An Integrated Strategy for Natural Biotoxin analysis - Waters Corporation - Food Safety

©2015 Waters Corporation 1

Integrated Screening & Confirmatory

Strategy for the Analysis of Natural

Biotoxins

©2015 Waters Corporation 2

Overview

Natural biotoxins & their significance?

Challenges & analytical requirements?

Screening assays

Routine quantitative method for 12 regulated mycotoxins – Benefits of mass detection?

– ACQUITY QDa mass detector

Advanced MS functionality – Confirmatory analysis

– Xevo TQ-S for large scale multi-toxin analysis

– Dealing with complex matrices (feedingstuffs)

LC-MS/MS based phycotoxin analysis (TQ-S)

Summary

©2015 Waters Corporation 3

41%

11%6%

12%

6%

6%

6%

12%

Mycotoxin Contaminantion Incidents by Commodity (2011 -2013) Nuts (Brazil, Cashew, Peanut,

Walnut, Chestnut, Almond) Alfatoxins

Coffee beans Ochratoxin A

Animal feed Aflatoxins

Spices (paprika & chilli) Aflatoxins & Ochratoxin A

Peanut butter Aflatoxins

Apples Patulin

Dried figs Aflatoxins

Confectionary Aflatoxins

Natural toxins – significance?

Natural toxins are chemicals that are naturally produced by living organisms. These toxins are not harmful to the organisms themselves but they may be toxic to other creatures, including humans, when eaten “They represent one of the most important and sensitive problems for our world and our life, as various many products we normally use in our diet are exposed to their contamination” MycoRed FP7 Project http://www.mycored.eu/

©2015 Waters Corporation 4

What are mycotoxins?

Mycotoxins are secondary metabolites produced by fungi that are toxic to humans & animals consuming the products

Mycotoxins are dangerous for feed & food chains as they can create contamination in pre- and post-harvest processes

Resistant to decomposition, digestion high or low temperature degradation & remain in the food

Toxic Effects – Aflatoxin B1 is a known carcinogen and immunotoxic

– Fusarium toxins, especially fumonisins are neurotoxic and possible carcinogens, trichothecenes (type A&B) are immunotoxic and zearalenone is estrogenic

– Ochratoxin A is a nephrotoxin, possibly carcinogenic to humans and associated with Balkan Endemic Nephropathy

©2015 Waters Corporation 5

Foodstuffs affected by mycotoxin & contamination?

Tree nuts

Peanuts

Grains

Cereals

Animal feeds

Coffee & tea

Fruits

Vegetables

Fruit juices

Honey

Beer

Wine

Dairy produce

Preserved meat

Farmed fish

Rice

Botanicals

Spices

Snack Foods

Processed foods

©2015 Waters Corporation 6

Food Legislation – which mycotoxins are regulated?

Maximum permitted levels for the major mycotoxins, aflatoxins

(AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), fumonisins

(FB1, FB2), deoxynivalenol (DON), zearalenone (ZEA) & patulin

are included in the European legislation

1881/2006/EC, 1126/2007/EC

Indicative maximum levels for the sum of T-2 & HT-2 have

been recently issued (Recommendation 2013/165/EU)

Although not regulated yet, attention is paid to the occurrence of

nivalenol (NIV), another Fusarium toxin that frequently

contaminates cereals in combination with DON

EFSA Opinions (emerging toxins e.g. enniatins, beauvericin)

©2015 Waters Corporation 7

Current mycotoxin screening strategies?

Performance criteria

Technique

LC-UV/FL Immuno-diagnostic (ELISA/LFD)

TLC (old technology but still

relevant in some geographies)

Ability for multi-mycotoxin screening

Methods are available for a large number of

mycotoxins.

Multiple detectors required to detect all target

compounds

Diverse physiochemical properties mean an array of kits are required to cover all

the regulated mycotoxins

Methods are available for a large number of

mycotoxins. Detection and identification procedures

have been developed making use of molecular

properties or reactions with spray reagents

Detection capability

LODs vary by analyte Post-column derivatisation

required to achieve detection limits for

alfatoxins

Typically very sensitive <<permitted limits

Typically sensitive <permitted limits

Time-to-result Longer turnaround times Quick turnaround times Rapid - quick turnaround

times

Flexibility (extension to other toxins)

Reliant on UV/FL chromophore

Depends on antibody cross-reactivity?

Reliant on UV/FL chromophore or

chromogenic reagent

Quantitative performance?

Quantitative Semi-quantitative Quantitative

©2015 Waters Corporation 8

Immunodiagnostic assays for mycotoxins

IAC; LFD; strip tests

©2015 Waters Corporation 9

Immunoaffinity chromatography

Immunoaffinity chromatography (IAC) is a type of LC in which the

stationary phase consists of an antibody (or antibody-related reagent)

IAC represents a sub-category of affinity chromatography, in which a

biologically related binding agent is used for the selective purification

or analysis of a target compound

The selectivity & affinity of antibodies for their given targets has

made these agents of great interest for many years as immobilized

ligands in affinity chromatography

©2015 Waters Corporation 10

Semi-quantitative test kits IAC columns with fluorometer detection

©2015 Waters Corporation 11

VICAM rapid screening solutions - Immunoaffinity columns and strip tests

• AflaTest

• AflaTest WB

• Afla WB SR

• Afla M1 HPLC

• AflaOchra HPLC

• AOZ HPLC

• Myco6in1

• CitriTest HPLC

• DONtest

• DONtest WB HPLC

• DON-NIV WB

• FumoniTest

• FumoniTest WB

Fluorometeric Tests AflaTest

Afla B

Afla M1 FL+

FumoniTest

FumoniTest 200

OchraTest

ZearalaTest

HPLC/UPLC/LC/MS Tests

using IAC

Aflatoxins, DON, NIV,

T-2, HT-2, OTA,

fumonisins,

zearalenone

New 6 in 1 IAC

Qualitative Strips AflaCheck DONCheck Quantitative Strips Afla-V DON-V Fumo-V

http://vicam.com/products

©2015 Waters Corporation 12

Current analytical strategies - aflatoxin analysis

Routinely analyzed using RP HPLC with FL detection

• Reverse phase eluents quench the fluorescence of aflatoxins

B1& G1

• Derivitization is needed to enhance the response

Derivitization methods for aflatoxins include;

• Post-column iodine addition

• Electrochemically generated bromine using a Kobra Cell®

• Photochemical Reaction for Enhanced Detection (PhCR)

Post-column derivatisation can interfere with FL detection of

other mycotoxins in multi-toxin analysis!

Limits sample throughput

©2015 Waters Corporation 13

Aflatoxin Analysis Kit

Vicam AflaTest® WB provides selective extraction for aflatoxins using wide-bore immunoaffinity columns (IACs)

Waters UPLC method uses the ACQUITY™ Fluorescence Detector

Provides higher sensitivity than HPLC methods

Uses a specialized flow cell and mercury/xenon lamp, avoids requirement for post-column derivatization

Use of UPLC ternary mixing allows chromatographic separation to be optimized (analysis time reduced 12 to 4 min)

©2015 Waters Corporation 14

Aflatoxin analysis kit – chromatographic separation

AF spiked milk powder

Minutes1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80

5

4

3

1

2

Aflatoxins

1 Aflatoxin M1

2 Aflatoxin G2

3 Aflatoxin G1

4 Aflatoxin B2

5 Aflatoxin B1

ACQUITY FLR Detector with large volume flow cell FL detection; Ex 365 nm and Em 429

AF B1 & G1 signal quenching

Allows detection of the aflatoxins at < permitted limits without need

for derivatisation

Improved separation, sensitivity and speed

©2015 Waters Corporation 15

Wide range of analytes of interest: Data rich spectra

Often complex matrixes: Superior selectivity offered by Single Ion

Recording (SIR)

Regulatory requirements and limits: Increased sensitivity

High throughput of routine samples: Increased analytical capability & scope

Ease of method development: Increased peak capacity (mass resolution of

chromatographic co-elutions)

Enhanced consumer safety

Benefits of Mass Detection for screening analysis?

©2015 Waters Corporation 16

What is a Mass Spectrometer?

1. Sample Introduction

2. Ion Source 3. Mass Analyser

4. Detector

5.Data System

LC, GC etc. Mass Spectrometer

©2015 Waters Corporation 17

What is a Mass Spectrometer?

Ion Source Mass Analyser (quadrupole) Detector

©2015 Waters Corporation 18

Analysis of regulated mycotoxins

using single quadrupole MS

(ACQUITY QDa)

Simple protocol & consolidated method

Potential for expansion of scope (emerging

natural toxins)

Collaboration with Veronica Lattanzio ISPA CNR, Bari, Italy

©2015 Waters Corporation 19

QDa

PDA

Mass detection

− m/z 30 to 1250

− ESI positive & negative

Modular, small footprint

Minimal maintenance

− Pre-optimised source ESI ±

− Consumable cone aperture

Minimal user- intervention

− Push button

− Fast warm up/ internal check

− Run samples

“If you can use a PDA, you can use a QDa”

ACQUITY QDa – routine screening tool

©2015 Waters Corporation 20

Extraction protocol and clean-up procedure – wheat & maize

10 g sample + 40 mL water

Extraction by blending for 2 min

Add 60 mL methanol

Extraction by blending for 2 min

Filter the extract through paper filter

5 mL of extract & evaporate until reduce the volume to approx 2mL

Add 5 mL phosphate buffer (pH 7.4)

Pass the sample through the Myco6in1+ column

Wash the column with 10 mL water

Elute the toxins with 3 mL methanol followed by 2 mL water

Dry the eluate

Reconstitute the residue with an appropriate volume of LC mobile phase

sequential extraction with water and

methanol

immunoaffinity column clean up

sample

analysis

©2015 Waters Corporation 21

Experimental -1 Multi-mycotoxin screening method

Sample preparation

UPLC conditions

Time %A %B

Initial 99.0 1.0

7.00 50.0 50.0

10.0 1.0 99.0

11.5 1.0 99.0

11.6 99.0 1.0

14.00 99.0 1.0

Parameter Setting

UPLC Acquity I Class

Column Cortecs UPLC C18 1.6 μm, 2.1x100 mm

Temperature (oC) 40

Flow rate (ml/min) 0.4

Injection volume (μl) 10

Mobile phase A composition

Aq 0.2% acetic acid & 1 mM ammonium acetate

Mobile phase B composition

MeOH 0.2% acetic acid & 1 mM ammonium acetate

Run time (min) 14

©2015 Waters Corporation 22

Experimental -2 Multi-mycotoxin screening method

Parameter Settings

Mode Performance (rotary pump)

Mass range (m/z) 150 to 800

Acquisition mode SIR

Ionisation mode (ESI) Pos & neg

Desolvation temperature (oC)

600 (default)

Cone voltage 10 to 20 (analyte dependent)

Source temperature (oC) 150 (default)

Capillary voltage (kV) 0.8 (default)

Sampling frequency (scan/s)

5 (default)

©2015 Waters Corporation 23

Overlay SIR Chromatograms for aflatoxins at permitted limits in wheat matrix

AFG2 5.85

16216

AFG1 6.19

25728

AFB2 6.54

44160

AFB1 6.86

49509 Aflatoxin Spiked conc

μg kg-1

AFB1 2

AFB2 1

AFG1 1

AFG2 1

©2015 Waters Corporation 24

Overlay SIR Chromatograms for 12 regulated toxins at permitted limits in wheat matrix

1.NIV

2.DON

3.AFG2

4.AFG1

5.AFB2

6.AFB1

7. HT-2

8. FB1 9. T-2

Mycotoxin R.T

(min) S:N

Spiked conc μg kg-1

1. NIV 2.21 1188 750

2. DON 2.97 20955 750

3. AFG2 5.85 5149 1

4. AFG1 6.19 18272 1

5. AFB2 6.53 1967 1

6. AFB1 6.86 12766 2

7. HT2 8.16 430 50

8. FB1 8.38 2682 800

9. T-2+NH4 8.64 64641 50

10. OTA 8.81 993 3

11. Zer 8.86 6682 100

12. FB2 9.00 1967 200

10. OTA

11. Zer

12. FB2

Normalised view

©2015 Waters Corporation 25

Quantitative, confirmatory method

*Analyst familiarisation

Specificity (analyte and matrix)

Calibration curve

Recovery or trueness

Repeatability (r)

Within-laboratory reproducibility

Reproducibility (R)

Decision limit (CCα)

Detection capability (CCβ)

Ruggedness (applicability)

6 month storage stability (solution and matrix)

Qualitative screening method

*Analyst familiarisation

Specificity (analyte and

matrix)

Decision limit (CCα)

Detection capability (CCβ)

Specificity (analyte and

matrix)

Ruggedness (applicability)

6 month storage stability

(solution and matrix)

Method Validation – requirements

under 2002/657/EC for VDRs

*Not a mandatory requirement

©2015 Waters Corporation 26

Experimental outline for this study

% analyte recoveries at maximum permitted levels (ML)

Repeatability & reproducibility

Detection & quantification limits (from matrix assisted calibration

graphs)

Evaluation of matrix effects by comparison of standard & matrix assisted calibration curves

Robustness of repeated injections (response stability)

Compliance with acceptability criteria for MS detection (criteria

established for screening & confirmation cf CD 657/2002/EC & SANCO 12571/2013)

©2015 Waters Corporation 27

Example linearity in processed corn matrix

Compound name: AFB1

Correlation coefficient: r = 0.998746, r^2 = 0.997494

Calibration curve: 764.675 * x + 22.6578

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

3-0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Re

sp

on

se

-0

5000

10000

3

Re

sid

ua

l

-5.0

0.0

Compound name: AFG1

Correlation coefficient: r = 0.999043, r^2 = 0.998087

Calibration curve: 398.47 * x + -57.5865

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Re

sp

on

se

-0

1000

2000

Conc

Re

sid

ua

l

-5.0

0.0

5.0

Compound name: T2 + NH4

Correlation coefficient: r = 0.998341, r^2 = 0.996685

Calibration curve: 374.918 * x + 2861.71

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375

Re

sp

on

se

-0

50000

100000

Conc

Re

sid

ua

l

-0.00

5.00

Compound name: DON

Correlation coefficient: r = 0.998656, r^2 = 0.997313

Calibration curve: 41.7541 * x + 11070.9

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500

Re

sp

on

se

-0

100000

200000

Conc

Re

sid

ua

l

-2.50

0.00

2.50

©2015 Waters Corporation 28

Matrix matched standards

©2015 Waters Corporation 29

Multi-mycotoxin method performance in matrix spiked at permitted limits (n=3)

Cornflake matrix

MycotoxinRetention time

(min)ESI Mode

Spiking

concentration

(ug/kg)

% Recovery & (%RSD)

spikes n=3

Correlation

coefficient

(R2)

Slope

Nivalenol 2.2 Pos 750 95 (6.3) 0.9945 63.0

Deoxynivalenol 3.0 Pos 750 104 (5.3) 0.9960 8062.3

Aflatoxin G2 5.8 Pos 2.5 87 (5.0) 0.9957 125.6

Aflatoxin G1 6.2 Pos 1 97 (2.8) 0.9921 62.0

Aflatoxin B2 6.5 Pos 1 104 (4.3) 0.9969 102.1

Aflatoxin B1 6.9 Pos 2 104 (1.8) 0.9946 68.8

HT-2 8.2 Pos 50 102 (6.4) 0.9900 12.8

T2* 8.6 Pos 50 108 (5.5) 0.9976 932.6

Fumonisin FB1 8.4 Pos 800 94 (4.3) 0.9937 3589.4

Ochratoxin A 8.8 Pos 3 60 (10.9) 0.9943 24.0

Zearalenone 8.9 Neg 100 93 (5.9) 0.9986 3118.0

Fumonisin FB2 9.0 Pos 200 66 (5.3) 0.9370 971.1

*NH4 adduct monitored

Maize snack matrix

MycotoxinRetention time

(min)ESI Mode

Spiking

concentration

(ug/kg)

% Recovery & (%RSD)

spikes n=3

Correlation

coefficient

(R2)

Slope

Nivalenol 2.2 Pos 750 97 (5.0) 0.9993 58.8

Deoxynivalenol 3.0 Pos 750 98 (0.7) 0.9973 1107.9

Aflatoxin G2 5.8 Pos 2.5 95 (10) 0.9965 89.4

Aflatoxin G1 6.2 Pos 1 87 (1.2) 0.9981 57.6

Aflatoxin B2 6.5 Pos 1 95 (2.4) 0.9984 6.03

Aflatoxin B1 6.9 Pos 2 89 (1.3) 0.9975 22.7

HT-2 8.2 Pos 50 112 (2.0) 0.9954 19.9

T2* 8.6 Pos 50 106 (1.1) 0.9967 2861.7

Fumonisin FB1 8.4 Pos 800 85 (3.5) 0.9897 64899.6

Ochratoxin A 8.8 Pos 3 101 (4.5) 0.9690 12.7

Zearalenone 8.9 Neg 100 100 (0.6) 0.9830 823.9

Fumonisin FB2 9.0 Pos 200 101 (2.0) 0.9954 2994.5

*NH4 adduct monitored

©2015 Waters Corporation 30

Large scale multi-mycotoxin method

using tandem quadrupole MS

(Xevo TQ-S)

Regulatory compliance

Extended scope (>35 toxins +)

Enhanced sensitivity

Applicable for challenging matrices

Advanced MS functions (RADAR)

©2015 Waters Corporation 31

MRM – Multiple Reaction Monitoring

More selective & sensitive than SIR

– Specific transition needed for response

– Less interference by background ions of

the same mass

A selected ion is transmitted through the first quadrupole

(precursor ion), fragmented in the collision cell, and a

specified fragment ion is then

transmitted through the second

quadrupole (product ion).

©2015 Waters Corporation 32

Confirmatory Methods

Majority of reference methods currently used for quality control purposes are based on immunoaffinity columns (IAC) with UV, FL or PDA detection Kobra cell in combination with FL is reference for aflatoxins

For unequivocal confirmation of chemical identity mass spectrometric detection is required

Identification criteria established for other residue analysis (cf CD 2002/657/EC)

– Precursor ion (quasi molecular ion)

– Diagnostic fragments

– Ion ratio (q:Q)

– Ion ration tolerances

– Retention time tolerances

Triple quadrupole MS in MRM mode can easily achieve this criteria

standards vs samples

©2015 Waters Corporation 33

CD 2002/657/EC Identification Point (IP) System

Requirement: mass fragments being measured using MS-MS

techniques e.g. Selected Reaction Monitoring (SRM)

Group A of Annex I (96/23/EC) 4 POINTS

Group B of Annex I (96/23/EC) 3 POINTS

S/N ratio for each diagnostic ion >3:1 A minimum of 1 ion ratio shall be measured Ion ratio tolerances (based on relative ion intensity)

©2015 Waters Corporation 34

Experimental TQ-S multi-mycotoxin confirmatory method

A generic and simplified sample extraction protocol using 84:16 (v/v) acetonitrile: acidified water

Parameter Setting

Instrument Xevo TQ-S

Ionisation mode ES (pos/neg switching)

Capillary (kV) 3.4

Source temperature (o C) 150

Desolvation temperature (o C)

400

Cone gas flow (L/hour) 150

Desolvation gas flow rate (L/hour)

800

©2015 Waters Corporation 35

MRM Acquisition TQ-S multi-mycotoxins confirmatory method

Time scheduled MRM acquisition mode

ES (pos/neg) switching

>12 data points across the peak

Quanpedia database of UPLC & MS parameters

Ion ratio tolerances automatically calculated (TargetLynx)

©2015 Waters Corporation 36

Total Ion Chromatogram (TIC) Mycotoxins spiked in almond extract

Time2.00 4.00 6.00 8.00 10.00 12.00

%

14

Alfatoxin B1 Alfatoxin B2 Alfatoxin G1 Aflatoxin G2 Ochratoxin A Deoxynivalenol Citrinin Fumonisin B1 Fumonisin B2 Nivalenol Diacetoxyscirpenol H2 toxin HT2 toxin 3-acetyl-DON 15-acetyl-DON Zearalenone (Zen) Penicillic acid Fusarenon X Ergotamine Roquefortin Β-Zearalanol Α-Zearalanol Cyclopiazonic acid Sterigimatocystin Various dwell times & time windows employed to achieve

12 data points across each peak

Nivalenol

Cyclopiazonic acid

©2015 Waters Corporation 37

TQ-S Confirmatory method Quantitative performance & linearity

Mycotoxin LoD (ng/ml)*

NIV 1

DON 1

AFB1 0.015

AFB2 0.015

AFG1 0.05

AFG2 0.05

T2 5

HT-2 8

ZEA 0.5

OTA 1

FB1 2

FB2 0.5

*LOD determined in feed matrix extracts

©2015 Waters Corporation 38

The challenge - matrix complexity & co-contamination

Feed extract (neat) background BPI full scan

Simultaneously acquired MRM transitions for enniatins B1, A1, A, B2

©2015 Waters Corporation 39

TQ-S sensitivity - reduction in ion suppression Mixed mycotoxin spiked feed extract

Matrix matched standard comparison to S/Std

Ability to inject a smaller amount or dilute the sample

helps reduce matrix effects

=

Ion suppression is

effectively reduced

©2015 Waters Corporation 40

TQ-S Analysis of naturally contaminated feed samples Extract dilution 1:10

U1 / cattle

feed

U2 / pig

feed

U3 / maize

gluten

U4 / Diva L

Vital pig

feed

U5 /Alpha

Maximal pig

feed

U6 / RyeU7 /

Barley

U8 /

Wheat

U9 /

Oats

U10 /

Maize

U11 /

Sunflower

oil

U12 / Pig

feed

15-acetyl-deoxynivalenol 0.5 nd nd 152.8 nd nd nd 13.2 33.4 nd nd nd nd

Aflatoxin B1 0.001 nd nd nd nd nd nd nd nd nd nd 0.2 nd

Aflatoxin B2 0.001 nd nd 0.8 nd nd nd nd nd nd nd 0.1 nd

Aflatoxin G1 0.001 nd nd nd nd nd nd nd nd nd nd 0.1 nd

Aflatoxin G2 0.001 0.3 nd nd nd nd nd nd nd nd nd nd nd

Alternariol 0.06 nd 3.2 nd nd nd 5.3 nd nd 7.6 2.6 10.0 nd

DON 0.13 nd 21.2 283.6 13.2 18.4 nd nd nd 4.8 nd 0.3 nd

Enniatin A 0.01 59.3 6.3 1.4 15.7 39.9 9.7 11.7 0.4 3.2 nd nd 50.5

Enniatin A1 0.01 148.6 17.1 3.2 40.1 19.0 14.2 34.1 0.5 4.9 nd nd 122.4

Enniatin B 0.01 125.2 43.3 5.8 65.3 53.3 92.8 52.9 0.4 9.0 nd nd 116.1

Enniatin B1 0.01 263.0 41.8 5.5 72.1 32.3 42.8 64.0 0.5 9.9 nd nd 238.2

Fumonisin B1 0.01 0.3 0.7 18.9 nd 4.0 nd nd nd 0.4 92.8 nd 1.7

Fumonisin B2 0.01 0.1 nd 3.1 nd 0.8 nd 0.2 nd nd 16.0 nd 0.3

HT-2 Toxin 0.25 nd nd nd nd nd nd nd nd 3.9 nd nd nd

Ochratoxin A 0.006 0.1 nd nd 0.1 nd 0.2 2.8 nd nd nd nd 0.1

Roquefortine 0.003 nd 0.3 0.3 0.2 0.1 nd nd nd nd nd nd nd

Sterigmatocystin 0.003 nd 0.1 0.4 0.2 nd 10.7 nd nd nd nd 0.1 0.2

Zearalenone 0.2 nd 1.6 84.0 nd 4.9 31.2 nd 6.1 nd nd nd nd

8 10 12 8 9 8 7 6 8 3 6 8

*Concentration determined against a solvent calibration series

Number of mycotoxins found

MycotoxinLOD

(ng/g)

Measured Concentration in animal feed extract diluted 1:10 (ng/g)*

Animal feed sample identity and type

©2015 Waters Corporation 41

Analysis of marine biotoxins using

Xevo TQ-S

©2015 Waters Corporation 42

Marine Biotoxins (phycotoxins)

Certain pytoplankton spp produce toxic

allelopathic secondary metabolites

Under favourable conditions unicellular

algae can proliferate termed “blooms” and

toxins can bioaccumulate in filter-feeding

bivalve molluscs

Ingestion of contaminated seafood is

estimated to cause 20% of all foodbourne

illness in the USA with around 1.5%

mortality rate globally

Over the past 3 decades the frequency and

global distribution of toxic algal incidents

have increased & human intoxications from

novel algal sources have occurred

©2015 Waters Corporation 43

Classification of the toxins

Toxins vary in hydrophilicity and are

classed by their effects:

– Amnesic Shellfish poisoning (ASP)

Domoic acid

– Paralytic Shellfish poisoning (PSP)

Saxitoxins

– Neurotoxic Shellfish poisoning (NSP)

Brevetoxins

Diarrhetic Shellfish poisoning (DSP)

Okadaic acid (OA) group,

dinophysistoxin (DTX)

Yessotoxins (YTX)

Pectenotoxins (PTX)

– Azaspiracid Shellfish poisoning (AZA)

Azaspiracids

Hyd

ro

ph

ilic

Lip

op

hilic

©2015 Waters Corporation 44

Lipophilic Toxins

Structures of a) EU regulated toxins and b) non-regulated cyclic imines

Toxin R1 R2

Okadaic acid CH3 H

Dinophysistoxin-1 CH3 CH3

Dinophysistoxin-2 H CH3

Toxin R1 R2

Azaspiracid-1 H CH3

Azaspiracid-2 CH3 CH3

Azaspiracid-3 H H

Toxin R1

Pectenotoxin-1 OH

Pectenotoxin-2 H

Toxin R1 n

Yessotoxin H 1

Homo Yessotoxin H 2

45OH Yessotoxin OH 1

45OH Homo Yessotoxin OH 2

a) b)

Toxin R1 R2 R3 R4

Pinnatoxin-E H OH CH3

Pinnatoxin-F H OH CH3

Pinnatoxin-G O

H

H H

13-desmethyl spirolide C

Gymnodimine

©2015 Waters Corporation 45

Worldwide Regulations / Procedures Lipophilic Toxins

European Union – Most types of lipophilic marine toxins can be found in shellfish and as a result EU

legislation covers OA, DTXs, PTXs, YTXs and AZAs

USA

– FDA –via the FDA no routine monitoring programs for these toxins have been established yet, legislation exists for OA and DTX1

Canada

– CFIA Regions must have in place a program to adequately monitor marine biotoxins to ensure that shellfish areas are closed when toxin levels reached proscribed levels

Chile – The National Health Service is responsible for detecting toxicity using a bioassay at 40

stations using monthly samples

– The Fisheries Research Institute monitors toxicity in conjunction with universities

– Programmes include measures of phytoplankton to understand more than just toxicity

– PSP & DSP toxins have had the most severe public health and economic impact in Chile

©2015 Waters Corporation 46

EU methods for official control purposes

Pre-2011

Official method of control was mouse or rat bioassay (Yasumoto et al 1978)

ESFA have noted the following shortcomings

– 24 hour observation time

– Insufficient detection capability; high variability & limited specificity

– Sacrifice of a large number of animals is involved

Other assays including LC-F, LC-MS, immunodiagnostic and functional assays

New regulations established in 2011 (15/2011)

Since July 2011, the official method for control of shellfish for the presence of

lipophilic marine biotoxins has been LC-MS/MS

– Fixed extraction procedure

– Separation using LC – either an acidic mobile phase or alkaline mobile phase

– Quantitative detection by tandem quadrupole MS

©2015 Waters Corporation 47

LC-MS/MS Method Development Aims

Produce a faster method using alkaline

conditions

– HPLC = 15 mins

– UPLC = 5 mins

Develop the method for regulated and

some non-regulated cyclic imines

compounds

Optimise method for different matrices

Generate single day lab validation data

©2015 Waters Corporation 48

Sample Extraction

Homogenized whole flesh shellfish tissue (1 g) was

extracted with methanol

Extract was vortex-mixed and centrifuged

Supernatant was transferred to a 10 mL volumetric

flask and made up to 10 mL with methanol

Filter crude shellfish extract prior to spiking / analysis

For DTX3 (ester forms of OA, DTX1 and -2)

– Extracts also subjected to alkaline hydrolysis using 2.5 M sodium

hydroxide

– Heat alkaline mixture for 40 min at 76oC, cool to RT and neutralise

using 2.5 M HCl

©2015 Waters Corporation 49

ACQUITY UPLC Method Alkali Method

Routine analysis » » rapid

analysis

Need good separation of

compounds » » high

resolution chromatography

Time (min)

% A % B

0.00 75 25

4.50 0 100

6.00 0 100

6.10 25 25

8.00 25 25

Mobile Phase A 100% H2O + 2 mM NH4HCO3 (adjusted to pH 11 with NH4OH)

Mobile Phase B

90% MeCN:10% H2O + 2 mM NH4HCO3 (adjusted to pH 11 with NH4OH)

Flow 0.6 mL/min

Column ACQUITY BEH C18 100 x 2.1mm, 1.7μm

Colum temp 30ºC

Inject. volume 2.5 µL

©2015 Waters Corporation 50

MRM Transitions ESI Negative

Compound name Parent

(m/z)

Daughter

(m/z) Ionisation

Dwell

(s) Cone (V)

Collision

(eV)

Standard

available

trinor YTX 550.4 396.4 - 0.003 75 30

No 550.4 467.4 - 0.003 75 30

YTX 570.4 396.4 - 0.003 75 30

Yes 570.4 467.4 - 0.003 75 30

homoYTX 577.4 403.4 - 0.003 75 30

No 577.4 474.4 - 0.003 75 30

45OH YTX 578.4 396.4 - 0.003 75 30

No 578.4 467.4 - 0.003 75 30

45OH Homo YTX 585.4 403.4 - 0.003 75 30

No 585.4 474.4 - 0.003 75 30

COOH YTX 586.4 396.4 - 0.003 75 30

No 586.4 467.4 - 0.003 75 30

COOH OH YTX 593.4 396.4 - 0.003 75 30

No 593.4 403.4 - 0.003 75 30

COOH Homo YTX 593.4 467.4 - 0.003 75 30

No 593.4 474.4 - 0.003 75 30

OA/DTX2 803.5 113.1 - 0.003 80 60

Yes 803.5 255.2 - 0.003 80 45

DTX1 817.5 113.1 - 0.003 80 60

Yes 817.5 255.2 - 0.003 80 45

©2015 Waters Corporation 51

MRM Transitions ESI Positive

Compound name Parent

(m/z)

Daughter

(m/z) Ionisation

Dwell

(s) Cone (V)

Collision

(eV)

Standard

available

GYM 508.2 162.2 + 0.003 60 55

Yes 508.2 490.2 + 0.003 60 40

SPX1 692.5 164.3 + 0.003 60 55

Yes 692.5 444.2 + 0.003 60 40

PnTX-G 694.5 164.3 + 0.003 60 55

Yes 694.5 676.5 + 0.003 60 40

20-Me SPX G 706.5 164.3 + 0.003 60 55

No 706.5 346.2 + 0.003 60 40

PnTX-F 766.5 164.3 + 0.003 60 55

Yes 766.5 748.5 + 0.003 60 40

PnTX-E 784.5 164.3 + 0.003 60 55

Yes 784.5 766.5 + 0.003 60 40

AZA3 828.5 658.4 + 0.003 35 40

Yes 828.5 792.5 + 0.003 35 30

AZA6 842.5 658.4 + 0.003 35 40 Yes

AZA1 842.5 672.4 + 0.003 35 40 Yes

AZA1/6 842.5 824.5 + 0.003 35 30 Yes/No

AZA4 844.5 658.4 + 0.003 35 40 No

AZA5 844.5 674.4 + 0.003 35 40 No

AZA4/5 844.5 826.5 + 0.003 35 30 No

AZA2 856.5 672.4 + 0.003 35 40

Yes 856.5 838.5 + 0.003 35 30

PTX12 874.5 213.1 + 0.003 40 30

No 874.5 821.5 + 0.003 40 30

PTX2 876.5 213.1 + 0.003 40 30

Yes 876.5 823.5 + 0.003 40 30

PTX11 892.5 213.1 + 0.003 40 30

No 892.5 839.5 + 0.003 40 30

PTX2sa 894.5 213.1 + 0.003 40 30

No 894.5 805.2 + 0.003 40 30

©2015 Waters Corporation 52

MRMs of Matrix Matched Standard Mussel extract

©2015 Waters Corporation 53

Single day validation results

Compound

Concentration

(µg/kg)

Recovery

(%)

RSDr

(%)

RSDrl

(%)

CCα

(µg/kg)

OA 160 99 2.7 4.1 171

DTX1 160 99 7.6 12.2 192

DTX2 160 102 2.6 4.1 171

YTX 1000 100 2.5 4.0 1070

AZA1 160 98 1.3 2.1 166

AZA2 160 98 1.9 3.0 168

AZA3 160 99 1.9 3.0 168

PTX2 160 103 8.7 13.9 197

GYM 200 99 3.9 6.3 221

SPX11 100 108 14.6 23.4* 141

SPX12 100 104 12.8 20.4 135

PinE 200 122 23.1 36.9* 347

PinF 200 91 5.1 8.1 224

PinG 50 102 3.9 4.8 54

©2015 Waters Corporation 54

Summary

Complete solution for targeted natural toxin analysis applicable for complex matrices

Immunodiagnostic assays & core detectors

– Point-of-control testing

– Cost-effective

ACQUITY QDa – accessible MS suited for routine screening

– Increased scope & selectivity

– Screening “plus” (in-source CID)

Xevo TQ-S – ultimate sensitivity MS suited for robust confirmation

– Confirmatory technique (MRM)

– Enhanced sensitivity

– Overcome challenges? (matrix interferences; requirement for labelled internal standards; low sample volume)