SESSION 3: NEW TECHNIQUES, CONFIRMATORY ANALYSIS … · KEYNOTE LECTURE 5 CHEMICAL RESIDUE & CONTAMINANT TESTING AN OVERVIEW OF LATEST ADVANCED TECHNOLOGICAL OPTIONS FOR RESIDUE ANALYSIS

1

Laboratoire d’Étude des Résidus et Contaminants dans les AlimentsONIRIS - France - www.laberca.org

Bruno LE BIZEC , Pf, Dr, HDR

KEYNOTE LECTURE 5

CHEMICAL RESIDUE & CONTAMINANT TESTING

AN OVERVIEW OF LATEST ADVANCED

TECHNOLOGICAL OPTIONS FOR RESIDUE ANALYSIS

Fabrice MONTEAU, Emmanuelle BICHON, Loïc HERPIN, Stéphanie PREVOST, Ludivine SEREE,

Gaud DERVILLY-PINEL

Egmond aan Zee, The Netherlands, 23-25 May 2016

SESSION 3: NEW TECHNIQUES, CONFIRMATORY ANALYSIS

2

g

• Extremely low concentration levels

mg 10-3

µg 10-6

ng 10-9

pg 10-12

fg 10-15

• Requiring unambiguous identification

100

1.1 What are fit-for-purpose analytical methods?

• Covering most matrices involved in MPs1. INTRODUCTION

• Accurate and precise

• Competent whatever the residue chemistryLow MW

Aliphatic

Charged

Polar

-

+ Aromatic

Hydrophobic

3

SAMPLE INTRODUCTIONIONIZATION

ION CHARACTERIZATION

IONDETECTION

A+ m/z IA, B, C

a1+ a2

+ a3+

1. INTRODUCTION

1.2 Outline of the talk

Ambient Ionization Techniques

DART, DESI, PSI, ASAP

Chromatographic Techniques

GC-APCI, SFC-API

High Resolution Mass Spectrometers

TOF, ORBITRAP, FT-ICR

ION MOBILITY, HANDHELD DEVICES

Other Trends

4

SAMPLEINTRODUCTIONAmbient Ionization Techniques

DART, DESI, PSI, ASAP

Chromatographic TechniquesGC-APCI, SFC-API

5

Paper Spray Ionization (+high throughput, low cost, ↘

polar compounds, quantitation, sensitivity)

� MEL (milk), VET DRUGS (meat), DYES (chili pepper)

Desorption electrospray ionization (+no sample prep, G/L/S matrices,

gentle ionization, ↘ powder, volatile, sensitivity, selectivity)

� HORMONES (Inj Site), MEL (FCM)

Ambient Solid Analysis Probe (+no sample prep,

L/S matrices, volatile/semi-volatile, ↘ sensitivity)

� HORMONES (preparation), DYES (spices)

Direct Analysis in Real Time (+no sample prep,

L/S matrices, small MW, no alkali adduct, no n+,

less Ion Supp.)

� VET DRUGS and HORMONES (food)

Guo et al., Mass Spectrometry Reviews, 2015, 9999, 1–27

6

ASAP

7

�Utilizes the heated N2 desolvation gas to vaporize the

sample and a Corona discharge for sample ionization.

�Rapid direct analysis of volatile/semi-volatile, solid and

liquid samples. No need for sample preparation.

2. SAMPLE INTRODUCTION

2.1 ASAP: Basic Principle

Ambient Solid Analysis Probe

McEwen, R.G. McKay, B.S. Larsen, Anal. Chem ., 2005, 77, 7826-7831.

8


2.1 ASAP: application to steroids

� Linear T°C gradient from 100 to 600 °C in 15 s, held at 600 °C.

� In positive mode, the Corona current was set at 3 μA, cone voltage to

45 V, and source offset to 70 V.

� In negative mode, the Corona current was set at 5 μA, cone voltage to

35 V, and source offset to 80 V.

Anal. Chem. 2014, 86, 5649−5655

9


NON ESTER STER-H20 �� Neutral Loss 96


FISHING THE STEROID

NUCLEUS

DETERMINING THE NATURE

OF THE ESTER

GETTING THE STEROID

ESTER MW

NATURE OF THE ESTER �� Enanthate

10


FISHING THE STEROID

NUCLEUS

DETERMINING THE NATURE

OF THE ESTER


GETTING THE STEROID

ESTER MW

BOLDENONE

391

BENZOATE

BOLDENONE-17-BENZOATE

[M+H]+

11

DART

12


2.2 DART: Basic Principles

� DART patent � first granted in 2005 by Cody & Laramee

� Two pivotal papers were published in 2005 (Cody et al.)

� DART introduced in 2005 as a commercial product

� DART is used for rapid ionization of small molecules under

ambient conditions

� Good suitability in analyzing most liquid, solid, and gas

samples consisting of either polar or non-polar compounds

� Analysis of food samples with simple/no sample treatment

Direct Analysis in Real Time

13


2.2 DART: Basic Principles

� Multiple acceptable ionization mechanisms, mainly Penning ionization (PI), proton transfer (PT) in

the positive mode and electronic capture (EC) in the negative one

� When He is used as the carrier gas, He* collides with an atmospheric pressure water molecule and

ionizes it. The ionized water molecule then undergoes several reactions with other neutral water

molecules resulting in the formation of a protonated water cluster. The water cluster then interacts

with the analyte molecule (S) generating a protonated molecule.

(He, N2, Ar)

14

� DART-Standardized Voltage and Pressure (SVP) ion

source (IonSense, Saugus, MA, USA)

� He flow: 3.5 L.min-1, Gas T°C: 300 °C;

� Discharge needle voltage: - 5 kV , Grid electrode: 350 V.

� Transmission module was moved at 0.5 mm.s-1


2.2 DART: application to steroid esters

m/z 443T Dec

SUSTANON PREPARATION

Reduced sample size (below < 10 μL)

High throughput (10 samples < 5 min)

Fully automated process

15


2.2 DART: application to vet drugs in feed

�Ionization of coccidiostats and benzimidazoles � [M+H]+ or [M–H]– for most targeted analytes

�Exception � polyether ionophores which provided the sodium adduct [M+Na]+ as the most intense peak

monensin

narasin

salinomycin

clopidol

maduramicin

robenidine

Extracted ion chronogram and MONENSIN MS medicated feed at 100 mg kg–1.

� Combination of a QuEChERS-like sample treatment with DART-orbitrap � improvement of ionization efficiency

� LCLs obtained at levels of 1–10 mg.kg-1 for benzimidazoles in milk and 0.25– 0.5 mg.kg-1 for coccidiostats in chicken feed

m/z 693.417

16

� DART-(MS/)HRMS

ambient hair scanning.

� Data-dependent product

ion scan + full-scan high-

resolution data acquisition.

� No need for time-

consuming and laborious

analysis of hair segments.

� LOD for cocaine in hair

was found to comply with

the cutoff value of 0.5

ng/mg recommended by

the Society of Hair Testing


2.2 DART: application to drugs in hair

CHRONOGRAMS

m/z 304.1543

0.04 ngcocaïne.mg-1

0.4 ngcocaïne.mg-1

BLANK

SPIKED

SPIKED

INCURRED

17

DESI

18


2.3 DESI: Basic Principle

� No sample preparation

� Effective for polar/non-polar molecules

� Charged droplets produced from a

pneumatically-assisted ESI and directed

onto a surface to be analyzed (at PATM)

� Resulting mass spectra similar to ESI

� Composition of the spray solvent, the gas

flow rate, applied voltage, impact angle,

spray alignment… to be optimized

� Incorporation of DESI in hand-held MSn

devices would move the pre-screening in

food control from the laboratory to the

sampling site.

Desorption Electrospray Ionization

19


2.3 DESI: 1st example of application

DESI

REACTIVE DESI

� Direct analysis of raw urine samples spiked with steroids

� Porous PTFE substrate with 10 µL of raw urine spiked with 8 ng of androsterone hemisuccinate, 20 ng of 5α-

androstan-3β,17β-diol-16-one and 8 ng of epitestosterone (around 1 ppm level)

20


2.3 DESI: 2nd example of application

Part A, 30:6 (2013) 1012-1019

� Image of the m/z values of scan number 6 (out of 10) of a slice

of bovine tissue incurred with Sustanon

� Intensity of the m/z values from HIGH to LOW

� One extracted mass spectrum (T Dec)

� Spatial resolution of DESI is not as high as that of MALDI (down

to 10–20 μm) or SIMS (1 μm–100 nm) � down to 200–40 μm

TESTOSTERONEDECANOATE

21

3D image of the injection site for the selected m/z value 443

Image of injection site and distribution profile for selected m/z value 443.

(a)First slice (surface), Second slice (2 mm depth), Third slice (4 mm depth).


2.3 DESI: 2nd example of application

22

PAPER SPRAY

23


2.4 Paper Spray: Basic principle and application

� Ion chronograms (signal versus time) for propanolol,

500 ng deposited on paper and eluted by allowing

solvent to wick through the paper continually

� Simple technique for introducing unprocessed

samples of fluids to the MS.

� Combines paper chromatography and ESI to

enable direct analysis of dried fluids without off-

line reconstitution or liquid chromatography.

24

SFC

25


2.5 Supercritical Fluid Chromatography: basic principle

� SFC is a form of NP chromatography

� CO2 is used as mobile phase

� CO2 has a polarity similar to n-heptane at its critical point, but the solvent strength can be

increased by increasing density or using a polar co-solvent = modifier.

� As a rule-of-thumb “…any molecule that will dissolve in MeOH is compatible with SFC…”

� As the supercritical phase represents a state in which L and G properties converge, SFC =

convergence chromatography

26

ajout

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

0

100

20140819007 13: MRM of 3 Channels ES+ TIC (Mabuterol)

6.36e75.13

12

3

4

56

7

8

9

10

11

1213

14

15

1617

18

1920

21222324

26

27

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

0

100

20150213-023 5: MRM of 18 Channels ES+ 368.2 > 294 (Bambuterol)

3.01e8

5.35

19

26

25

2420

17

222328

13

4 9611

87

16

1012

27518

2 3

1514

1

UHPLC

USFC (UPC2)

Feed sample spiked with ββββ-agonists at 25 µg/kg2. SAMPLE INTRODUCTION

2.5 Supercritical Fluid Chromatography: 1st illustration

ISOMETA

SALBU

27

HYDROXYFLUTAMIDE

OSTARINE

BICALUTAMIDE

ANDARINE

UPLC-QqQ

USFC (UPC2)-QqTOF

Selective Androgen Receptor Modulators2. SAMPLE INTRODUCTION

2.5 Supercritical Fluid Chromatography: 2nd illustration

28

GC-APCI

29

Ionization processDesign of the interface

2.6 GC and APCI: principle and illustration

Transfer line temperature was set at 380°C with a sheath nitrogen gas at 31 psi (constant pressure). Source temperature

was 150 °C. Auxiliary and cone gas flow rates were 300 and 0 L h-1. APCI �� positive dry mode.


30

100 fg on-column


2.6 GC and APCI: illustration

m/z 370

m/z 241

m/z 370

m/z 256

@ 20 eV@ 10 eV

5ξ-androstane-3ξ,17ξ-diols (n=8)

as di-TFAA derivatives

EI

APCI

TFAA derivative

TFAA derivative

31

ION SEPARATION

High Resolution Mass SpectrometersTOF, ORBITRAP, FT-ICR

Other TrendsION MOBILITY, HANDHELD DEVICES

32

TOF/ORBI

33

� Multi-residue method for

vet drugs screening in urine

(UPLC-TOF)

� > 100 analytes belonging to

different vet drugs families

� Urine samples � diluted,

injected unfiltered into the

UPLC–TOF

3. ION SEPARATION

3.1 HRMS: accurate mass/generic ions

SULFONAMIDES

ESI+

C6H6NO2S

156.012

GENERIC DAUGHTER ION

BLANK URINE

SPIKED URINE (1 mg.L-1)

WITH ≠SULFONAMIDES

� Trend towards full-scan accurate mass spectrometry (MS) either

based on TOF or FT-Orbitrap

� Retrospective analysis of the full-scan datasets of signals from

novel metabolites or illegal designer substances was presented as a

major step forward.

34

3. ION SEPARATION

3.2 HRMS and multi-residue methods

� HPLC–ToF-MS � shown to be suitable for about

100 veterinary drugs in meat, fish and egg.

� Median mass error for vet drugs at concentrations

ranging from 4 to 400 µg.kg-1 � median 2.5 ppm

� The SigmaFit value (isotope pattern matching) ↘

when […] ↗ and ↗ when matrix complexity ↗

� R2 > 0.99 for 92% of the compounds

� The study showed multi-compound/multi-matrix

capability.

� The highest deviations in terms of mass

measurement error � found in chromatographic

regions where most interferences elute

� HPLC–ToF-MS can not compensate for inadequate

sample preparation.

� Meat sample spiked at the validation level.

� Extracted mass chromatograms (± 0.01u)

BLANK MEAT

STANDARD

EIC

TIC

TIC

EXTRACT

SigmaFit value Mass error

35

3. ION SEPARATION

331.2 ± 0.5 u

331.2268 ± 10 ppm

331.2268 ± 5 ppm

TAc in hair at 5 ng.g-1 UHPLC-Orbitrap, 60,000 RP

3.3 HRMS: mass extraction window and selectivity

10 µµµµg.g-1 DICLOXACILLIN in muscle

470.034 u

Clean extracts

10 ppm

40 ppm

120 ppm

36

Pirimicarb in animal feed matrix at 10 ng.g -1; RP: 100,000 (Orbi)

m/z 239.1503

1 u

± 100 ppm

± 5 ppm

m/z 345.1809

Salmeterol, urine at 30 ng.mL -1, RP 25,000 (ORBI)

± 2 ppm

± 5 ppm

(J Am Soc Mass Spectrom 2009, 20, 1464–1476)

37

m/z minIsopyrin, 25 ng/g in animal feed

XICs 246.1601 with 5 ppm MEW

246.160 u

246.160 u

246.160 u

246.160 u

RP

3. ION SEPARATION

3.3 HRMS: mass extraction window and selectivity

Several data points showed mass deviations that

exceeded the mass tolerance window of the XIC (5ppm)

For a RP of 25,000 and below,

assignment of masses is not reliable

Complete loss of

analyte response � FN

� Major difficulty is caused by endogenous compounds present in much higher

concentrations than target analytes

ISOPYRIN INTERFERENCE

(J Am Soc Mass Spectrom 2009, 20, 1464–1476)

10 mmu

38

IMS

39

DRIFT GAS

ENTRY

EXITIONS

Ion separation is driven by E (Rf + Travelling voltage) applied to an ion mobility

cell which contains a neutral gas at a controlled pressure (mbar)

3. ION SEPARATION

3.5 Ion Mobility spectrometry � a renewed interest

Relation between ion

mobility K and geometry

40

QTOFIM

Possible fragmentation

by CID in TRAP or/and

TRANSFER

cccc

3. ION SEPARATION

3.5 Ion Mobility spectrometry � tested instrument

41

20130618003.raw : 1

EXCESSIN DRIFT TIME

DEFECTIN DRIFT TIME

m/z

DRIFT TIME (ms)

COMPACT

CHEMICAL STRUCTURES

ELONGATED

CHEMICAL STRUCTURES

3. ION SEPARATION

3.5 Ion Mobility spectrometry � β-agonists, defect/excess in drift time

42

5α-androstane-3β,17α-diol

5β-androstane-3β,17α-diol

5β-androstane-3β,17β-diol

5β-androstane-3α,17α-diol

5α-androstane-3α,17α-diol

5α-androstane-3α,17β-diol

5α-androstane-3β,17β-diol

5β-androstane-3α,17β-diol

3. ION SEPARATION

3.5 Ion Mobility spectrometry � androgenic steroids, diastereoisomers

43

3. ION SEPARATION

3.5 Ion Mobility spectrometry

44

DIRECT DETECTION WITHIN 30 s OF MELAMINE USING PORTABLE MASS SPECTROMETER

3. ION SEPARATION

3.6 Handheld Mass Spectrometer

5 mg.mL-1 melamine in whole milk

5 mg.g-1 melamine in milk powder 1 mg.mL-1 melamine in urine

Standard in solvent

Multiple stage MS analysis (n=5) starting with 20

ng/µµµµL clenbuterol ([M + H]+ at m/z 277).

45

170

m/z

Int.LRMS (Q) �� 170

UHRMS (FTICR) �� 170.0329 reduced number of EC

M170T143

OBSERVED THEORETICAL

3. ION SEPARATION

3.7 Ultra High Resolution Mass Spectrometer171

172

HRMS (TOF/ORBI) �� 170.033 numerous EC possibilities

46

5. CO

NC

LUS

ION

SP

ER

SP

EC

TIV

ES

47

4. CONCLUSION AND PERSPECTIVES

4.1 Different options

MIRRORBALL

48

LABORATORIES

INSPECTION

YEARS

PERFORMANCE

FILL THE GAP…

4. CONCLUSION AND PERSPECTIVES

4.2 Observation

1980 20202000

49

AN AMBIENT IONIZATION

UNIVERSAL (Ɣ POL)

EFFICIENT

QUANTITATIVE (Ɣ S/L/G)

NO ION SUPPRESSION

THE PERFECT DETECTOR

RAPID, SENSITIVITY

LARGE DYNAMIC RANGE

LONG LIFE

ADAPTED TO FIELD CONSTRAINTS

A HANDHELD MULTIDIMENSION MS

1st DIM �� FULL SCAN HRMS, FAST, SENSITIVE

COLL CELL �� MULTI-EVENTS, NO CROSS-TALK

2nd DIM �� FULL SCAN HRMS, FAST, SENSITIVE

QUANTITATIVE, SCAN ACQUISITION > 50 Hz

A LEARNING MACHINE

AUTO CALIBRATION

AUTO RECORDING

AUTO DATA-ANALYSIS

AUTO DIAGNOSTIC

Vision of the future miniature mass spectrometer designed for on-site analysis

50

[email protected]

PhD

BOSS

Post

Doc

QO

51

Laboratoire d’Étude des Résidus et Contaminants dans les AlimentsONIRIS - France - www.laberca.org

Bruno LE BIZEC , Pf, Dr, HDR

KEYNOTE LECTURE 5

CHEMICAL RESIDUE & CONTAMINANT TESTING

AN OVERVIEW OF LATEST ADVANCED

TECHNOLOGICAL PTIONS FOR RESIDUE ANALYSIS

Fabrice MONTEAU, Emmanuelle BICHON, Loïc HERPIN, Stéphanie PREVOST, Ludivine SEREE,

Gaud DERVILLY-PINEL

Egmond aan Zee, The Netherlands, 23-25 May 2016

SESSION 3: NEW TECHNIQUES, CONFIRMATORY ANALYSIS … · KEYNOTE LECTURE 5 CHEMICAL RESIDUE & CONTAMINANT TESTING AN OVERVIEW OF LATEST ADVANCED TECHNOLOGICAL OPTIONS FOR RESIDUE ANALYSIS

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