportunities, one of

which is to identify differential metabolites across

sample groups. While instrumentation and

appropriate methods for collecting metabolomics

SWATH acquisition data are well-established, the

lack of software tools for processing large-scale

metabolomics SWATH acquisition studies remains

a challenge for wide-spread adoption of the

workflow in laboratories. The OneOmics suite, a

cloud- based solution for SWATH acquisition data

processing, enables fast and confident

identification of differential metabolites across

experiment groups in large-scale metabolomics

SWATH acquisition studies. The entire processing

workflow, from meta data management to

examining biological significance of results, is

supported in the platform.

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POSTERS

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A general analytical platform and strategy for the screening and determination

of pyrrolizidine alkaloids in food matrices with high risk of contamination using

high-resolution mass spectrometry (Q-Orbitrap)

S. Rizzo, R. Celano, L. Rastrelli, A. L. Piccinelli

Dipartimento di Farmacia, Università degli studi di Salerno, Via Giovanni Paolo II, 84084 Fisciano (SA), Italia.

Summary: A general analytical platform for the screening of pyrrolizidine alkaloids in food matrices with

high risk of contamination was developed in this study. The platform includes a first highly selective extraction

procedure (SALLE) and a second step of analysis (UHPLC-MS/MS), allowing to determine 108 pyrrolizidine

alkaloids in the studied matrices.

Keywords: pyrrolizidine alkaloids, high-resolution mass spectrometry, targeted screening analysis

1. Introduction Pyrrolizidine alkaloids (PAs) are a large group of

naturally occurring phytotoxins recently regarded

as undesirable substances in plant-derived food

products due to their genotoxic and carcinogenic

activities [1-2]. A high-throughput screening and

confirmatory method combining salting-out

assisted liquid-liquid extraction (SALLE) with

ultra-high performance liquid chromatography

coupled with high resolution tandem mass

spectrometry (UHPLC-MS/MS) for the detection

of pyrrolizidine alkaloids in food matrices with

high risk of contamination (beehive products,

herbal infusions, and plant-based dietary

supplements) was developed in this study [3-4].

Firstly, an in-house diagnostic product ion filtering

strategy was developed and applied to naturally

PAs-producing plants to build a spectral library to

be used for the screening of a huge number of real

samples. A total number of 108 pyrrolizidine

alkaloids were analysed, including

30 reference standards, 45 PAs identified and

characterized in nine PAs-producing plants from

two different families: Asteraceae (Eupatorium

cannabinum, Lithospermum officinale, Petasites

hybridus, Senecio vulgaris, Tussilago farfara) and

Boraginaceae (Anchusa officinalis; Borago

officinalis, Echium italicum, Symphytum

officinale), based on their exact mass and

fragmentation pattern and confirmed with well-

known chemotaxonomic data and online databases,

and 33 tentatively identified using the diagnostic

product ion filtering strategy and literature

information. The mass spectrometer operated in

positive ion mode and full-HRMS- data-dependent-

MS2(full-HRMS-dd-MS2) acquisition mode at a

resolution of 70 000 (full- HRMS) and 17 500 (dd-

MS2). The screening method was applied and then

validated in food matrices with high risk of

contamination (beehive products, herbal infusions,

and plant-based dietary supplements), which differ in

the nature of co- extractants and may cause a variable

matrix effect [5]. The method proved to be linear up

to a concentration of 50 µg L–1 (R2 > 0.99). The limits

of quantification (LOQs) of the reference standards

were lower than 1 µg kg–1 for all the tested matrices.

Recoveries were in the range of 70-120% and

repeatabilities, expressed as relative standard

deviations (RDSs) lied in the range of 1- 11%.

2. Experimental and Results A wide database of PAs (617 molecules) was built

by drawing on EFSA and literature data; the

database was implemented by collecting

information about the collecting molecules such

as CAS number, elemental composition, molecular

weight and m/z ratio of precursor and product ions.

The collected PAs were then grouped into different

classes according to the necine base type

(retronecine, heliotridine, platynecine, otonecine,

supinidine and trachelanthamidine) and the

esterified necic acid type (monoester, open-chained

and cyclic diester) to develop an in-house

diagnostic product ion filtering strategy to be used

for the rapid screening and determination of PAs in

food matrices with high risk of contamination.

The crucial part of the platform building was the

development of the diagnostic product ion filtering

strategy as it allowed us to first build and then

continuously implement the HRMS spectral library

through the characterization of PAs- producing

plants and finally detect (and sometimes identify)

unknown PAs through the screening analysis of

real samples. Literature studies were fundamental

for the development of the strategy; however,

important clarifications also arose during the

screening analysis of real samples thanks to the

comparison of detected

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spectra with those present in online spectral

libraries (the most consulted was the MoNA

online library). The strategy was divided into

two subsets since the literature studies

immediately allowed to delineate the different

behavior of PAs and PANOs regarding their

fragmentation patterns. The PA and PANO’s

subsets of the strategy are shown in the Figures

1 and 2 respectively.

Figura 1. PAs’ subset of the strategy.

Figura 2. PANOs’ subset of the strategy.

The analytical screening method was successfully

applied to the analysis of a huge number (250) of

real samples (including honeys, pollen, food

supplements, black and green teas, and herbal

infusions) proving to be effective in the screening

and detection of both target and unknown PAs.

Overall, the percentage of contamination resulted

to be high in herbal infusions composed by mixed

plants (95.5 %), honey samples (89.0 %), black

teas (77.3 %), solid plant-based dietary

supplements (62.9%), and pollen samples

(50.0%); however, the percentage of samples

exceeding the MLs was only worrying for pollen

(33.3 %), herbal infusions composed by mixed

plants (21.6 %), black teas (16.6 %) and to a lesser

extent honey (6.9 %). No one of the solid dietary

supplements exceeded the MLs established by the

2020/2040/EU Regulation. Regarding herbal

infusion labelled as dietary supplements, green

teas, and herbal infusions composed by single

plant, the percentage of contamination resulted to

be low and no one of the samples exceeded the

MLs. The results of the present study highlight the

need to extent the monitoring studies beyond the

list of the most relevant PAs (28) to be monitored

in food and herbal products proposed by the

CONTAM Panel of EFSA to increase the amount

of data available on this topic and thus preserve the

health of consumers.

3. Conclusions A general platform for the simultaneous

screening and determination of 108 hepatotoxic

pyrrolizidine alkaloids in matrix with high risk of

contamination has been developed for

monitoring purpose in the present study. Target-

PAs could be detected at sub to low ppb levels

within a fast analysis of 30 min, including the

easy and cheap salting-out assisted liquid-liquid

extraction and the chromatographic separation.

High resolution mass spectra were acquired in

full MS data dependent MS2 acquisition mode,

which allowed to verify the identity of the target-

PA within the HRMS spectral library or to

further search before deciding if a confirmatory

analysis is needed. Furthermore, the method can

be easily and continuously expanded to

accommodate additional target compounds and it

can be even re- interrogated without having to re-

analyse the samples to search for PAs not target

at the moment of the analysis but discovered at

a future time.

References 1. EFSA CONTAM Panel (2016) EFSA Journal 14 (8), 4572.

2. EFSA CONTAM Panel (2017) EFSA Journal 15 (7), 4908.

3. Rizzo, S., Celano, R., Campone, L., Rastrelli, L., & Piccinelli, A. L. (2022). Journal of Food Composition and

Analysis, 108, 104457.

4. Chung, S. W., & Lam, C. H. (2018). Journal of agricultural and food chemistry, 66(11), 3009- 3018.

5. Magnusson B. & Örnemark U. (2014). Eurachem Guide: The Fitness for Purpose of Analytical Methods

120.0808 ≈60% 120.0808 bp

138.0910 ≈40% 138.0910 <5%

83.0491 ≈20%

220.1332 <5%

PANOs type R or H P or S

open or cyclic P P open or cyclic monoesters

diesters monoesters diesters

136.0757 <50%

R open diesters

H open diesters

214.1074 bp 254.1387 bp 214.1074 >50% 254.1387 >50%

O7-Acetyl O7-Angelyl O7-Acetyl O7-Angelyl

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Determination of sugars in olive leaves in response to water deficit

C. Benincasa, R. Nicoletti, M. Pellegrino, F. Carbone, A. Salimonti

CREA - Research Centre for Olive, Fruit and Citrus Crops, 87036 Rende (CS), Italy

Summary: This work presents the results of the composition of carbohydrates in olive leaves from plants

irrigated with different water regimes to study the role of sugars in the response to water deficit. Quantitative

analysis of glucose, fructose, galactose, and mannose was performed by cesium attachment electrospray

tandem mass spectrometry.

Keywords: Mass spectrometry, Sugar analysis, Olive leaves

1. Introduction Green plants produce carbohydrates through the

process of photosynthesis. These compounds are

used either as energy sources for vegetative growth

and development or as precursors in the

biosynthesis of a wide range of molecules,

including lipids, proteins, and polysaccharides.1

Selected plant species appear to have evolved the

biochemical processes and physiological

conditions necessary for with standing unfavorable

environmental variables, including drought,

flooding, salinity, irradiance, and both high and

low temperatures.2,3 This study describes the

evaluation of sugars, glucose, fructose, galactose,

and mannose, in olive leaves whose plants were

irrigated with different water regimes to study the

role of sugars in the response to water deficit. In

fact, understanding the mechanisms by which olive

plants tolerate water deficit is essential for

selecting more tolerant plant cultivars. Quantitative

analysis of sugars was performed by cesium

attachment electrospray tandem mass

spectrometry.

2. Experimental Materials. The experimental material consisted in

leaves of six cultivars of olive plants: Arbequina,

Coratina, Frantoio, Itrana, Leccino, and Moraiolo,

reased under controlled conditions. A certain

number of replicas, for each experimental thesis,

were subjected to a treatment with commercial

mycorrhizae, which would seem to contribute to

the improvement of the resilience of the olive trees

in response to water stress. Olive leaf samples

were collected and freeze dried until analyses.

Chemicals. Cesium iodide, acetonitrile, glucose,

fructose, galactose, and mannose were purchased

from Sigma Aldrich (Italy). Distilled water was

produced in situ by means of a Milli-Q plus system

(Millipore, Bedford, MA, USA).

Extraction procedure. Approximately 0.2 g of

dried leaf was extracted with 100 ml of distilled

water by sonication (15 min) and further

centrifugation (10 min). The filtered supernatant

solution was analysed by electro spray ionization

tandem mass spectrometry (ESI-MS/MS). The

extraction was done in triplicate.

Quantitative analysis. Standard stock solutions

were prepared dissolving the sugar standards in

distilled water. Aliquots of these solutions were

further diluted to obtain five calibration standards

at concentrations range between 0.625 and 10

g/mL. Calibration curves were built using a least-

squares linear regression analysis with correlation

coefficients between 0.9996 and 0.9999. The

accuracy and precision were evaluated at two

concentration: 3.25 and 6.50 g/mL (recovery

between 94 and 109 %).

LC–MS/MS analysis. Measurements were

performed by using a MSD Sciex Applied

Biosystem API 4000 Q-Trap mass spectrometer.

The LC–MS was operated in the positive ion mode

using multiple reaction monitoring (MRM): for

each analyte, the transition of the deprotonated

molecular ion [Sugar-H]+ was scanned on the first

quadrupole and its main fragments on the third one

[Sugar-Cs]+. The experimental conditions were as

follow: ionspray voltage (IS) 5500 V; curtain gas

15 psi; temperature 100 °C; ion source gas (1) 35

psi; ion source gas (2) 45 psi; collision gas

thickness (CAD) medium; entrance potential (EP),

declustering potential (DP), entrance collision

energy (CE) and exit collision energy (CXP) were

optimized for each transition monitored. The

analytes were separated on a Chromegabond

carbohydrate column [(5 m particle size, 15 cm

length and 2.1 mm i.d.) at a flow rate of 300

mL/min with an injection volume of 10 L. A

binary mobile phase consisted of acetonitrile (A)

and CsCl H2O 54 M (B). The total elution time

was 10 min per injection.

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3. Results and Conclusions The sugars profile of olive leaves can be

conveniently determined by following the gas

phase chemistry of their cesium adduct. In this

work, for each cultivar studied, the highest sugar

content was recorded in the leaves belonging to

plants that have undergone water stress. For the

experimental theses subjected to commercial

mycorrhizae treatment, it wasn’t observed a

significantly contribute to the improvement of the

resilience of olive trees in response to water stress.

The results obtained can be explained by

considering that the accumulation of the sugars in

olive leaves is a strategy to provide a long-term

protection against future periods of water deficit.

Acknowledgments This work was supported under the Progect

“ALIVE” (D.M. MiPAAF 93880 del 29/12/2017).

References

1. C.M. Duffus and J.H. Duffus; Carbohydrates: Structure, Location, Function. By Carbohydrate

Metabolism in Plants; Longman House: Burnt Mill, Harlow Essex, UK, 1984.

2. H.K Lichtentaler; J. Plant Physiol. 1995, 148, 4.

3. J. Ingram, D. Bartels; Plant Mol. Biol. 1996, 47, 377.

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Development of mass spectrometric methodologies for the quality assessment of

bergamot-based food products in the framework of the activities of the

QUASIORA laboratory

I. Santoro2, L. Bartella1,2, F. Mazzotti1,2, L. Di Donna1,2

1 Department of Chemistry and Chemical Technology, University of Calabria, Via P. Bucci, Cubo 12/D, Rende, CS,

87036, Italy 2 QUASIORA Laboratory, Agrinfra Research Net, University of Calabria, Via P. Bucci, Cubo 12/D, ,

Rende, CS, 87036, Italy

Summary: The key mission of the QUASIORA laboratory is to provide technological support to the small and

middle enterprises operating in the agri-food district, through the setting up of innovative research

methodologies which aim at qualifying the food products.

Keywords: QUASIORA laboratory, mass spectrometry, agri-food products, bergamot

1. Introduction The QUASIORA (Quality, Safety, and Origin of

Food) laboratory was established in 2009 at the

Department of Chemistry and Chemical

Technologies of the University of Calabria

(UNICAL) in accordance to the project Action 2,

FAP - Scientific Research and Technological

innovation, funded by Calabria Region.

The main activity of the laboratory relies on the

development of new and accurate analytical

methods, based on mass spectrometry, for the

assessment of the quality of foods. The

QUASIORA lab is involved in the following fields:

• Quality and safety of foods: The technical

expertise present in the lab allows the design

and the development of protocols and

methodologies addressed to the quali-

quantitative assay of molecular markers in

typical foods of the territory

• Nutraceutical: development and

characterization of “medical foods” and “novel

foods”

• Determination of the food origin and product

qualification through molecular fingerprint.

By 2015, UNICAL has filed the trademark

QUASIORA at the Chamber of Commerce in

Cosenza; in 2020, the QUASIORA laboratory

received a new grant from Calabrian Region in

order to realize its main mission, which is to

valorise the typical food products of the region,

through the application of those innovative

methods, helping the local companies to grow-up

in those markets which require high-quality

products [1-6].

Currently, QUASIORA operates with companies,

through i) collaboration in research grants such as

the case of “Terre Grecaniche”, a consortium of

wine producers, or “Azienda Pratticò” a local

producer of bergamot-based beverages and the

“Cedro di Calabria Consortium”, which promotes

the production and the use of Citron, a typical

Calabrian citrus fruit, or ii) by simple third-party

activities as in the case of Esserrepharma, a

company which produces bergamot-based food

supplements.

Here are shown some of the activities of the

researchers of the QUASIORA laboratory who

developed a new method, based on LC_MSMS, for

the characterization of quality markers molecules

and to prevent safety issues in bergamot foods.

Fig. 1 The laboratory logo-trademark registered at the

Chamber of Commerce of Cosenza

2. Experimental Coumarines and flavonoids were directly analyzed

in food supplements from Esserrepharma and food

beverages from Azienda Pratticò after dissolution

(if needed), filtration and centrifugation. High

resolution HPLC/MS (Thermo Orbitrap Exploris

120) operating in positive mode was used for the

analysis of coumarines. The assay was performed

in SIM mode following the exact mass of

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Bergapten and Bergamottin, the two main

coumarines in bergamot. The HPLC run was

obtained by a gradient of H2O (0.1% Formic Acid)

and Acetonitrile eluents. The total run time was 16

minutes. The coumarines were quantified through

an external calibration curve that was built

sampling five standard solution in triplicate at

different concentration (25, 50, 100, 200, 400

µg/L).

Instrumental determination of flavonoids was

carried out by High Performance Liquid

Chromatography (Thermo Accela) coupled with a

Triple quadrupole mass spectrometer (Thermo TSQ-Vantage) operating in negative ionization

mode using MRM scan mode. Pure neoeriocitrin,

neoesperidin, naringin, peripolin, brutieridin and

melitidin standards were used for the assay

together with caffeic acid as internal standard. A

calibration curve has been built sampling five

standard solution in triplicate at different

concentration (25, 50, 100, 200, 400 µg/L) and the

internal standard (caffeic acid) concentration was

set at 100 µg/L. The HPLC run was obtained by a

gradient of H2O (0.1% Formic Acid) and Methanol

eluents. The total run time was 26 minutes.

3. Results The methods developed were applied to different

juices and extracts from Bergamot. The analyzed

samples were divided into 28 liquid extracts of

bergamot, 10 solid extracts, 10 commercial juices

(1 juice sweetened with stevia). The concentration

of flavonoids ranged between 260-1000 mg/L in

the commercial juices, 28300 mg/l in the liquid

extracts, 30% w/w in solid extract.

The concentration of coumarins present in the

extract samples varies from between 0-140 mg/L

for the liquids and 0.003-0.4% w/w in solids.

4. Conclusions The application of the methods developed by the

QUASIORA laboratory to the agri-food products

can support the small and middle enterprises to

qualifying their products, facilitating their access to

the market worldwide, and pushing forward the

business initiative and job growth.

References 1 L. Bartella, F. Mazzotti, ,A. Napoli, G. Sindona, L. Di Donna. Analytical and Bioanalytical Chemistry 410(8) (2018)

pp. 2193-2201

2 L. Bartella, F. Mazzotti, ,A. Napoli, G. Sindona, L. Di Donna. Food and Chemical Toxicology 136 (2020).

3 L. Di Donna, L. Bartella, L. De Vero, M. Gullo, A.M. Giuffrè, C. Zappia, A. Caridi. European Food Research and

Technology. 246(10) (2020) pp. 1981-1990.

4 L. Di Donna, D. Taverna, S. Indelicato, A. Napoli, G. Sindona, F. Mazzotti. Food Chemistry 229 (2017) pp. 354-357

5 G. Sindona, L. Di Donna. Toxic Chemical and Biological Agents (2020) pp. 1-10

6 L. Di Donna, G. De Luca, F. Mazzotti, A. Napoli, R. Salerno, D. Taverna, G. Sindona. Journal of Natural Products

72(7) (2009) pp.1352-135

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Identification of sinapoylquinic acids in coffee by “surrogate standards”

fingerprinting: a win-win strategy

S. Colomban 1, E. Guercia1, E. De Angelis1, L. Navarini2

1Aromalab illycaffè S.p.A., AREA Science Park, Padriciano 99, 34149 Trieste, Italy

2illycaffè S.p.A., via Flavia 110, 34147 Trieste, Italy

Summary: The focus of this paper is to identify sinapoylquinic acids (SiQA) in coffee extracts using an

extract of Gardenia Fructus as surrogate standard for the characterization of the 3 isomers.

Keywords: sinapoylquinic acids, coffee, UHPLC‐ESI‐MS/MS

1. Introduction Chlorogenic acids (CGAs) are a large class of ester

formed between quinic acid and hydroxycinnamic

acids. They are present in coffee as a complex

mixture of positional and geometric isomers, where

caffeoylquinic acids (CQA) are the most abundant,

followed by dicaffeoylquinic acids (diCQA) and

feruloylquinic acids (FQA) [1]. Sinapoylquinic

acids are the most widely distributed of these less

common cinnamic acids, being reported in

Gentianales [2] (the order with the greatest range),

Aquifoliales, Asterales, Caprifoliales, and

Solanales. The lack of synthetic standards or the

high cost for authentic commercial standards for

LC/MS identification and quantification could be

the weak spot in the metabolites identification

process, but a different strategy proved to be

effective: the use of surrogate standards supported

by LC/MS data providing retention time, UV-vis

spectrum, molecular mass and mass fragmentation

data [3]. This strategy allows the fingerprinting of

a specific class of compounds that, in most cases,

can be identified unequivocally at minimal cost.

2. Experimental Gardenia Jasminoides fruits (zhi zi) extract and

food supplement containing gardenia fruits and

artemisia capillaris thumb. (yin chen hao tang)

were purchased from Qiu Tian Srl (Acquaviva, San

Marino Rep.), Arabica and Robusta lyophilized

coffee extracts were prepared in our lab from an

aqueous ethanolic extract. Sample were extracted

with water/ethanol (30/70 v/v) for 30 min at 60°C

in an ultrasound cleaning bath Sonorex RK100

(Bandelin), the ratio of sampling weight to

extraction solution volume was 20. After extraction

the sample was centrifuged (5 min, 8602× g RCF

at 20°C, Allegra 64R Centrifuge, Beckman

Coulter, Indianapolis, IN, USA) and filtered with

regenerated cellulose (RC) membrane 0.20 µm

(Reliaprep Ahlstrom-Munksjö Oyj, Helsinki,

Finland) and diluted with water if needed. Analyses

were performed with UHPLC-DAD and UHPLC-

MS/MS as previously reported in literature [4].

3. Results All data for chlorogenic acids presented in this

manuscript use the recommended IUPAC

numbering system. Generally, peak assignments

have been made on the basis of the structure-

diagnostic hierarchical keys published in literature

[5], supported by examination of the UV spectrum

and retention time relative to 5-caffeoylquinic acid.

The three isomers of sinapoylquinic acids are

characterized by precursor ion at m/z 397, so we

focused only on compounds producing a precursor

ion of m/z 397. Fragmentation was recorded and

compared with literature. We decided to start our

sinapic acid derivatives exploration with

lyophilized Gardeniae Fructus (ZhiZi and Yin

Chen Hao Tang) extract, available on the market at

an affordable price. When identification of

sinapoylquinic acids in these extracts was

successful we optimized the identification method

with a Multiple Reaction Monitoring mode (MRM)

to identify the regioisomers of sinapoylquinic acid

in coffee. Three isomers were detected with

precursor ion at m/z 397, these eluted after 5-

caffeoylquinic acid. The first sinapoylquinic acid

was assigned as the 3-isomer (3-SiQA). The most

strongly retained isomer was assigned to 4-

sinapoylquinic acid (4-SiQA) and has a

fragmentation characteristic of 4-acyl chlorogenic

acid. The last isomer elutes between 3- and 4-

sinapoylquinic acid, and has a fragmentation at m/z

191, characteristics of the 5-acyl chlorogenic acid,

so it was assigned to 5-sinapoylquinic acid (5-

SiQA).

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Once the three regioisomers were successfully

identified we added the specific transitions of

SiQAs to the MRM method we routinary apply for

the determination of chlorogenic acid profile in

coffee, and we were able to identify SiQAs on

different samples: food supplement containing Zhi

zi and coffee (both Arabica and Robusta).

4. Conclusions In conclusion we proved that using a matrix that is

particularly rich in secondary metabolites of

interest as a standard to identify and optimize an

LC-MS/MS method is an effective and cheap

strategy to fulfil characterization of those

compounds in other matrices.

Once the identification of the sinapoylquinic acids

is performed we observed that this chlorogenic acid

derivatives are present in both Arabica and Robusta

coffee, and further investigations and

quantification of these compounds are needed.

We strongly believe that the approach used to

identify new compounds on coffee matrix is

effective and can help research team to improve the

characterization of minor compounds already

described in literature without the need of time

consuming organic synthesis or expensive

commercial standards purchase.

References

1. A. Farah, J. De P. Lima, The Royal Society of Chemistry, Coffee, London (2019) pp 584–610.

2. M.N. Clifford, W. Wu, J. Kirkpatrick, R. Jaiswal and N. Kuhnert, Rapid Commun. Mass Spectrom, 24 (2010), pp

3109–3120.

3. H. Čížkova, V. Soukupova, M. Voldřich, R.Sevcik, Journal of Food and Nutrition Research, 46 (2007), pp 28-34.

4. S. Colomban, E. Guercia and L. Navarini, J Mass Spectrom, 55 (2020), e4634.

5. M. N. Clifford and N. E. Madala, J Agric Food Chem, 65 (2017), pp 3589-3590.

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Stable isotope analysis to detect differences in four compartments of Simmental

cull cows fed on C3 and C4 diets

S. Pianezze1,2, M. Corazzin2, L. Bontempo3, A. Sepulcri2, E. Saccà2, M. Perini1, E.

Piasentier2

1Centro Trasferimento Tecnologico, Fondazione Edmund Mach, San Michele all’Adige (TN), Italy 2Dipartimento di Scienze Agroalimentari, Ambientali e Animali, University of Udine, Udine (UD), Italy

3Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all’Adige (TN), Italy

Summary: The isotopic analysis of the fat (EA-IRMS) and the fatty acids (GC-C-IRMS) of 13 multiparous

Italian Simmental cull cows made it possible to discriminate between animals fed on different feeding regimes

(either C3-based or C4-based) and to collect information about the metabolic pathway of the FAs in the bovine

organism.

Keywords: fatty acids, isotope ratio mass spectrometry, gas chromatography

1. Introduction Fatty acids (FAs) are long chain carboxylic

acids that can be found in the adipose tissues and

the muscles of bovines. They may derive from

the diet only, as for the essential FAs (such as

linoleic and linolenic acid), and/or from de novo

endogenous synthesis [1]. The FAs metabolic

pathway starts in the rumen, where the

hydrolysation of dietary complex fats into long

chain FAs occurs [2]. Then, the FAs released

during this process are converted into saturated

FAs through biohydrogenation reactions [3].

The FAs reach the liver carried by the blood,

whose flow, together with the FAs

concentration, influences their supply to this

organ [4]. The deposition of the FAs into the

animal tissue can be considered as the final step

of their metabolism.

2. Results and Discussion The aim of this study was to discriminate

between two groups (nTOT = 13) of Italian

Simmental cull cows fed on different diets and

to widen the knowledge about the FAs

metabolic path in the bovine organism. The first

group was fed on a C3 products-based diet

(δ13CC3_BULK_DIET = -32.55‰) while the

second one was fed on a C4 products-based one

(δ13CC4_BULK_DIET = -18.74‰). Beside the

diet, three compartments of the animals were

considered: rumen, liver and meat. The fat

extracted from the four matrices was both

analysed through EA-IRMS (elemental

analysis-isotope ratio mass spectrometry), as a

bulk sample, and through GC-C-IRMS (gas

chromatography-combustion-isotope ratio mass

spectrometry), after a derivatization process

which made it possible to measure the δ13C of

five FAs (C16:0, C18:0, C18:1n-9, C18:2n-6

and C18:3n-3). A good discrimination between

C3 and C4 groups was achieved. Moreover,

different trend of δ13C passing from the diet to

the loin were found as for C3 and C4 groups.

3. Conclusions The δ13C analysis of the bulk fat and the FAs

helped in shedding light on the metabolic path

that the FAs follow in the bovine organism and

on how this path changes depending on the diet.

Nevertheless, the metabolic path of the FAs is

still far from being completely understood.

Therefore, more studies focusing in particular

on the various chemical reactions taking place

into the rumen, should be carried out.

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References 1. C. Galli, P. Risé; European Journal of Lipid Science and Technology, 108 (2006), pp 521–525.

2. Buccioni, M. Decandia, S. Minieri, G. Molle, A. Cabiddu; Animal Feed Science and Technology, 174 (2012),

pp 1–25.

3. W.W. Christie, X. Han; Lipid Analysis, (2012), pp 3–19.

4. J.F. Hocquette, D. Bauchart; Reproduction, Nutrition, Development, 39, (1999), pp 27–48.

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LC-MS/MS characterization of phenolic compounds in olive oils obtained with

different extraction techniques

C. Benincasa1, M. Pellegrino1, G. Lacertosa2, G. Montanaro3

1 CREA - Centro di Ricerca Olivicoltura, Frutticoltura, Agrumicoltura, Rende (CS), Italy 2 ALSIA - Centro Ricerche Metapontum Agrobios, Metaponto (MT), Italy

3 Università degli Studi della Basilicata, Dipartimento delle Culture Europee e del Mediterraneo (DICEM), Matera

(MT), Italy

Summary: The objective of this study was the evaluation of the physical-chemical and organoleptic

parameters of the oils obtained with different extraction techniques, with particular emphasis to the

determination of phenolic compounds performed by tandem mass spectrometry.

Keywords: Extra virgin olive oil, Oil extraction process, Phenolic compounds.

1. Introduction Olive oil represents the main source of fat in the

Mediterranean diet and represents a valuable food

product from both nutritional and economic point

of view. The nutritional value and the health

promoting effects of extra virgin olive oil (EVOO)

rely on its favorable nutrient composition,

including oleic acid as the most abundant fatty acid

and fat-soluble vitamins and the presence of

phenolic compounds1–3. The latter are recognized

in contributing to the positive health effects related

to the consumption of extra virgin olive oil4, 5 and

can be affected by the production process involving

crushing the olives into a paste, mixing the pastes

in order to progressively favor the coalesce of oil

drops and separating the oil from the aqueous

phase5–7. The legal term "Extra virgin olive oil of a

superior category obtained directly from olives and

solely by mechanical procedures" does not give the

right emphasis to the production process where

important biochemical processes take place. In

fact, if not kept under control, the extraction system

can lead to a deterioration in the quality of the

product. By favoring the goal of maximum yield

and extraction efficiency by adopting

hyperoxidative methods and olive paste

temperatures above 30 °C, the quality of the oil

produced is compromised. On the contrary, by

adopting non-invasive extraction technique, as the

uses a module capable of conditioning the

temperature of the paste before it is subjected to

malaxation, it can guarantee the reduction and

degradation of phenolic substances, tocopherols,

phytosterols, pigments, ensuring the integrity of the

glycerine component as well. The objective of this

study was, therefore, the evaluation of the physical-

chemical and organoleptic parameters of the oils

extracted with different types of olive mills

techniques, with particular emphasis to the

determination of phenolic compounds.

2. Experimental Olive samples of Coratina, Frantoio e Leccino

cultivar were collected in the crop year 2019–2020.

The extraction systems utilised consisted of

hammer and knife crushers, two and three-phase

decanters with malaxer cooling temperatures at 10-

12 °C and 26-27 °C. Free fatty acids, peroxide

values, UV absorption characteristics, and fatty

acid methyl esters were determined as described by

the official method of Europaean Union

Commission Regulation 2568/9119. Phenols were

determined by following extraction procedures

optimised at CREA laboratory. The crude

methanol extracts containing the phenolic fraction

were analyzed by electro spray ionization tandem

mass spectrometry (ESI-MS/MS) using a MSD

Sciex Applied Biosystem API 4000 Q-Trap mass

spectrometer in the negative ion mode using

multiple reaction monitoring (MRM). The analytes

were separated on an Eclipse XDB-C8-A HPLC

column (5 μm particle size, 150 mm length and 4.6

mm i.d.) (Agilent Technologies, Santa Clara, CA,

USA) at a low rate of 300 μL/min with an injection

volume of 10 μL. A binary mobile phase made up

of 0.1% aqueous formic acid (A) and methanol (B)

was gradient programmed to increase B from 10 to

100% in 10 min, hold for 2 min and ramp down to

original composition (90% A and 10% B) in 8 min.

The total elution time was 20 min per injection.

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3. Results and Conclusions The different types of mills did not significantly

affect the oil yields and the quality parameters such

as the content of methyl esters of fatty acids, free

acidity, the number of peroxides and the

spectrophotometric constants (K232, K270 and

△K). The extraction conditions, on the other hand,

resulted in a significant difference in the content of

the antioxidant component. The oils that were

found to be richer in phenolic compounds were

those produced with a two-phase extraction system

equipped with a malaxer cooling at temperatures of

10-12 °C. In fact, the rapid cooling of the pasta was

able to produce an increase in the phenolic

concentration probably thanks to the inhibiting

effect of the polyphenoloxidase which is

deactivated at these temperatures. Similar situation

with regard to the content of alpha tocopherol.

Furthermore, the oils produced with a two-phase

extraction system equipped with a malaxer cooling

at temperatures of 10-12 °C were found to have

superior organoleptic properties.

Acknoledgements The research activities were performed under the Action 4 of the Project “Ottimizzazione della Redditività e

della Gestione degli OLIveti e dei processi produttivi dell’Olio LUCANO - O.R.G.OLI.O. LUCANO” (PSR

Basilicata 2014-2020 - Bando Misura 16 - Sottomisura 16.2 - "Sostegno a progetti pilota e allo sviluppo di

nuovi prodotti, pratiche, processi e tecnologie" – EX D.G.R. 26 Settembre 2018 N. 976 E SS.MM.II.).

References 1. E. Rhouma et al.; Natural Product Research, (2019) https://doi.org/10.1080/14786419.2019.1637867.

2. S. Lupinacci et al.; J. Nephrol. (2016), DOI 10.1007/s40620-016-0368-4.

3. Martinez-Gonzalez et al.; Circ. Res. (2019), 124, 779–798.

4. Visioli et al.; Br. J. Pharmacol. (2020), 177, 1316–1330.

5. Clodoveo et al.; Ultrasonics Sonochemistry, (2013a), 20 (5), 1261-1270

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Tuna Fish wastes valorisation: green production and triacyclglycerols

characterization of Tuna fish oils by HPLC/HRMS and GC/MS

S. Indelicato, D. Bongiorno, G. Avellone, V. Di Stefano, L. Ceraulo, Mirella Vazzana, M. Mauro, V. Arizza

Department of Biological, Chemical and Pharmaceutical Science and Technology (STEBICEF), University of Palermo,

Via Archirafi, 90123 Palemo, Italy;

Summary: The wastes obtained from various commodities are often characterized by a content of bioactive

compounds that justifies their recovery. In this work a “green” procedure to obtain a tuna-fish oil rich in

polyunsaturated fatty acids has been developed and the final product characterized.

Keywords: fatty acids, tuna fish oil, mass spectrometry

1. Introduction Food waste generated in great quantities worldwide

is a current point in the view of a more sustainable

circular economy. Depending on the type of food a

different degree of wastes is generated, taking into

consideration both food chain and industrial

processing. The residues produced by the various

supply chains in several cases are still

characterized by a good content of bioactive

molecules which, properly recovered, have the

potential to be reintroduced at different stages of

the production cycles of other products, often

reinforcing their intrinsic value. For this reason, are

always welcome the development of new protocols

to recover molecules of nutritional/nutraceutical

interest from by-products and waste of the agri-

food and industrial production chain. The simplest

and less invasive recovery procedures are of course

preferable as they should do not introduce

bioactive principles modification, and reduce the

presence of xenobiotic substances.

2. Experimental Sample preparation of fatty acids methyl esters

(FAMEs) for the analytical determination by

GC/MS has been carried out dissolving

approximately 0.1 g of each oil in a 5 mL screw top

test tube with 0.2 mL KOH (2 N) in methanol

solution and 1 mL n-hexane. The solutions were

vigorously shaken for 30 min and left to stratify

until the upper solution became clear. For GC

analysis, a Thermo Fisher TSQ 8000 was used,

equipped with a Trace 1300 GC. Samples

preparation for the analytical determination of

TAGs by LC/HRMS has been simply made by

dissolving 0.5 μL of oil in 1.5 mL of methanol. The

samples thus prepared were placed in an ultrasonic

bath for 5 min, in order to facilitate the dissolution.

For LC-APCI/MS, analysis used a Waters Q-Tof

Premier equipped with a thermostatic autosampler.

The chromatographic details are reported in [1].

3. Results The chemical characterization of the extracted oil

has been performed determining fatty acids methyl

esters analysed by GC/MS, and triglycerides

(TAGs) determined by an HPLC/HRMS approach

based on a ballistic gradient. Both the results

obtained have been compared with literature data

[2] based on commercial tuna fish oil, and are in

good agreement. On the other hand, several other

TAGs have been identified in our study, and, in a

semi quantitative approach, their relative

abundances have been used to determine fatty acid

composition by means of an in-silico

saponification approach [3].

4. Conclusions The described approach allowed to individuate and

determine 84 different TAGs. The in-silico

saponification, based on the identified TAGs and

their relative abundance, gave relative percentual

of fatty acids that are in fair agreement with the

fatty acid percentages determined by the traditional

FAMEs protocol, suggesting a good and correct

identification/quantification of the TAGs.

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Fig. 5 Total ion current HPLC/MS trace of the tuna fish oil. L=Linoleic acid;P=palmitic acid, Po= palmitoleic acid;

O=oleic acid; S=Stearic acid; Ec=Eicosenoic acid; Ep=eicosapentenoic acid;M=myristic acid; De= Docosahexaenoic

acid; Er= erucic acid

References 1. P. Agozzino, G. Avellone, D. Bongiorno, L. Ceraulo, S. Indelicato, S. Indelicato, K. Vèkey; J Mass Spectrom. (2010),

45, pp: 989–995.

2. C. Baiocchi, C. Medana, F. Dal Bello, V. Giancotti, R. Aigotti, D.Gastaldi; Food Chemistry (2015), 166, pp 551–

560.

3. S. Indelicato, D. Bongiorno, L. Ceraulo, C. Emmanuello, F. Mazzotti, C. Siciliano, D. Piazzese; Food Anal. Methods (2018), 1, pp 873–882

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Are you wake yet? Mapping the spatial metabolome of coffee beans with MS-

imaging

A. Smith1, G. Bindi1, P. Crisafulli2, L. Navarini2, and F. Magni1

1 Clinical Proteomics and Metabolomics Unit, Department of Medicine and Surgery, University of Milano-Bicocca,

20854, Vedano al Lambro, Italy 2 illycaffè S.p. A., via Flavia 110, 34147, Trieste, Italy

Summary: this work shows the possibility to map the spatial metabolome of seven different Coffea species

using MALDI MS-imaging, highlighting some metabolomic differences within the main tissues of the coffee

beans as well as among the different species studied Keywords: coffee, spatial metabolomics, mass spectrometry imaging

1. Introduction Coffee is among one of the main consumed

beverages worldwide due to its desirable aromatic

and phytochemical properties. However, each

coffee species possesses differences in terms of

metabolomic content which may have an impact

upon their quality and utility as a commercially

available coffee product. For this reason, many

studies have focused on characterising the bean

chemical content of different coffee species [1], but

little is known regarding the spatial distribution of

the main compounds inside the coffee bean tissues

[2]. Techniques as MALDI MS-imaging which

also consider the spatial distribution of chemical

compounds represent important tools for their

metabolic characterisation and may uncover

further insights related to specific phytochemical

properties of different coffee bean species.

2. Experimental Beans of 7 Coffea species (Coffea arabica, C.

brevipes, C. eugenioides, C. arabica var. laurina, C.

liberica, C. canephora, and C. sessiflora) were

cryo-sectioned at a thickness of 10µm and thaw-

mounted onto conductive indium tin oxide glass

slides. 40mg/mL 2 5-dihydroxybenzoic acid in

70% MeOH w/0.1% trifluoroacetic acid was

deposited onto each coffee bean section using a

HTX TM-Sprayer. Mass spectra were acquired in

both positive-ion and negative-ion mode for each

coffee bean, within the m/z 100 to 700 range, using

a rapifleX MALDI Tissuetyper™ (Bruker Daltonik

GmbH, Bremen, Germany) MALDI-TOF/TOF

MS equipped with a Smartbeam 3D laser operating

at 10 kHz frequency. MALDI-MS images were

acquired with a beam scan setting of 46 µm and a

raster sampling of 50 μm in both x and y

dimensions.

3. Results Initially, the coffee bean distribution of known

target metabolites which constitute to the known

phytochemical properties of coffee beans were

evaluated, including Trigonelline (m/z 138.05,

[M+H]+), Theobromine (m/z 181.06, [M+H]+),

Caffeine (m/z 195.19, [M+H]+), Quinic acid (m/z

191.15, [M-H]-), and Caffeoylquinic acid (m/z

353.08, [M-H]-), which were mostly localised to

the hard portion of the endosperm. Moreover, the

presence of 16-O-methylcafestol (m/z 331.52,

[M+H]+]), a diterpene that is considered an

authenticity marker for Robusta, was evaluated and

in fact was only found to be present in high

abundance within the Robusta coffee beans. To

further highlight the added value of performing

spatially resolved MS analysis, segmentation of the

spatial metabolomics datasets, in both positive-ion

and negative-ion mode, was performed. In both

instances, the segmentation maps were able to

reveal the different tissues present within the coffee

bean, including the hard external endosperm, soft

internal endosperm, as well as the embryo tissue.

In particular, the embryo was associated with a

rather distinct metabolomic profile that was

characterised by a large abundance of Sucrose (m/z

381.08, [M+K]+) with respect to the endosperm.

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Figure 1. Arabica coffee bean segmented using the spatial metabolomics data obtained in positive-ion (left) and

negative-ion (right)mode.

4. Conclusions The work presented highlights the possibility to

map the main metabolites content of coffee beans

as caffeine, trigonelline, sucrose, diterpenes and

some chlorogenic acids and detect metabolomic

differences among different species, thus

representing a highly promising tool to discover

new properties of this important food product.

References

1. Gutiérrez Ortiz AL, Berti F, Solano Sánchez W, Navarini L, Colomban S, Crisafulli P, Forzato C. T. Food

Chem. 2019 Jul 15;286:459-466. 2. Li N, Dong J, Dong C, Han Y, Liu H, Du F, Nie H.. J Am Soc Mass Spectrom. 2020 Dec 2;31(12):2503-2510.

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Risk in glyphosate residue quantification in cereal products:

the 2-amino-3-phosphopropionic acid case study

L. Sabatino, V. Pantò, G. Morabito, F. Lazzaro, M. Scordino, P. Traulo, G. Gagliano

Ministero delle Politiche Agricole Alimentari e Forestali - Dipartimento dell’Ispettorato centrale della tutela della

qualità e della repressione frodi dei prodotti agro-alimentari (ICQRF) - Laboratorio di Catania

Summary: About hundred samples of different dry-commodities (maize, wheat, flour, animal feed and spelt)

were analyzed for glyphosate residue using IC- Orbitrap mass analyzer. A molecule showing quite similar ion

transitions as glyphosate, co-eluting with glyphosate, was often observed. This analyte was identified as 2-

amino-3-phosphonopropionic acid (APPA), isomer of glyphosate.

Keywords: glyphosate, 3-phosphonoalanine, organophosphonate

1. Introduction Phosphonates are organophosphorus molecules

that contain a highly stable C – P bond and a

weaker C – O – P phosphate ester bond. The inert

nature of the C-P bond and the close resemblance

of the phosphonic acid part to the biologically

important phosphoric acid and carboxylic acid

functional groups determine that many natural

phosphonate products are potential enzyme

inhibitors. In fact, in recent years, the biological

properties conferred by the C – P bond have led to

the 1. Introduction into the environment of a vast

range of synthetic organophosphonates such as

insecticides, herbicides, fungicides, antibiotics and

chemical weapons. Examples are the widely used

herbicide glyphosate and glufosinate [1]. The

present work shows the risks associated with the

determination of the pesticide glyphosate in ion

chromatography coupled to a high-resolution

detector (IC-HRMS) due to the presence of 2-

amino-3-phosphonopropionic acid (APPA). This

molecule is a ubiquitous phosphonate amino acid

of biogenic nature [1], and it is isobaric and isomer

of the glyphosate molecule (Table 1). Therefore, as

already highlighted by Zoller et al. [2], its

behaviour in ion chromatography leads to a

possible interference in the identification and

quantification of the glyphosate molecule.

Table 2. Chemical structure and formula of glyphosate and 2-amino-3-phosphonopropionic acid (APPA)

Glyphosate 2-amino-3-phosphonopropionic

acid

Formula: C3H8NO5P Formula: C3H8NO5P

Exact Mass: 169.0140085 Exact Mass: 169.0140085

2. ExperimentalAnalytical Method: Quick method for the analysis

of numerous highly polar pesticides in food

involving extraction with acidified methanol and

LC-MS/MS measurement - food of plant origin

(QuPPe-PO-Method, V 12).

Instrument: IC-Dionex 6000 system with high

resolution analyser Orbitrap®, Thermo QExactive.

Materials:

- Solutions certified with a known title of single

active ingredients: APPA and glyphosate;

- Water at least "grade 3" ISO 3696 standard;

- Methanol for pesticide analysis;

- Acetonitrile for pesticide analysis;

- Formic acid 99%;

- 0.45 µm PTFE filters;

- Dionex IonPac AG19-4μm column.

Sample preparation:

- Weigh 5.0 g ± 0.5 g of sample in 50 ml falcon;

- Add 10 ml of water;

- Add 10 ml of methanol for pesticide analysis,

acidified to 1% with formic acid;

- Shake for 1.5 min at 3000 rpm in

Geno/Grinder®;

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- Filter with a 0.45 µm filter into plastic vials;

- Take 100 μl of filtrate and add 1000 μl of water

in vials.

Gradient: The water flow rate was 0.25 mL/min

with a gradient from 15 mM OH- for 5 min; from

15 mM OH- to 60 mM OH- in 7 min; 60 mM OH-

for 6 min; from 60 mM OH- to 75 mM OH- in 0.5

min; 75 mM OH- for 1.5 min; from 75 mM OH- to

15 mM OH- in 0.5 min; with a cycle time of 20.5

min. The OH- eluent (0.5 ml/min) was neutralized

using a Dionex ADRS 600 2 mm electrolytically

regenerated suppressor (Thermo). Desolvatation

post column with acetonitrile at 0.150 ml/min. The

injection volume was 100 μL of the extract diluted

with 1000 l water.

Mass analyser for identification and

quantification: Full scan (FS) was conducted in

ESI negative mode in the range 50-400 m/z and

product reaction monitoring (PRM) acquisition

was focused on m/z 168.00673 ion.

External calibration: Stock solution of APPA and

glyphosate at concentration of 40 g/l were utilized

to prepare external standard calibration. A 5-point

calibration curve corresponding to a range of 0.5–

7 ug/l was constructed.

3. Results and Conclusions Glyphosate residues analysis on cereal samples

have often shown the presence of an interferent

which coelutes with glyphosate and exhibits

similar fragmentation pattern. This substance has

been identified as APPA, a molecule with identical

composition, same carboxylic and phosphonic

functional groups of glyphosate but differs in the

position of nitrogen of the amino group (Table 1).

This compound was found in 50 of samples of

maize and spelt, origin Moldavia, in the

concentration range of 0.05–0.4 mg/kg; it was also

found in 17 different samples (wheat, flour, animal

feed). To avoid the risk of misstatement in

qualitative and quantitative results when analysing

glyphosate, it is necessary to check whether APPA

is properly distinct from glyphosate in

chromatograms. Figure 1 shows TIC and PRM

fragments of the two analytes. Glyphosate shows

the PO2- fragment at m/z 62.964 which is almost

undetectable in APPA and fragment at m/z 124.017

[M- CO2]- due to the CO2 loss. Instead, the ion at

m/z 77.975 is characteristic of APPA. The ions

corresponding to the molecular ion [M]- at m/z

168.00673 and those relating to the loss of water

[M- H2O]- at m/z 149.996 are common to both

substances. The SANTE/11312/2021 guidelines

for the identification and quantification of

molecules indicate that in the case of the use of a

high-resolution detector, a minimum of two ions

per analyte as required; the selection of the ion at

m/z 62.964 is essential for a correct identification

of glyphosate. For a correct quantification, it is

necessary to evaluate adjustments of

chromatographic gradient to allow better

separation or the use of a different technique such

as HPLC with HILIC columns.

Fig. 1. Total ion chromatograms (TIC) and Products reaction monitoring (PRM) of glyphosate (A) and APPA (B)

References

1.J.P. Quinn, A.N. Kulakova, N.A. Cooley, J.W. McGrath; Environmental Microbiology, 9-10 (2007), pp 2392–2400.

2.O. Zoller, P. Rhyn, H. Rupp, J.A. Zarn, C, Geiser; Food Additives & Contaminants: Part B, 11-2 (2018), pp 83–91.

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Untargeted screening of perfluoroalkyl and polyfluoroalkyl substances (PFASs)

in air of Italian e-waste recycling facilities

C. Barola1, S. Moretti1, F. Buiarelli2, F. Lucarelli3, L. Goracci4, S. . Lorenzetti5, P. Di Filippo6, F. Paoletti1, R. Galarini1

1 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ”Togo Rosati”, Perugia, Italy;

2Department of Chemistry, University "La Sapienza", Rome, Italy; 3Department of Physics, University of Florence and

I.N.F.N., Sesto Fiorentino, Florence, Italy; 4Department of Chemistry, Biology and Biotechnology, University of

Perugia, Perugia, Italy; 5Department of Food Safety, Nutrition and Veterinary Public Health, Italian National Institute of

Health (ISS), Rome, Italy; 6DIT INAIL, Rome, Italy

Summary: Per- and polyfluoroalkylated substances (PFASs) are a wide group of environmental

contaminants used in several industrial products. An untargeted procedure was applied to detect new PFASs

in particulate matter collected in two e-waste recycling plants using LC-Q-Orbitrap. Three possible PFASs

were tentatively identified and one was confirmed by standard comparison.

Keywords: Per- and polyfluoroalkylated substances (PFASs), e-waste, LC-Q-Orbitrap

1. Introduction

Waste of Electrical and Electronic Equipment

(WEEE) also known as e-waste is commonly

considered a secondary raw material for the

recovery of valuable components such as

precious metals, plastics, glass and ceramics.

Nevertheless, the occurrence of hazardous

organic and inorganic chemicals in e-waste

components poses environmental and

occupational risks. Therefore, the issues

regarding the impact of e-waste recycling

processes are becoming a global problem [1].

Suspended particles present in e-waste material

can contain, among others, per- and

polyfluoroalkylated substances (PFASs), a wide

group of synthetic molecules produced since the

late 1940s. PFASs are a family of thousands of

compounds used in various industrial

applications including electric and electronic

equipments. As several long-chain PFASs have

been banned from production and use, short-

chain and new PFASs are synthetized as

alternatives. The aim of this work was the

development of an untargeted analytical

procedure in order to detect the presence of

possible unknown PFASs in particulate matter

collected in e-waste facilities through liquid-

chromatography coupled to high-resolution

mass spectrometry (LC-HRMS) [2].

2. Experimental

Fine and coarse particulate matter fractions

were collected over a workday in different areas

(dismantling and shredding) of two Italian e-

recycling facilities in December 2020 and May

2021. The analyses were performed by liquid-

chromatography coupled to Q-Orbitrap (LC-Q

Exactive, Thermofisher Scientific, San Jose,

CA, USA) applying the LC conditions

described in [3]. Three different acquisition

modes were applied: full MS/dd-MS2 (TopN),

full MS/AIF/NL dd-MS2 and full MS/DIA. In full

MS/dd-MS2 (TopN), the selection of fragment

ions is based only on their intensity. This

approach is useful to identify unexpected

compounds also when specific neutral losses are

unknown. On the other hand, in the Full

MS/AIF/NL dd- MS2 experiment, the full scan

is followed by an "All Ion Fragmentation" (AIF)

and, therefore, only ions producing known

neutral losses (NLs) were fragmented. In our

case, more than fifty diagnostic NLs were

included (HF, C4HF5O4, C6ClF11O2 etc.) that

were capable of triggering the relevant dd-MS2

experiments. Finally, in the Full MS/DIA

experiment, full scan is followed by a data

independent analysis with an isolation window

set at m/z 60 covering a mass range m/z 120-

1500. With respect to AIF experiment, the

advantage of DIA was to minimize the post-

interface ion suppression phenomenon,

improving sensitivity [4]. For data analysis,

different features of Compound Discoverer

package (Thermo Fisher Scientific) were used

in order to obtain independent information

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2

about the identity of the detected substances. In

addition to specific NLs, more than 80

characteristic fragments (C2F5-, C5F9O-,

C3ClF6O- etc.) were monitored. Along with the

predicted compositions generated by

Compound Discoverer based on m/z values

and isotopic patterns, two different mass lists

containing both formula and exact m/z of more

than 6000 PFASs were used. One of these was an

in-house custom list created by injecting about

one hundred available PFAS reference

materials; this list included also

chromatographic retention times. The second

list was based from the database available at the

website of the US Environmental Protection

Agency [5]. Moreover, standard, relative and

Kendrick mass defects were calculated for each

compound using as Kendrick formula four

repetitive units: CF2, C2F4, C2F4O and

C2HF3. Finally, the automatic search in

mzCloud, mzVault and Chemspider databases

was performed

3. Results

Using the described approach three potential

PFASs were detected in both e-recycling

facilities: bistriflimide (1,1,1-Trifluoro-N-

((trifluoromethyl)sulfonyl)methanesulfonamide),

1,1,2,2,3,3,4,4,5,5,6,6,7,7,7, penta- decafluor-1-

heptan-sulfonamide and pentafluorooctansulfinic

acid. The most frequently found was

bistriflimide, especially in one of the two plants.

Later, the identity of bistriflimide was confirmed

purchasing its commercial reference material. In

the Figure, the chromatogram and spectra of

bistriflimide together with the output of

Compound Discoverer package are shown.

Fig. 1– Compound Discoverer output of the peak at 5.8 min (left side). The acquired full scan and MS2 spectra are

shown in the central and right figures, respectively. The predicted formula (C2HF6NO4S2) was found in ChemSpider

database and attributed to bistriflimide. In MS2 spectrum, the characteristic fragment ions at m/z 77.9639 (NSO -) and

m/z 82.9592 (FSO2-) were recognised (green points) thanks to their inclusion in the in-house fragment database.

4. Conclusions The untargeted protocol developed here

combines several MS experiments and database

resources and it has been shown to be able to

detect unexpected PFAS molecules. A further

improvement of this approach can be obtained

implementing the databases with further specific

fragment ions and neutral losses belonging to

novel and emerging PFAS compounds.

Acknowledgments This work was supported by INAIL BRiC 2019 – ID13 (“Environmental assessment and health impact

of organic emergent contaminants such as bromine flame retardants and perfluoroalkylated substances

and inorganic toxic contaminants in occupational environments.)

References 1. O. Alabi, Y. Adeoluwa, X. Huo, X. Xu, A. Bakare; Journal of Environmental Health Science and

Engineering 19, (2021) pp. 1209–1227.

2. Y. Liu, L.A. D’Agostino, G. Qu, G. Jiang, J.W. Martin; Trends in Analytical Chemistry, 121, (2019), 115420.

3. C. Barola, S. Moretti, D. Giusepponi, F. Paoletti, G. Saluti, G. Cruciani, G. Brambilla, R. Galarini;

Journal of Chromatography A, 1628, (2020), 461442.

A. Kaufmann, M. Widmer, M. Kaden Rapid Communications in Mass Spectrometry 24(14) (2010) pp 2162-2170

4. https://comptox.epa.gov/dashboard/chemical_lists/epapfasrl

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Presence of novel perfluoro-polyether carboxylic acids in poultry eggs

C. Barola1, S. Moretti1, R. Avolio2, F. Paoletti1, S. Sdogati1, D. Giusepponi1, M. C. Abete2, A. Stramenga3, T. Tavoloni3, A. Piersanti 3, R. Galarini1

1 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ”Togo Rosati”, Perugia,

Italy; 2Istituto Zooprofilattico Sperimentale del Piemonte, della Liguria e della Valle d’Aosta,

Torino, Italy; 3Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ”Togo

Rosati”, Ancona, Italy

Summary: In the last years, new per- and polyfluoroalkylated substances (PFASs) have been produced to

replace some old compounds potentially toxic. In this work, we report for the first time the presence of these

substances in poultry eggs collected in an industrial area of Northern Italy.

Keywords: Per- and polyfluoroalkylated substances (PFASs), eggs, LC-Q-Orbitrap

1. Introduction

Per- and polyfluoroalkylated substances

(PFASs) are a wide group of synthetic

molecules produced since the late 1940s and

used in various commercial products. As

several long-chain PFASs have been banned

due to their potential toxicity, short-chain and

new PFASs are synthetized as alternatives.

Recently, novel chloro- perfluoro-polyether

carboxylates and related PFAS have been

detected in environmental samples collected

in New Jersey (USA) [1,2]. This presence has

been attributed to industrial facilities using

and/or manufacturing PFASs located in that

area. The aim of this work was the

development of an analytical procedure to

detect novel perfluoro-polyether

carboxylates in eggs collected near an

industrial plant producing PFASs in

Northern Italy.

2. Experimental

Three poultry eggs samples were collected in

the vicinity of an industrial plant producing

fluorinated polymers (Solvay Specialty

Polymers Italy S.p.A., Spinetta Marengo,

AL, Italy), whereas other four egg samples

were collected from a different area. Two

grams of homogenised sample was extracted

and purified as described in Barola et al. [3]

with slight modifications. The analyses

(suspect screening) were performed by

liquid-chromatography coupled to Q-

Orbitrap mass analyser (LC-Q Exactive,

Thermofisher Scientific, San Jose, CA,

USA). Negative ionization mode was

applied performing t-SIM-ddMS2

acquisition. In the inclusion list the exact m/z

of the precursor ions of hydro- and chloro-

perfluoro-polyether carboxylates were

inserted on the basis of data published by

Washington et al. [1] and McCord et al. [2].

3. Results

The general structure of the searched

perfluoroether carboxylic acids is reported in

Figure 1 [1,2]. The different congeners of

chloro (ClPFPECA) and hydro

perfluoroether carboxylic acids (HPFPECA)

are classified with the relevant number of

perfluoroethyl (e) and perfluoropropyl (p)

units.

Fig. 1. General structure of the perfluoroether carboxylic acid oligomeric series (R=Cl: ClPFPECA; R = H:

HPFECA). The R group position is likely variable [1,2]

Both ClPFPECAs and HPFPECAs were

detected in all three egg samples collected near

the Solvay Specialty Polymers plant, while

they were not detected in any of the four

samples taken far from this industrial plant.

Some of these compounds could be traced

back to the mixture of telomers (CAS 329238-

24-6) produced by Solvay with the trade name

ADV 7800 [4]. In absence of reference

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materials, the analyte detection was performed

without the knowledge of RTs and therefore

the t-SIM-ddMS2 experiment was carried out

all along the chromatographic run. Since

ClPFPECAs and HPFPECAs fragment in

source, the monitored precursor ions were [M-

CF2-CO2-H]-. In Figure 1, the LC-MS

chromatograms and MS2 spectra of some

ClPFPECAs found in one of the analysed eggs

are shown. All the measured m/z were

generally within 5 ppm error. In the same egg

sample also five hydro perfluoroether

carboxylic acids were detected (HPFPECA-

0,1, HPFPECA-2,0, HPFPECA-1,1,

HPFPECA-0,2 and HPFPECA-1,2).

Fig. 2. LC-HRMS chromatograms and relevant MS2 spectra of some ClPFPECAs found in one of the contaminated egg

samples

4. Conclusions To the best of our knowledge, this is the first

evidence of the presence in poultry eggs of

the novel perfluoro- polyether carboxylates

described for the first time by Washington et

al. [1]. Although no reference materials are

currently available, the presumptive

identification of these compounds is

strengthened by the co-presence of several

congeners belonging to the ClPFPECA and

HPFECA series shown in Figure 1.

References 1. J. W. Washington, C. G. Rosal, J. P. McCord, M. J. Strynar, A. B. Lindstrom, E. L. Bergman, S. M. Goodrow,

H. K. Tadesse, A. N. Pilant, B. J. Washington, M. J. Davis, B. G. Stuart, T. M. Jenkins. Science. 2020,

368(6495): 1103– 1107.

2. J. P. McCord, M. J. Strynar, J. W. Washington, E. L. Bergman, S. M. Goodrow. Environmental Science &

Technology Letters 2020 7(12): 903–908.

3. C. Barola, S. Moretti, D. Giusepponi, F. Paoletti, G. Saluti, G. Cruciani, G. Brambilla, R. Galarini; Journal

of Chromatography A, 1628, (2020), 461442.

4. EFSA Scientific Opinion. EFSA Journal 2010; 8(2):1519

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Combining passive sampling and LC-MS/MS for the study of emerging

pollutants in seawater

E. Magi, M. Di Carro, B. Benedetti, H. MacKeown, C. Scapuzzi, M.

Baglietto

Department of Chemistry and Industrial Chemistry, University of Genoa, via Dodecaneso, 31, 16146, Genoa, Italy

Keywords: seawater passive sampling, tandem mass spectrometry, emerging pollutants

1. Introduction and Contents Emerging Contaminants (ECs) in marine waters

include different classes of compounds, such as

pharmaceuticals and personal care products,

showing “emerging concern” related to the

environment and human health. Their

measurement in seawater is challenging mainly due

to the low concentration levels and the possible

matrix interferences. Mass spectrometry combined

with chromatographic techniques represents the

method of choice to study seawater ECs, due to its

sensitivity and versatility. Nevertheless, these

powerful instrumental techniques need suitable

sample collection and pre-treatment. Conventional

spot sampling may not allow sufficient sensitivity;

moreover, due to the complex dynamics of the

coastal marine environment, discrete samples may

not offer a representative picture of contaminant

concentrations.

Passive sampling represents a powerful approach

to overcome these problems. Passive samplers are

generally deployed in water for several days or

weeks, allowing an in-situ pre-concentration of the

contaminants, which can improve sensitivity [1].

Also, in the case of integrative samplers, the

relatively long deployments permit an estimation

of time-weighted average (TWA) concentrations,

rather useful for the assessment of marine water

quality. Marine applications for passive samplers

were first mainly for traditional and mostly

hydrophobic contaminants such as PAHs, PCBs

and organochlorine pesticides. In the past two

decades, more and more work has been conducted

on passive sampling of polar compounds (1<

logKow< 4) in marine waters. The most popular

devices are the Polar Organic Chemical Integrative

Samplers (POCIS), the Chemcatcher and the

organic Diffusive Gradients in Thin film

technology (o-DGT). Figure 1 shows the number

of publications for each methodology over the last

21 years, highlighting that most published works

on seawater monitoring use the POCIS [2].

Fig. 6. Cumulative publications on integrative passive samplers (POCIS, Chemcatchers, o-DGTs and others)

for polar emerging contaminants since the first application in marine waters.

POCIS was designed to sample rather polar

compounds, characterized by a logKow comprised

between 0 and 5 [3]. It consists in a sorbent phase

sandwiched between two microporous membranes,

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usually made of polyethersulfone (PES), which are

kept together by two stainless rings.

Our research group has a large experience in the

use of POCIS for water studies, initially focused on

freshwater monitoring and, later, on seawater

applications [4]. Detection and quantification of

ECs in marine water is challenging due to the low

concentration levels (sub µg L-1), the variety of

their physicochemical properties and the

complexity of the matrix. We developed a reliable

multiresidue LC-MS/MS method, taking also into

account the possible matrix effect (ME) that the

seawater matrix may cause, thus avoiding an

overestimation or underestimation of the analyte

concentration, as well as improving detection

limits. The analytical method allows the

determination of several emerging contaminants at

trace levels: pharmaceuticals, UV-filters,

estrogens, artificial sweeteners, perfluoroalkylic

compounds, bisphenol A, triclosan, caffeine and

paraxanthine. The analytical method was applied to

various POCIS deployed in different locations of

the Ligurian coast (Italy, Mediterranean Sea) and

provided information about the quality of waters

subjected to different anthropic impacts. Few

examples of these case studies will be presented

and discussed.

References

1. B. Vrana, I.J. Allan, R. Greenwood, G.A. Mills, E. Dominiak, K. Svensson, J. Knutsson, G. Morrison; TrAC - Trends

Anal. Chem. 24 (2005) 845–868

2. H. MacKeown, B.Benedetti, M. Di Carro, E. Magi; Chemosphere 299 (2022) 134448.

3. D. Alvarez, S. Perkins, E. Nilsen, and J. Morace, Science of the Total Environment 484 (2014) 322

4. M. di Carro, E. Magi, F. Massa, M. Castellano, C. Mirasole, S. Tanwar, E. Olivari, and P. Povero, Marine Pollution

Bulletin 131 (2018) 87

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Fast and Easy Methodology for the Determination of Formaldehyde in Indoor

Working Environments by Derivatization Coupled with GC-MS

P. Avino, C. Di Fiore, A. Iannone, F. Carriera, I. Notardonato

University of Molise, Department of Agriculture, Environmental and Food Via De Sanctis s.n.c. 86100 Campobasso

(Italy)

Summary: the analysis of formaldehyde from indoor air sample was generally performed using HPLC system,

after a proper derivatization reaction. In this paper, an easier methodology has been set up to improve its

monitoring in indoor air of workplaces, achieving limit of detection in the order of ppb.

Keywords: formaldehyde, derivatization, GC-MS.

1. Introduction

In recent years, the attention to formaldehyde has

increased due to its broad application in several

production sectors, such as manufacturing, textile,

chemical and medical ones. This compound, in

fact, occurs both in outside and inside air,

particularly in workplaces [1]. Exposure to

formaldehyde, in fact, can lead to several diseases

such as irritation of the eyes, nausea and allergic

skin reactions. Furthermore, in 2004, formaldehyde

has been recognized as carcinogen substance by

International Agency Research of Cancer (IARC).

For this reason, its determination in indoor

environment using easier and faster analytical

methodologies is a matter of urgency to protect the

public health. Traditionally, the quantification of

formaldehyde in air indoor samples is carried out

by means of high-pressure liquid chromatography

(HPLC), after a passive or active sampling using

cartridges or tubes impregnated with 2,4-

dinitrophenylhydrazine (DNPH) [2]. However, this

method allowed to achieve limit of detection in the

order of mg kg-1. In this paper, an innovative, fast,

reliable and sensitive method is proposed for the

analysis of formaldehyde from indoor air samples.

2. Experimental

In this study, formaldehyde is sampled using

atmospheric samplers coupled with Solid Phase

Extraction (SPE) Carbograph 1 cartridges, washed

and conditioned prior to use. The analyte is then

desorbed from the SPE cartridge using acetonitrile.

After, formaldehyde is derivatized using an

innovative approach. Briefly, trifluoroacetic

anhydride is used as derivatization agent combined

with an iron-based artificial catalyst for promoting

the reaction. Derivatized formaldehyde is then

injected in the GC-MS system and analysed. This

new methodology allows to lower the detection

limit compared to the HPLC one; with the new

method, the detection limit is of the order µg kg-1.

Fig. 7. Derivatization reaction of formaldehyde using

trifluoroacetic anhydride

3. Results

From the findings resulted that the methodology

proposed is more sensitive than the traditionally

one. The limit of detection of the new method is of

0.009 µg mL-1, with a linear dynamic range which

ranged between 0.05 and 0.50 µg mL-1. The limit

of quantification is of 0.039 µg mL-1 whereas R2

value obtained is of 0.9981, confirming the

goodness of the method. On the basis of the results,

the use of this method can be proposed to determine

the formaldehyde present in traces in the

workplace, significantly expanding the protection

of workers.

Fig. 2. Mass spectrum of derivatized formaldehyde

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The percentage recoveries reported in Table 1 are

obtained using the proposed method. Recoveries

were determined by analysing three solutions at

two known concentrations. The goodness of the

methodology is confirmed by the standard

deviation values that range between 8.6 and 11.3.

Table 3. Percentage recoveries of derivatized

formaldehyde at known concentrations.

Recovery

0.1 µg mL-1 ±SD

Recovery

0.3 µg mL-1 ±SD

89.2 11.3 94.1 8.6

4. Conclusions

The determination of formaldehyde in workplaces

is a concern of scientific community from years.

Since this compound is generally present in traces

in indoor air, sensitive analytical methodologies

are necessary to detect it. Based on this

consideration, a new protocol is set up to lower the

limit of detection, and analyse the presence of

formaldehyde in a faster, easier and more sensitive

way. However, this is a preliminary study for the

setting up of the methodology.

All the experimental tests were carried out only on

laboratory indoor air, but further tests will be

performed on real samples to analysed the presence

of formaldehyde in air samples of workplaces.

References

1. W. Rosenberg, B. Beckmann, R. Wrbitzk; Journal of Chromatography B, 1019 (2016), pp.117-127.

2. D. Bourdin, V. Desauziers; Analytical and Bioanalytical Chemistry, 406 (2014) pp. 317-328

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Unravelling the proteomic content and spatial distribution of amyloid causative

proteins in renal biopsies using MALDI-MSI

G. Bindi1, A. Smith1, F. Pagni2, F. Magni1, V. L’Imperio2

1 Clinical Proteomics and Metabolomics Unit, Department of Medicine and Surgery, University of Milano-Bicocca 2 Department of Medicine and Surgery, Pathology, University of Milano-Bicocca, San

Gerardo Hospital, Italy

Summary: This study concerns the feasibility of MALDI-Mass Spectrometry Imaging to detect the spatial

distribution of amyloid precursor proteins within formalin fixed paraffin embedded renal biopsies amyloidosis.

Keywords: Renal Amyloidosis, Mass Spectrometry Imaging, Spatial Proteomics

1. Introduction

Renal amyloidosis is a group of diseases

characterised by an abnormal extracellular

deposition of amyloid fibrils, composed of a

variety of misfolded proteins depending on the

classification of the disease. At the present time,

the diagnostic gold standard for amyloidosis is

Congo Red staining combined with polarisation

microscopy, although it remains insufficient for

confirming the amyloid nature of protein

aggregates [1]. Matrix Assisted Laser Desorption

Ionization - Mass Spectrometry Imaging (MALDI-

MSI) is a mass spectrometry-based technique used

to visualise the spatial distribution of compounds

within tissue sections. In this study, we assess the

feasibility of MALDI-MSI as a tool to detect a

characteristic proteomic signature for secondary

amyloidosis (AA), light chain amyloidosis (AL)

lambda, and AL kappa and rare non-AL non-AA

amyloidosis in formalin-fixed paraffin-embedded

(FFPE) renal biopsies.

2. Experimental design FFPE renal biopsies of patients diagnosed with

amyloidosis AA, AL lambda and AL kappa, and

non-AL non-AA, as well as a healthy control and

undiagnosed renal biopsies, were sectioned and

mounted onto conductive indium tin oxide (ITO)

glass slides. The day of the analysis, slides were

deparaffinized and rehydrated by performing

consecutive washes with toluene (3×5 min), 100%

ethanol (2×5 min), 70% ethanol (1×3 min) and

HPLC grade H20 (2x2 min). Antigen retrieval was

performed in a bath of 10 mM citric acid buffer at

97°C for 45 min before hydrating the tissue by

washing in HPLC grade H2O (1x2 min) prior to

trypsin application (100ng/µl) using an

iMatrixSpray sprayer. α-CHCAmatrix (10mg/ml

HCCA, 70%ACN, 1%TFA) was deposited onto

sections using a HTX TM-Sprayer. Mass spectra

were acquired in positive-ion mode within the m/z

700 to 2000 range, using a rapifleX MALDI

Tissuetyper™ mass spectrometer (Bruker

Daltonics, Bremen, Germany). MALDI-MS

images were acquired with a beam scan setting of

6 µm and a raster sampling of 10 μm in both x and y dimensions.

Data processing was carried out using SCiLS Lab

software for the analysis of tryptic peptides whose

mass was derived from in silico digestion

(PeptideMass, www.expasy.org) of proteins of

interest, including amyloid precursor proteins.

3. Results In this study we explored several amyloidosis

cases including four classes of amyloid, searching

for amyloid precursor proteins in situ. For all

tryptic peptides, ions were considered absent

when the absolute intensity (A.I.) of peaks in the

mean spectrum was lower than 1 A.I. Figure 1

shows an example of distribution of amyloid

precursor Immunoglobulin Lambda Variable 2-

11 (IGLV2- 11, m/z 968.51) within an AL lambda

renal biopsy, where the higher intensity within the

glomeruli region suggests that MALDI-MSI is

feasible for the detection of amyloid aggregates

with a high specificity and coherent spatial

distribution. For each class of amyloidosis, a

panel of in silico digested proteins was considered

to characterise the targeted proteomic content.

Additionally, we considered amyloid cases whose

diagnosis was challenging and were able to assign

these to one of the amyloidosis classes based on the

panel of target proteins detected. For instance, in

Figure 2 we show the distribution of amyloidosis

AA precursor protein, SAA1 (m/z 894.44), in the

biopsy of an undiagnosed class of amyloidosis

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104

Figure 1. Spatial distribution of IGLV2-11

(m/z 968.51) in a AL lambda amyloidosis

renal biopsy: a) Congo Red stained image; b)

MALDI-MS image; c) Overlay

.

Figure 2. Spatial distribution of SAA1 (m/z

894.44) in an undiagnosed amyloidosis

renal biopsy: a) Congo Red stained image;

b) MALDI-MS image; c) Overlay

4. Conclusions This study highlights the possibility to unravel the

proteomic content of renal biopsies, taken from

patients diagnosed with various classes of

amyloidosis, in its native spatial context and detect

characteristic amyloid precursor proteins. MALDI-

MSI was found to be capable of detecting amyloid

causative proteins on tissue with a high spatial

resolution and this is key when considering that

Congo Red, the current diagnostic gold standard, is

unable to detect the type of amyloid aggregates.

The application of this spatial proteomics approach

may enable the detection of renal amyloidosis with

greater specificity

.

References

1. Yakupova, E. I., Bobyleva, L. G., Vikhlyantsev, I. M. et al. (2019). Congo Red and amyloids: history and

relationship. Bioscience Reports (Vol. 39, Issue 1). Portland Press Ltd.

2. Gupta, N., Kaur, H., & Wajid S. (2020) Renal amyloidosis: an update on diagnosis and pathogenesis.

Protoplasma 257:1259–1276

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105

Suspect screening of Novel Psychoactive Substances using QTrap Technology

G. Di Francesco1, M. Croce1,2, F. Vincenti1, C. Montesano1, M. Sergi3, R. Curini1

1 Department of Chemistry, Sapienza University of Rome, Rome, Italy

2 Department of Public Health and Infectious Disease, Sapienza University of Rome, Rome, Italy 3Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100

Teramo, Italy

Summary: Novel psychoactive substances are a group of synthetic substances, for which reference standard

are not available, for this reason the main problem is their identification. Suspect screening, for which

standards are not required, is a promising approach since the only information needed is about the

structural information findable in literature.

Keywords: Suspect Screening, Novel Psychoactive Substances, Mass Spectrometry

1. Introduction Novel psychoactive substances (NPS) are a wide

group of substances, principally of synthetic

production, characterised by pharmacological and

toxicological properties extremely dangerous for

the users. These substances can be catalogued in

different classes, according to their biological

activities (e.g., hallucinogens, psychostimulants,

sedatives, hypnotics) or to their chemical structures

(e.g., synthetic cannabinoids, synthetic stimulants,

phenethylamines, and synthetic opioids) [1]. These

designer drugs are produced to simulate similar

effects of traditional recreational drugs but with the

possibility to circumvent legislative measures due

to their different chemical structures hindering the

possibility of understanding the real diffusion of

these compounds in national and international

borders [2]. In 2020 an increase of emergency

hospital admissions associated to benzodiazepine’s

consume was registered; during the same years the

notifications of intoxication following the

consumption of cannabis adulterated with synthetic

cannabinoids notably increased [3]. As of

December 2020, more than 1000 NPS have been

reported to the UNODC Early Warning Advisor

(EWA) from 126 countries. The aim of this work

was the creation of a robust and versatile suspect

screening method for traditional drugs and NPS

detection, starting from a target method based on

97 standard substances.


The target analytes selected in this study belonged

to different classes, including synthetic and

cannabinoids (28 drugs), cathinones (8 drugs),

alkaloids (5 drugs), phenethylamines (14 drugs),

arylcyclohexylamines (2 drugs), opioids (6 drugs)

and tryptamine (7 drugs).Initially a target method

was created with these substances; to this aim each

one of the 97 standards was optimized with a single

manual injection using a UHPLC-MS/MS

apparatus that combined an Exion LC AD

chromatographic system with a QTRAP 6500 mass

spectrometer equipped with ESI's Turbo V source.

The best chromatographic separation was obtained

with a Kinetex 2.6 μm F5 100 Å column, 100x2.1

mm, using 75:25 ACN-MeOH + 0.1% formic acid

(phase B) and 5 mM ammonium formate in water

(phase A). The chromatographic gradient

(duration: 15.50 minutes) was selected on the basis

of different experiments, in order to obtain a good

separation of the different analytes. For what

concerns the MS detection, scheduled advanced

MRM (AsMRM) algorithm and polarity switching

were used. Specific parameters (e.g. Trigger

Threshold, Dwell Weight) were inserted to obtain

a better investigation of all the MRM transitions. A

suspect screening method was then created by

using the same chromatographic conditions and

adding new MRM transitions of common NPS for

whom standards were not available. Q1 and Q3

values were obtained from HighResNPS database

[4], while the other MS parameters were achieved

by analogy with drugs belonging to the same

chemical class. Retention time was predicted by

using a Quantitative Structure Retention

(Chromatographic) Relationship (QSRR)

specifically developed for the purpose. IDA

(Information Dependent Acquisition) acquisition

mode that coupled an EPI (Enhanced Product Ion)

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acquisition to an AsMRM as survey scan was used as

acquisition mode. Finally, the performance of the

developed suspect screening method was evaluated

with a standard mixture containing 21 NPS, that were

not included among the 97 initial drugs and with real

samples arising from seizures.

3. Results and discussion In this study a new method for target and suspect

screening of known illicit drugs and NPS was

developed. Initially a target method was created for

97 selected drugs, this first step was crucial to

establish the chromatographic conditions and to set

the AsMRM conditions. The method created

included in a single AsMRM run two transitions for

every analytes as well as deuterated internal standard.

Reached this point, the target method was used as the

basis for the creation of the suspect screening

method. This was accomplished by adding new

MRM transitions, based on existing databases and

literature data. To include these new transitions in the

AsMRM method, retention times (Rt) were predicted

by using a specific QRSS method engineered on the

97 standard substances. The approached used in the

prediction model is linear multiple regression (MLR),

which is based on the calculation of a set of molecular

descriptors correlated with rT; they are then

normalised, correlated and the best used in the

calculation of the prediction equation.

Limits of detection were calculated by analysing

standard solutions at decreasing concentrations and

range between 0.007 ng/mL for 25-C-NBOMe and

0.4 ng/mL for morphine, making the method suitable

for toxicological screening and quantification in

biological matrices with dilute and shoot approaches.

Oral fluid (OF) was considered for testing purposes,

excellent results in term of accuracy, precision and

matrix effect were obtained. For suspect screening, a

sMRM-IDA-EPI method was developed; this

approach allowed to combine quadrupole and ion trap

functions, allowing simultaneously detection of a

compound by MRM transitions and identification on

the basis of automatically acquired MS/MS ion

spectra recorded at a fixed area threshold setting.

Screening capabilities are then improved and putative

identification of drugs by library matching may be

possible even if standards are not available. As a

proof of concept, the performance of the suspect

method was evaluated by analysing a mixture

containing 21 reference standards not included in the

initial dataset; 19 out of 21 analytes were correctly

detected. Predicted Rt were generally in accordance

with experimental Rt, with an average shift of about

13%. Finally, the suspect screening method was

applied to four seizures. MXPR and 3-MMC were

identified in these samples, showing that this

approach is able to detect NPS even in real samples

4. Conclusion and future prospective Suspect screening was made possible thanks to the

possibility of combining the AsMRM with IDA/EPI

scan, which provided the fragmentation spectra. The

greatest issue in creating a suspect screening model

was trying to optimise all the parameters present in

the target method to extend to real samples. In this

way, libraries of illicit substances (especially NPS)

are enriched. New transitions can be added to the

method based on new NPS identified on the market.

References

1. King LA and Kicman AT Drug Test. Anal. 3, 401–403 (2011)

2. Salomone et al. Ital J Add 4-1,85-91 (2014)

3. https://www.emcdda.europa.eu/edr2021_en

4. https://highresnps.forensic.ku.dk/

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Untargeted UHPLC-HRMS approach for the identification of rotten defect

markers in hazelnuts

F. Spataro1, F. Rosso2, G. Genova2, A. Caligiani1

1 Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma,

Parco Area delle Scienze 27/A, 43124 Parma, Italy 2 SOREMARTEC ITALIA S.r.l., Piazzale Ferrero 1, 12051, Alba, Cuneo, Italy

Summary: The rotten defect is one of the main defects of the hazelnut during the harvest and post-harvest

phases. The aim of this study is to identify the main common markers between different origins of commercial

hazelnuts. Approximately 30 potential markers were highlighted following an untargeted approach using

UHPLC-HRMS.

Keywords: Rotten, Hazelnuts, Untargeted UHPLC-HRMS.

1. Introduction The rotten defect is the strongest sensory off-note

of hazelnuts, which every year threatens their

availability and marketability, inducing yield

losses as a consequence of non-compliance with

the quality standards required for their industrial

use [1][2]. The term "rotten hazelnuts" refers to

hazelnuts with necrotic spots and / or internal

browning, which at worst give a black nut. The

incidence of the rotten defect on harvested

hazelnuts generally ranges between 1% and 15%,

but even a small presence of damaged fruit could

be negative for the organoleptic properties [2][3].

A study conducted by Battilani et al. (2018) [1]

identified the genus Diaporthe as the main one in

the rotten hazelnuts in the Caucasian region, as well

as other fungal species (such as Alternaria spp.,

Cladosporium spp., Fusarium spp., And

Colletotrichum spp.). Another common defect of

hazelnuts, sometimes related to rotten, is the

“cimiciato” due to a bug attack [4].

Therefore, the aim of this study is to determine the

main compounds responsible for the rotten defect

in different cultivars and hazelnut origins through

an untargeted approach using UHPLC-HRMS in

order to subsequently be able to develop a valid

instrumental method for the detection and

quantification of defect.

2. Experimental The UHPLC-HRMS system used for the study

consists of a UHPLC UltiMate 3000

(ThermoFisher, USA) equipped with a C-18 Luna

Omega Polar 1.6 µm column (Phenomenex, USA)

and an Orbitrap Q-Exactive Focus (ThermoFisher,

USA) with H-ESI ion source (positive ionization

for this study). Hazelnuts from 3 different

geographical areas were used for experimentation,

divided as follows: Piedmont (Italy), Ordu (Turkey)

and Akcackoca (Turkey). Hazelnuts from Piedmont

are the only mono-cultivars, specifically “Tonda

Gentile Trilobata”, while Turkish hazelnuts are a

blend of different cultivars of those areas.

Each hazelnut origin is divided into 5 subgroups of

visual defects: control, weak rotten, strong rotten,

rotten with “cimiciato” defect and rotten with

mold. From each subgroup hydroalcoholic

solutions were prepared containing gradual

percentages of rotten hazelnuts. Solutions

containing 100%, 10%, 4% and 2% of hazelnuts

were prepared.

Compound Discoverer 3.3 (ThermoFisher, USA)

and SIMCA 17 (Sartorius, Germany) software

were respectively used for the identification and

characterization of potential markers and for the

multivariate analysis, such as PCA and OPLS-DA

techniques. Before processing in SIMCA 17 the

.raw files were processed with XCMS software

(XCMS, USA) [5].

3. Results Using Compound Discoverer 3.3, a discriminant

analysis was performed between control samples

and rotten samples on one cultivar at a time (Figure

1), considering each subgroup and setting the

following parameters: p-value <0.05,

Log2FoldChange > 4 and Retention Time (RT)

<24 min.

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Fig. 1 Volcano Plot of discriminant analysis for 10% rotten samples vs. control samples in Piedmont “Tonda Gentile

Trilobata” (p-value <0.05, Log2FoldChange >4 and RT < 24’).

The results were crossed between the 3 origins

and about 30 potential common markers related

to the rotten defect were identified in both 10 and

2% rotten sample solutions.

Furthermore, as reported in Figure 2, through

OPLS-DA (Orthogonal Partial Squares

Discriminant Analysis) an observed vs. predicted

model was obtained (internal validation through

Permutation Plot), which shows how even at low

concentrations of rotten, 2 and 4%, the main

variables (VIP>1, Variable Influence in

Projection) can be discriminated between one

level of rotten and another, thus indicating the

potential for quantitative analysis.

Fig. 2 OPLS-DA: observed vs predicted (VIP>1) for control, 2 and 4% rotten samples (Piedmont, Ordu and Akcackoca).

4. Partial conclusions and next aims The first studies conducted have encouraged the

good potential of the UHPLC-HRMS system,

both the discriminating analysis of control

samples vs. rotten samples and the multivariate

analysis have shown that some compounds can be

potential markers of the rotten defect. Being a

multi-component food matrix, identifying a group

of "only" 30 compounds is a good starting

point for all further evaluations. The formulas

and molecular structures when proposed

seemed congruent to the literature [6] and

therefore it will be interesting to investigate the

assignment of molecular structures for the

selected compounds. For this purpose, the MS-

MS fragmentation will allow to improve the

interpretation of the spectra and can also

represent an important tool in the screening and

selection process of a few units of markers.

References

1. Battilani, P., Chiusa, G., Arciuolo, R., Somenzi, M., Fontana, M., Castello, G., & Spigolon, N. (2018). Diaporthe as the main

cause of hazelnut defects in the Caucasus region. Phytopathologia Mediterranea, 57(2), 320-333.

2. Arciuolo, R., Santos, C., Soares, C., Castello, G., Spigolon, N., Chiusa, G., ... & Battilani, P. (2020). Molecular characterization

of Diaporthe species associated with hazelnut defects. Frontiers in plant science, 1956.

3. Valeriano, T., Fischer, K., Ginaldi, F., Giustarini, L., Castello, G., & Bregaglio, S. (2022). Rotten Hazelnuts Prediction via

Simulation Modeling-A Case Study on the Turkish Hazelnut Sector. Frontiers in Plant Science, 13, 766493-766493.

4. Singldinger, B., Dunkel, A., & Hofmann, T. (2017). The cyclic diarylheptanoid asadanin as the main contributor to the bitter off-

taste in hazelnuts (Corylus avellana L.). Journal of Agricultural and Food Chemistry, 65(8), 1677-1683.

5. Tautenhahn, R., Patti, G. J., Rinehart, D., & Siuzdak, G. (2012). XCMS Online: a web- based platform to process untargeted

metabolomic data. Analytical chemistry, 84(11), 5035-5039.

6. Chepkirui, C., & Stadler, M. (2017). The genus Diaporthe: a rich source of diverse and bioactive metabolites. Mycological.

Rotten 4%

Rotten 2%

Control

Piedmont Ordu Akcackoca

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Phytochemical investigation and biological activity of Bakhtiari savory (Satureja

bachtiarica Bunge.) leaves.

M. R. Samani1,2, G. D’Urso1, M. Masullo1, S. Piacente1

1 Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 84084, Fisciano, SA, Italy. 2 Ph.D. Program in Drug Discovery and Development, Department of Pharmacy, University of Salerno.

Summary: Satureja bachtiarica (Lamiaceae) is an Iranian endemic plant, used as traditional medicinal

herb and spice. In this study, the definition of the chemical profile of S. bachtiarica was performed by LC-

ESI/LTQOrbitrap/MS/MSn and NMR analysis. Moreover, the evaluation of the antioxidant activity of the

extract of S. bachtiarica was carried out.

Keywords: Satureja bachtiarica, LC-ESI/LTQOrbitrap/MS/MS n, NMR analysis

1. Introduction Bakhtiari savory (Satureja bachtiarica Bunge.), also

known with the Persian name “Marzeh Bakhtiari”,

is a medicinal and culinary herb belonging to the

Lamiaceae family (Figure 1), widely distributed in

southwestern Iran. Even if S. bachtiarica is reported

to possess many biological and pharmacological

activities including antioxidant, antimicrobial, anti-

inflammatory, analgesic, antiseptic, antiviral, and

anti-nociceptive activities [1,2], little is known about

its chemical composition. For this reason, in the

present study, a phytochemical investigation of this

species was carried out.

2. Experimental The air-dried leaves of Satureja bachtiarica Bunge

were extracted by maceration at room temperature

using EtOH-H2O 7:3 v/v. The obtained extract was

subjected to n-butanol /water partition to remove

free sugars. In order to have a preliminary metabolite

profiling of S. bachtiarica, the butanol extract was

chromatographically separated on a Luna C18 5 μm

(150x2.1mmm) (Phenomenex Aschaffenburg,

Germany) column. The analyses were performed in

negative ion mode by using a Linear Ion Trap-

Orbitrap hybrid mass spectrometer (LTQ-Orbitrap

XL, Thermo Fisher Scientific, Bremen, Germany)

with electrospray ionization. The structural

determination of compounds has

been performed by integrated data from 1D and 2D-

NMR experiments and HRMSn analysis. Different

classes of natural compounds have been identified.

3. Results The LC-MS profile of butanol extract of S.

bachtiarica whole plant guided the isolation of

several compounds reported in Figure 2, whose

structures were unambiguously elucidated by NMR

analysis. In this way specialized metabolites of

different classes were identified, mainly belonging

to monoterpenes (1, 5), flavonoids (3,9-16, 18-20,

25-26, 28-29, 31-33 and 37-42), organic acids (4, 7),

indoles (6), phenylpropanoids (22 and 35-36),

phenolics (8, 23), lignan (27), coumarin (30), fatty

acid (34), and biphenyls (43-45). Moreover, the total

flavonoids content and the antioxidant activity of S.

bachtiarica hydroalcoholic extract have been

evaluated by using the allumine chloride

colorimetric assay and DPPH assay, respectively. S.

bachtiarica extract showed an interesting

antioxidant activity. The DPPH assay, revealed that

the antiradical activity of the extract was 85.31

μg/mL, using ascorbic acid as a reference compound

(1.35 μg/mL). This result is in agreement with the

high content of flavonoids (162.33μg/g of plant

extract), expressed in rutin equivalents.

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Fig.1. Satureja bachtiarica Bunge

Fig. 2. LC-MS profile in negative ion mode of S. bachtiarica hydroalcoholic extract.

4. Conclusions This is the first chemical investigation of S.

bachtiarica by an advanced technique like HR-

LC-ESI-Orbitrap- MS along with NMR

spectroscopy that provides an unambiguous

structural elucidation of compounds. The present

study highlights the extract of S. bachtiarica as a

source of phenolic compounds with antioxidant

activity. The obtained results provide evidences

for further investigation to promote the use of

plant extract of

S. bachtiarica for pharmaceutical purposes.

References 1. S. M. Memarzadeh, A. Gholami, A. G. Pirbalouti, S. Masoum; Industrial Crops and Products, 154 (2020), pp

112719. 2. M. Rahimmalek, M. Afshari, D. Sarfaraz, M. Miroliaei; Industrial Crops and Products, 154 (2020), pp 112640.

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Target Discovery of the triterpenoid Myrianthic Acid through a Combined

Approach

A. Capuano1, E. Gazzillo1, G. D’Urso1, V. Samukha2, M. G. Chini2, M. V. D’Auria3,

G. Bifulco1, A. Casapullo1

1Dipartimento di Farmacia, Università degli Studi di Salerno, via G. Paolo II 132, 84084, Fisciano, Italia

2Dipartimento di Bioscienze e Territorio, Università degli Studi del Molise, Via F. De Sanctis 1, Campobasso, Italia 3Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italia

Summary: A Chemical proteomic approach based on limited proteolysis and Mass Spectrometry was combined

with in silico studies for the study of the interactome of a natural triterpenoid compound named Myrianthic

acid.

Keywords: Limited proteolysis, Mass Spectrometry, Myrianthic Acid.

1 . Introduction Natural products (NPs) represent a successful

source of potential drugs thanks to the great

diversity of their structural characteristics.

Identification of the protein target of NPs is

essential for the knowledge of their mechanisms of

action and for the development of new drugs.

Chemical proteomics offers several approaches for

these target identifications; Activity-Based Protein

Profiling (ABPP) and Compound-Centric

Chemical Proteomics (CCCP) are conventional

methods based on the chemical modifications of

the ligand in order to use it as bait and fish its

targets. Unfortunately, the functionalization of the

molecule could alter its bioactivity and, therefore,

preliminary structure-activity relationship studies

(SAR) are necessary which lengthen experimental

time. Furthermore, a low chemical reactivity of the

compound could avoid its modification.

Label-free proteomic procedures overcome these

limitations, using the native (unmodified)

compound. Drug affinity responsive target

stability (DARTS, see figure)[2] and Limited

Proteolysis coupled to Multiple Reaction

Monitoring-Mass Spectrometry (LiP-MRM-

MS)[3] permit study of a ligand-protein target

complex directly in a complex protein mixture

(such as a crude cell lysate), using limited

proteolysis in native conditions. The ligand binding

stabilizes the target protein structure, which is less

sensitive to proteases, and the decreased

proteolysis can be detected by 1D-SDS- PAGE.

Combination of DARTS with LiP-MRM- MS

allows identification of the protein targets and

definition of the protein(s) region(s) interested in

the binding with a small molecule.

Fig.1 DARTS Workflow.

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2. Results and Conclusion A combined approach has been applied to the

study of the interactome of Myrianthic Acid

(MA, see figure) [4], a natural triterpenoid

with an interesting bioactive profile. DARTS

led to the identification of the Fatty Acid

Synthase (FAS) as the best MA partner, while

LiP-MRM-MS gave insights on some peptide

fragments located in different domains of the

enzyme, probably involved in the binding.

Molecular docking analysis was also

performed to confirm DARTS and LiP-MRM-

MS data.

Fig.2. Myrianthic Acid

.

3. Experimental

Both in DARTS and LiP-MS experiments

Myrianthic Acid was incubated with cell lysate

and then a limited proteolysis was performed

on proteins. The Mass Spectrometric analysis

was performed according to bottom-up

approach; thus, proteins were also digested

with trypsin. For DARTS experiment the

analysis was performed on the Q-Exactive

Hybrid Quadrupole-Orbitrap Mass

Spectrometer (ThermoFisher Scientific,

Bremen), while the LiP-MS experiment was

analysed trough Q-Trap 6500 (ABsciex).

References 1. X. Chen, et al. Signal Transduction and Targeted Therapy,2020,5,1 13.

2. B. Lomenick, et al. PNAS, 2009, 106, 21984–21989.

3. Y. Feng, et al. Nat. Biotecnology, 2014, 32, 1036-1044.

4. C. Festa, et al. Phytochemistry Letters, 2015, 13, 324- 329

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Development of a LC-MS/MS method for the quantification of multiple

vitamin D metabolites

G. Troiano1,2, C. LeGoff1, S. Peeters1, P. Montoro2, E. Cavalier1

1Department of Pharmacy, University of the Study of Salerno, via Giovanni Paolo II, 132, I-84084 Fisciano,

SA, Italy 2 Hospital Center Universitarie de Liege (CHU de Liege), Université de Liège, Belgique

Summary: The aim of the project was developing an analytical method to separate and measure

multiple vitamin D metabolites paying particular attention on 1,25-Dihydroxyvitamin D. Generally,

25-Hydroxyvitamin D is the preferred marker of vitamin D status, but 1,25-Dihydroxyvitamin D is

an important analyte in specific clinical situations.

Keywords:1,25-Dihydroxyvitamin, ultra-performance liquid chromatography, tandem mass

spectrometry.

1. Introduction Over the past decades, several skeletal and non-

skeletal diseases have been related to low levels

of circulating vitamin D. Indeed, it appears that

vitamin D plays a role in the pathogenesis of

auto-immune and cardiovascular diseases,

cancer, and diabetes as much as it is involved in

bone health.

Generally, 25-hydroxyvitamin D is the

preferred marker of vitamin D status, but it

seems clear that 1,25-dihydroxyvitamin D

(calcitriol), the active form of vitamin D, is an

important analyte in specific clinical situations.

Hence the need to separate and quantify

calcitriol.

Though, the accurate measurement of 1,25-

dihydroxyvitamin D results to be a challenge

due to its highly lipophilic nature and the

picomolar circulating concentrations. Besides,

it must be distinguished from 25-

hydroxyvitamin D, which circulates at 1000-

fold higher concentration, and from the vitamin

D metabolites coming from alternative

pathways, which are very similar structurally

and may represent isobars in chromato-graphic

resolution.

2. Experimental Synthetic standards of vitamin D metabolites

{25-hydroxyvitamin D3 -[25-OH D3], 3-epi-25-

hydro-xyvitamin D3 [3-epi-25-OH D3], 25-

hydroxyvita-min D2 [25-OH D2], 24,25-

dihydroxyvitamin D3 [24,25-(OH)2 D3], 23,25-

dihydroxyvitamin D3 [23,25-(OH)2 D3], and

1,25-dihydroxyvitamin D3 [1a,25-(OH)2 D3] }

were derivatized with the rea-gent DMEQ-TAD

{4-[2-(6,7-dimethoxy-4-methyl -3,4-

dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-

3,5-dione} to improve the ionization efficiency

relative to native metabolite.[1]

Chromatographic separations were achieved

using CORTECS-C18+ UPLC column (1,6 mm,

2.1x100 mm) and a water/methanol-based

gradient solvent system. The mobile phase A

was water, and the mobile phase B was

methanol, both supplemented with 2 nM of

ammonium acetate and 0.1% formic acid. The

initial conditions were 50:50 (v/v) mobile phase

A:mobile phase B at flow rate of 0,350 mL/min.

Mobile phase B was increased up to 67% over

11 minutes using linear gradient (curve 6), after

that the mobile phase B was stabilized on 67%

up to 20 minutes as an isocratic, before

returning to starting conditions for 5 minutes,

for a total run of 25 minutes. An extended

chromatography was involved to better resolve

of all the metabolites.[1]

The spectrometric analysis was performed by

using tandem mass spectrometry (MS/MS) and

the mass spectrometer was set on ESI positive

ionization mode. The conditions were the

capillary voltage at 1,10 kV, the desolvation

temperature at 650°C, the desolvation gas at

1000 L/h and the cone gas at 150 L/h. The

acquisition mode used to record the transitions

was the multiple reaction monitoring [MRM].

The analysis was performed by using Acquity

UPLC connected in-line with a Xevo TQ-S

mass spectrometer, products by Waters

Corporation.

The same method was tested on 200 l of serum

samples: protein precipitation was carried out

PO19

114 MASSA 2022


using zinc sulfate and methanol and the organic

extract-ion was achieved by solid phase

extraction [SPE]. The recovered vitamin D

metabolites were deri-vatized with DMEQ-

TAD and dried under a stream of prepurified N2

at 35°C. The extract was redis-solved in

methanol/water UPLC mobile phase.

3. Results The DMEQ-TAD adducts were

chromatographi-cally resolved as showed in the

table 1.

DMEQ-TAD adducts consisted of 6R and 6S

iso-mers and their chromatographic bands were

eluted as two peaks with the difference of 1 or 2

seconds. The more abundant 6S isomer,

correspondent to the more intense peak, was

used for quantification.

The major characteristic ions, as parent ions, for

derivatized metabolites were their molecular

ions [M+H]+ as shown in table 1. For what

concerns the daughter ions, the mass/charge

(m/z) ratio of 468 was chosen as the fragment

ion because of greater specificity and lower

background as compared with the fragment at

mass/charge (m/z) ratio of 247. Only for the

fragment ion coming from 1,25-(OH)2 D3 was

selected a very specific fragment at the

mass/charge (m/z) ratio of 484.

The results of the analysis carried out on the

deriva-tized synthetic standard of 1,25-

dihydroxyvita-min D revealed a limit of

detection of 10 pg/ml, a concentration lower

than the physiological values. However, the

same performance was not achieved on samples

from sera test due to the matrix effect and the

possible ionization suppression.

4. Conclusions The analytical method developed showed a

good resolution for all the metabolites tested.

The calci-triol peak is separated from the

potential interfe-rences constituted by the

catabolic metabolites with similar structures

and by 25-hydroxyvitamin D which in turn is

separated from its epimer in C3.

It remains to optimize the preparation of the

serum samples in order to clean-up and remove

common matrix effect interferences such as

salts, proteins and phospholipids, with the

perspective to apply the method on sera test and

obtain a better performance.

Fig. 8. MRM of 1a,25-Dihydroxyvitamin D in 10 pg/ml concentration.

Table 4. RT and transitions for vitamin D metabolites

Compound Retention Time (min) MRM (m/z)

24,25-(OH)2 D3 9,13 - 11,22 762,40 > 468,17 - 762,40 > 247,09

23,25-(OH)2 D3

1,25-(OH)2 D3

10,76 - 12,45

11,80 - 12,87

762,47 > 468,11 - 762,47 > 247,09

762,47 > 484,10 - 762,47 > 247,09

25-OH D3

3-epi-25-OH D3

25-OH D2

15,21 - 17,45

15,45 - 17,23

16,11 - 19,00

746,44 > 468,12 - 746,44 > 247,03

746,44 > 468,12 - 746,44 > 247,03

758,47 > 468,12 - 758,47 > 247,04

References

1. Kaufmann, M. et al. Journal of Clinical Endocrinology and Metabolism 99, 2567–2574 (2014);

2. Kaufmann, M. et al. Journal of Bone and Mineral Research 32, 1589–1596 (2017).

115 MASSA 2022


Summary ORAL PRESENTATIONS.........................................................................................................8

Applicazioni della spettrometria di massa per lo studio ed il controllo dei materiali

polimerici ……………………………………………………………………………………...8

PL1 Soft MS of synthetic polymers: a focus on bioplastics characterization…………....9

KN1 High resolution-mass spectrometry for safety assessment of food packaging

materials……………………………………………………………………………10

OR1 TGA-GC-MS and MALDI-TOF mass spectrometry: two promising tools for the

characterization of ultrathin polymer films…………………………………..….... 11

OR2 Optimization of polymer processing for the reduction of volatile organic compounds

(VOCs)……………………………...………………………………………….......13

La spettrometria di massa per lo studio di sostanze naturali …………………………….....14

PL2 Lipidomic analysis in food: The role of a detailed elucidation of intact lipids in

functional foods for investigating on nutritional aspects……………………….......15

OR3 Metabolomics in the study of Foeniculum vulgare. From the characterization of

bioactive compounds to cell metabolomics studies for the confirmation of their

activities....................................................................................................................16

OR4 Untargeted metabolomics approach to understand grapevine communication

mediated by volatile organic compounds against downy mildew……………...…...18

OR5 The development of a manually curated database of the metabolic proteins of

Triticum aestivum (TriMet_DB)…………………... …………...............................20

OR6 LC-MS for the Analysis of Psychoactive Substances in Archaeological Finds.........22

OR7 Determination of Carbazole Alkaloids in Murraya Koenigii leaves by means LC-

MRM/PI/IDA/EPI analysis …………………………………………......................24

OR8 Comprehensive two-dimensional liquid chromatography coupled to mass

spectrometry detection for characterization of bioactive compounds in food and

natural products.........................................................................................................26

OR9 Quantification of glyphosate in milled and brown rice in LC-ICP-MS/MS….……27

OR10 Raw materials in food manufacturing: a complexity ascertained by high-resolution

mass spectrometry…………………………..……………………………………...29

OR11 Use of native-mass spectrometry to elucidate the oligomeric state and substrate-

binding affinity of FrlB, a bacterial deglycase...........................................................31

OR12 Assaying vegetable specimens by FT-ICR mass spectrometry, an election technique

in metabolomic studies………………………………………..................................33

La spettrometria di massa nelle Scienze della vita…………………….……………..………....35

PL3 Metabolomics, analytical tools for the systems-level analysis of metabolism….......36

OR13 Proteomic profile of extracellular vesicles secreted by astrocytes using high

resolution mass spectrometry………………………………………………………37

OR14 Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Profiling

of Commercial Enocianina and Evaluation of Their Antioxidant and Anti-

Inflammatory Activity.……………………………………... …..…………………39

OR15 Emerging per- and polyfluoroalkylated substances (PFASs) in wild boar liver........41

OR16 Plasma proteome investigation of COVID-19 patients with different outcomes

through an untargeted label-free LC-MS/MS approach…………………………... 43

OR17 A novel Spatial Multi-omics mass spectrometry imaging workflow to assist clinical

investigations…………………………………………………................................45

116 MASSA 2022


OR18 The detection of new products by HPLC-HRMS validates a novel stoichio- kinetic

model for the reaction between the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•)

and common antioxidants…………………………………………………….……46

OR19 MS-based approaches in drug development: elucidation of RNA-ligand-protein

interactions for the investigation of bis-3-chloropiperidines targeting TAR……….47

OR20 Discovery and characterization of a novel lipid class via LC-ESI-MS & MSn analysis:

the case of Acyl-Glucuronosylglycerols………………….………………………..49

OR21 Quali-quantitative determination of toxic carbonyl compounds in the emissions of

both heated tobacco products (HTP) and conventional cigarettes……..…………...53

50 anni della DSM……………………………………………………………………………..55

PL4 The first 50 years of GSM – DSM…………………….……………..............................56

KN2 Mass Spectrometry in Sicily: a Historical Overview…………................................ 58

Validazione, qualità del dato e miscellanea …..……………………...………………..........61

PL5 Current state and future perspectives on validation of Mass Spectrometry

Methods……………………………………………………………………………62

OR22 Development and validation of a rapid and simple analytical method for the

simultaneous analysis of pyrrolizidine alkaloids and related N-oxides in beehive

products by salting out assisted liquid liquid combined with liquid chromatography-

tandem mass spectrometry…………………………………………………….…...63

OR23 Perfluoroalkyl and polyfluoroalkyl substances (PFASs) in honey by LC-Q- Orbitrap:

method development and validation………………………………..……………...65

OR24 Material screening of electronic devices by ICP-MS and HR-ICP-MS: towards a low

background for astroparticle physics experiments…………………………………67

OR25 Different applications of the Liquid Chromatography coupled to Isotope Ratio Mass

Spectrometry (LC-IRMS) ……………………………………………………........69

OR26 Validation of analytical method for the determination of cortisol in hair by UHPLC-

MS/MS……………………………….....................................................................71

OR27 Fast and sensitive high-throughput antineoplastic drugs surface contamination

monitoring by ultra-high performance liquid chromatography coupled with tandem

mass spectrometry……………………………………………………..…………...73

OR28 Rapid analysis and interpretation of metabolomics SWATH acquisition……...…...75

POSTER PRESENTATIONS..................................................................................................76

PO1 A general analytical platform and strategy for the screening and determination of

pyrrolizidine alkaloids in food matrices with high risk of contamination using high-

resolution mass spectrometry (Q-Orbitrap)……………………………………...…77

PO2 Determination of sugars in olive leaves in response to water deficit……………......79

PO3 Development of mass spectrometric methodologies for the quality assessment of

bergamot-based food products in the framework of the activities of the QUASIORA

laboratory..................................................................................................................81

PO4 Identification of sinapoylquinic acids in coffee by “surrogate standards”

fingerprinting: a win-win strategy……….................................................................83

PO5 Stable isotope analysis to detect differences in four compartments of Simmental cull

cows fed on C3 and C4 diets……………………………..………........................... 85

PO6 LC-MS/MS characterization of phenolic compounds in olive oils obtained with

117 MASSA 2022


different extraction techniques……………………………………………............. 87

PO7 Tuna Fish wastes valorisation: green production and triacyclglycerols

characterization of Tuna fish oils by HPLC/HRMS and GC/MS………………..... 89

PO8 Are you wake yet? Mapping the spatial metabolome of coffee beans with MS-

imaging……………………………………............................................................ 91

PO9 Risk in glyphosate residue quantification in cereal products:

the 2-amino-3-phosphopropionic acid case study…………………………….........93

PO10 Untargeted screening of perfluoroalkyl and polyfluoroalkyl substances (PFASs) in

air of Italian e-waste recycling facilities....................................................................95

PO11 Presence of novel perfluoro-polyether carboxylic acids in poultry eggs...................97

PO12 Combining passive sampling and LC-MS/MS for the study of emerging pollutants in

seawater ...................................................................................................................99

PO13 Fast and Easy Methodology for the Determination of Formaldehyde in Indoor

Working Environments by Derivatization Coupled with GC-MS.......................... 101

PO14 Unravelling the proteomic content and spatial distribution of amyloid causative

proteins in renal biopsies using MALDI-MSI.........................................................103

PO15 Suspect screening of Novel Psychoactive Substances using QTrap Technology... 105

PO16 Untargeted UHPLC-HRMS approach for the identification of rotten defect markers

in hazelnuts………………………………………...……………………………..107

PO17 Phytochemical investigation and biological activity of Bakhtiari savory (Satureja

bachtiarica Bunge.) leaves.....................................................................................109

PO18 Target Discovery of the triterpenoid Myrianthic Acid through a Combined

Approach………………………………………….…………...…………………111

PO19 Development of a LC-MS/MS method for the quantification of multiple vitamin D

metabolites………………………………………………………………………..113

118 MASSA 2022


Proceedings of the

MASSA 2022


All the scientific contributions of the symposium are collected in this

international volume with ISBN code

You can cite your work as follow:

N. Surname, N. Surname, … and N. Surname; <abstract title>, in Proceedings

of the MASSA 2022, , Ed.XXXX, ISBN: xxxxxxxxxx, <page number>, 2022,

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