analysis of mycotoxins by uplc and tandem quadrupole mass spectrometry - waters corporation food...

©2015 Waters Corporation 1

Analysis of Mycotoxins by Tandem

Quadrupole Mass Spectrometry


Overview

Mycotoxins and their significance?

– Mycotoxins & phytotoxins

Mycotoxin occurrence data

Analytical strategies for mycotoxins

– Vicam rapid screening methods

– Aflatoxin analysis with FL

– H-Class Xevo TQD

o Analysis of patulin in apple juice

o Multi-toxin method

– Xevo TQ-S

o Analysis of complex matrices (reduction of matrix effects)

Summary


What are mycotoxins & phytotoxins?

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

Phytotoxins are substances produced by plants e.g. alkaloids, terpenes, phenolics, herbicides

“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/

Mycotoxins are dangerous for feed and 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 carcinogen. It is immunotoxic and causes stunted growth in children

and growth retardation in animals

– Fusarium toxins, especially fumonisins are neurotoxic and possible carcinogens, trichothecenes are immunotoxic and zearalenone is estrogenic

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

http://www.mycored.eu/








Foodstuffs effected by mycotoxin & phytotoxin contamination

• Tree nuts

• Peanuts

• Grains

• Wine

• Coffee

• Cereals

• Feed

• Fruits and vegetables

• Fruit juices

• Honey

• Oats

• Ethanol

• Dairy

• Rice

• Botanicals

• Spices

• Snack Foods

• Pet Food


Global occurrence & significance of mycotoxins?

The occurrence of mycotoxins in food and feed typically shows a geographical pattern but can be affected by climatic changes…


Analytical requirements for mycotoxins & volume of testing

HR-MS

LC-

MS/MS

(QqQ)

LC +core detector Fluorometer

ELISA Strip Tests

Research and metabolomic based analysis

Academic, government, commercial

laboratories

Finished food products, biological samples, speciation, conjugated metabolites

High volume testing - field tests, short turnaround times Analysis conducted at receiving

points, transit points, processors sites, commercial labs

Quantitative laboratory based testing,

Commercial & official control laboratories

finished foods & feeds, complex matrices, multi-toxin analysis


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

http://vicam.com/products


Semi-quantitative test kits- IAC columns with fluorometer detection


PBS SAMPLE EXTRACTION

Extract sample with PBS

Centrifuge and remove 35ml PBS supernatant

70% METHANOL EXTRACTION

Add 35ml methanol to make a 70% solution

Extract sample, centrifuge and remove supernatant.

Dilute supernatant and filter through glass

microfibre filter

(Extract B)

AFFINITY CHROMATOGRAPHY STEP 1

Pass 50 mL Extract B over affinity column.

Wash column with PBS

AFFINITY CHROMATOGRAPHY STEP 2

Pass 5 mL Extract A over column.

Wash column with water.

Elute toxins.

FILTRATION

Filter through glass

microfibre filter

(Extract A)

Meets CEN criteria

Can be used with LC detectors

or LC-MS/MS

Myco6in1” Extraction Procedure


Aflatoxins analysis in foodstuffs

ACQUITY UPLC H-Class with direct

Fluorescence Detection

Collateral number: 720003286en


Introduction – 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


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

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

Provides higher sensitivity than HPLC methods

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


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 <EU permitted limits

without need for derivatisation

Improved separation, sensitivity and speed


Aflatoxin analysis kit – performance in

spiked matrices

Spiking levels: 4 μg kg-1 total G and B2, 1.5 μg kg-1 B1 and 0.05 μg kg-

1 M1 (cereal only)


Rapid MS/MS analysis of patulin

in apple juice

UPLC with TQD


Patulin - Introduction

Patulin is produced by Aspergillus and Penicillium fungi species on apples

• Damaged fruit more susceptible to infection

• Suspected genotoxicity

5-hydroxymethylfurfural (HMF) is formed from sugars during thermal

processing and shares UV chromophore with patulin at λ276 nm

Legislative limits in EU, US FDA and Japan for apple juice are 50 µg L-1,

infant apple juice 10 µg L-1 and for solid apple products 25 µg kg-1

Patulin and HMF


Why choose UPLC-MS/MS?

UPLC uses sub 2 μM particles, mobile phases

at higher linear velocities and operating at

higher pressures than HPLC =

Improved resolution for complex mixture

analysis

Enhanced resolution reduces ion suppression

by separating species that co-elute in HPLC

Shorter run times without compromising

chromatographic resolution, increasing sample

throughput

Narrower chromatographic peaks increase

concentration of analytes entering the MS source,

increasing signal intensity and improving LoDs

Speed Resolution

Sensitivity


MS/MS Detection

TQ Detector (TQD) designed to be;

• UPLC compatible (short inter scan &channel delays)

• Small foot print

• Multi-mode (ionisation /acquisition)

• Easier to operate

• IntelliStart tools

• Set up checks

• LC & MS method

• Monitor performance

34cm (~13”) of linear bench space


TQD Schematic

T-wave enabled collision cell allows short ion residence times = ensures sufficient data points across narrow UPLC peaks &allows multiple analytes to be monitored in parallel

MRM mode: MS1 and MS2 are both set to a static value giving enhanced selectivity for the analytes of interest


Experimental conditions

Extraction Protocol

SPE: Waters Oasis HLB 3cc /60mg

Condition step: 3 mL methanol

3 mL water

Load: 2.5 mL sample

Wash 1: 3 mL 1% NaHCO3 (1g/100mL)

Wash 2: 1 mL 0.1% acetic acid

Dry under vacuum

Elute: 2 x 1.5 mL 10% ethyl acetate in

methyl t-butyl ether (MTBE)

Reconstitute: 500 µL water


Instrument parameters

UPLC Conditions

Mobile phase: H2O – MeCN

gradient (pH 10)

Analytical column: BEH shield

RP18 2.1 X 100 mm 1.7 μm

Flow rate: 0.6 mL/min

Run time: 4.5 min

Injection volume: 20 µL (full

loop mode)

Acquisition: Multiple Reaction

Monitoring (MRM)

Collision gas: argon

Software: MassLynx v4.1 for

acquisition and TargetLynx for

data processing

MS/MS Conditions


Chromatographic performance –apple

juice

Time0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

0

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

0

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

0

1001161

541

7528

HMF

Patulin Patulin: 153>109

Patulin: 153>81

HMF: 129>95

16 data points across the peak

Reporting level 50 μg L-1

Rt = 1.1 min

Rt = 1.4 min


Linearity – apple juice

TargetLynx is used for

quantitation, QC checks and

ion confirmation

Linear range 1 – 1000 μg L-1


Multi-mycotoxin analysis in pistachio,

almond and cashew nuts

Collaboration with

• André de Kok

• Peter Rensen

• Martien Spanjer

UPLC with TQD


Aim

To evaluate the suitability of the ACQUITY TQD for the

simultaneous determination of multiple mycotoxins in

foodstuffs at EU legislative limits

Multiple reaction monitoring (MRM)

Mycotoxin extracts and solvent standards provided by the

Food and Consumer Product Safety Authority (VWA),

Amsterdam, Netherlands

• Pistachio

• Almond

• Cashew nut


Automated MRM scheduling functionality

MRM Method Development Streamlining the workflow – Quanpedia


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 and time windows employed to achieve

12 data points across each peak

Nivalenol

Cyclopiazonic acid


TargetLynx Browser


Ion Ratio Reproducibility

0

0.2

0.4

0.6

0.8

1

1 3 5 7 9 11 13 15

Injection number

Ion

rati

o

Fumonisin B1, 1.8% Fumonisin B2, 2.3%

Ergotamine, 2.1% Roquefortin, 3.5%


Peak Area Reproducibility

0

500

1000

1500

2000

1 3 5 7 9 11 13 15

Injection number

Peak a

rea

Penicillic acid, 4.2% RSD

3-Acetyl-Don, 8.9% RSD

Ergotamine, 5.6% RSD


Application of the Xevo TQ-S to

improve sensitivity and reduce matrix

effects


Xevo TQ Xevo TQ-S

Ion source sampling cone

How did we increase sensitivity?

Much larger sampling orifice and

modified vacuum system to deal with increased gas load


TQ-S Stepwave ion optics

Maximising signal

Minimising noise

Two types of T-wave enabled RF ion tunnels; On axis and off axis relative to the MS analyser - narrow ion tunnel conjoined to wide ion tunnel

Entrance to the Stepwave=captures all ions in the disperse ion cloud


Comparison of Xevo TQ-MS and TQ-S Spiked barley AF1 0.1 μg kg-1

Xevo TQ Xevo TQ-S

Quantitative transition

Confirmatory transition

Better peak shape and increased sensitivity (x40) reduces the number of manual integrations

Processed data becomes more consistent giving more accurate ion ratios increasing confidence in compound identification

Response of the secondary ion is stronger

40x sensitivity increase


Reduction in ion suppression Spiked feed extract

Matrix matched standard comparison to S/Std

Ability to inject a smaller amount or dilute the sample helps reduce matrix effects


Rapid electronics allow instrument to

switch between MRM and full scan (FS

takes 100ms)

– No loss of MRM data quality

– Added information can be gained from

full scan

o Monitor matrix

o Assess for possibility of matrix

effects

o Added information for sample prep

method development

o Search for significant non-targeted

compounds

RADAR functionality – Xevo TQ-S Simultaneous full scan & MRM

MRM

Full scan


Animal feeds – the challenge matrix complexity & co-contamination

Feed extract (neat) background BPI RADAR scan

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


Measured concentrations for mycotoxins identified in 12 different samples of animal feedingstuffs (diluted 1:10) using TQ-S

U1 / cattle

feed

U2 / pig

feed

U3 / maize

gluten

U4 / Diva L

Vital pig feed

U5 /Alpha

Maximal pig feed

U6 /

Rye

U7 /

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

Mycotoxin

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

Animal feed sample identity and typeLOD

(ng/g)

Number of mycotoxins found

Collaboration with University of Ghent

Matrix dilution and Labelled standards for quantitation


Summary

Natural toxins pose a significant threat to food security

– Stringent global regulations for contaminant control established

o New EU regulations expected (EFSA opinions 2012)

Occurrence often linked to climatic conditions

– Highest incident of RASFF alerts in EU

Wide variety of toxins & very different chemistries…

Variety of analytical strategies required for adequate control

Different customer testing requirements

– High volume (PoC) testing

– Semi/ quantitative testing (single toxin)

– Highly complex matrices & need for high sensitivity

– Multi-toxin testing

– Masked toxins and metabolites

Portfolio of different solutions from Waters & Vicam

analysis of mycotoxins by uplc and tandem quadrupole mass spectrometry - waters corporation food...

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