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Analysis of Mycotoxins by UPLC and Tandem Quadrupole Mass Spectrometry - Waters Corporation Food Safety

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Mycotoxins are are secondary metabolites produced by fungi and are dangerous for feed and food chains as they can create contamination in pre- and post-harvest processes. Many are highly toxic and as such levels in food products are regulated in Europe, the US, Japan and other countries. This presentation is an overview of the application of ultra-performance liquid chromatography combined with tandem quadrupole mass spectrometry to analyse various food products for mycotoxins in line with regulatory requirements.

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Analysis of Mycotoxins by UPLC and Tandem Quadrupole Mass Spectrometry - Waters Corporation Food Safety

  1. 1. ©2015 Waters Corporation 1 Analysis of Mycotoxins by Tandem Quadrupole Mass Spectrometry
  2. 2. ©2015 Waters Corporation 2 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
  3. 3. ©2015 Waters Corporation 3 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
  4. 4. ©2015 Waters Corporation 4 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
  5. 5. ©2015 Waters Corporation 5 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…
  6. 6. ©2015 Waters Corporation 6 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
  7. 7. ©2015 Waters Corporation 7 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
  8. 8. ©2015 Waters Corporation 8 Semi-quantitative test kits- IAC columns with fluorometer detection
  9. 9. ©2015 Waters Corporation 9 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
  10. 10. ©2015 Waters Corporation 10 Aflatoxins analysis in foodstuffs ACQUITY UPLC H-Class with direct Fluorescence Detection Collateral number: 720003286en
  11. 11. ©2015 Waters Corporation 11 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
  12. 12. ©2015 Waters Corporation 12 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)
  13. 13. ©2015 Waters Corporation 13 Aflatoxin analysis kit – chromatographic separation, AF spiked milk powder Minutes 1.70 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
  14. 14. ©2015 Waters Corporation 14 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)
  15. 15. ©2015 Waters Corporation 15 Rapid MS/MS analysis of patulin in apple juice UPLC with TQD
  16. 16. ©2015 Waters Corporation 16 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
  17. 17. ©2015 Waters Corporation 17 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
  18. 18. ©2015 Waters Corporation 18 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
  19. 19. ©2015 Waters Corporation 19 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
  20. 20. ©2015 Waters Corporation 20 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
  21. 21. ©2015 Waters Corporation 21 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
  22. 22. ©2015 Waters Corporation 22 Chromatographic performance –apple juice Time 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 % 0 100 0.60 0.80 1.00 1.20 1.40 1.60 1.80 % 0 100 1161 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
  23. 23. ©2015 Waters Corporation 23 Linearity – apple juice TargetLynx is used for quantitation, QC checks and ion confirmation Linear range 1 – 1000 μg L-1
  24. 24. ©2015 Waters Corporation 24 Multi-mycotoxin analysis in pistachio, almond and cashew nuts Collaboration with • André de Kok • Peter Rensen • Martien Spanjer UPLC with TQD
  25. 25. ©2015 Waters Corporation 25 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
  26. 26. ©2015 Waters Corporation 26 Automated MRM scheduling functionality MRM Method Development Streamlining the workflow – Quanpedia
  27. 27. ©2015 Waters Corporation 27 Total Ion Chromatogram (TIC) Mycotoxins spiked in almond extract Time 2.00 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 SterigimatocystinVarious dwell times and time windows employed to achieve 12 data points across each peak Nivalenol Cyclopiazonic acid
  28. 28. ©2015 Waters Corporation 28 TargetLynx Browser
  29. 29. ©2015 Waters Corporation 29 Ion Ratio Reproducibility 0 0.2 0.4 0.6 0.8 1 1 3 5 7 9 11 13 15 Injection number Ionratio Fumonisin B1, 1.8% Fumonisin B2, 2.3% Ergotamine, 2.1% Roquefortin, 3.5%
  30. 30. ©2015 Waters Corporation 30 Peak Area Reproducibility 0 500 1000 1500 2000 1 3 5 7 9 11 13 15 Injection number Peakarea Penicillic acid, 4.2% RSD 3-Acetyl-Don, 8.9% RSD Ergotamine, 5.6% RSD
  31. 31. ©2015 Waters Corporation 31 Application of the Xevo TQ-S to improve sensitivity and reduce matrix effects
  32. 32. ©2015 Waters Corporation 32 Xevo TQXevo 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
  33. 33. ©2015 Waters Corporation 33 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
  34. 34. ©2015 Waters Corporation 34 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
  35. 35. ©2015 Waters Corporation 35 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
  36. 36. ©2015 Waters Corporation 36  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
  37. 37. ©2015 Waters Corporation 37 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
  38. 38. ©2015 Waters Corporation 38 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
  39. 39. ©2015 Waters Corporation 39 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

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