Presentation: Mycotoxin Analysis Using SPE

© 2013 Sigma-Aldrich Co. All rights reserved.sigma-aldrich.com/analyticalMycotoxin Analysis Using SPEMichael Ye, Ken Espenschied, Emily Barrey,Matilal Sarker, Olga Shimelis, and Jennifer ClausSupelco, Div. of Sigma-AldrichBellefonte, PA 16823 USAApril 8, 2013T413127© 2013 Sigma-Aldrich Co. All rights reserved.2Agenda• What are mycotoxins?• Supel™ Tox SPE• Aflatoxin• DON and B-Tricothecenes• Future of mycotoxin analysis© 2013 Sigma-Aldrich Co. All rights reserved.3Mycotoxins• Toxic secondary metabolites produced by fungi• Exist in food as a result of fungal infection of crops• Resistant to temperature, such as cooking and freezing• Remain in the food chain in meat and dairy products• Health Risks:–Cancer–Weakened Immune System–Allergens or Irritants–Death© 2013 Sigma-Aldrich Co. All rights reserved.4Classes of Mycotoxins•Aflatoxins•Fusarium –Zearalenone (ZEA or ZON): – Trichothecenes– Deoxynivalenol (DON)– Fumonisins•Ochratoxin•Patulin© 2013 Sigma-Aldrich Co. All rights reserved.5Regulations• Over 100 countries have regulations regarding mycotoxins in the feed industry• Increasing awareness changing views on levels• Export/Import industries impacted• EU and FDA main bodies• Many based upon current available analytical methods© 2013 Sigma-Aldrich Co. All rights reserved.6*Source: Trilogy Labs, 2011© 2013 Sigma-Aldrich Co. All rights reserved.7Methods•Mycotoxin Analysis• HPLC•Most widely and frequently used•Quantitative•Requires extract cleanup•GC• Trichothecenes such as Type A•Requires extract cleanup• ELISA•No cleanup. Dilute and shoot•Matrix constituents may cause false positives• Test Strips(Lateral Flow)•No cleanup •Some quantitative•Matrix constituents may cause false positives•Sample Prep• Immunoaffinity Columns•Current industry standard•Bind and elute procedure: 3 steps •Specific columns for eachmycotoxin•Often require refrigeration so as not to denature protein•SPE• Interference removal: 1 step or Bind and elute procedure: 3 steps •Specific cartridges for eachmycotoxin•No refrigeration required© 2013 Sigma-Aldrich Co. All rights reserved.8Supel Tox SPE•Two Supel Tox SPE cartridges were tested in corn, wheat, and/or raw peanut paste• Multi-layer SPE cartridge for cleanup of aflatoxins andzearalenone (ZON) (Supel Tox AflaZea)• Carbon-based SPE for cleanup of Trichothecenes, type B, including DON (Supel Tox DON)© 2013 Sigma-Aldrich Co. All rights reserved.9AflatoxinsOOOO OOCH3Aflatoxin B1OOOO OOCH3Aflatoxin B2OOOOOOOCH3Aflatoxin G1OOOOOOOCH3Aflatoxin G2© 2013 Sigma-Aldrich Co. All rights reserved.10Aflatoxins•Aflatoxins are commonly present in:•Corn and corn byproducts•Tree nuts•Peanuts and byproducts•Spices/fruits© 2013 Sigma-Aldrich Co. All rights reserved.11Aflatoxins•Aflatoxin levels of concern:•FDA action levels – 20 ppb in finished products for human consumption– 200 ppb in swine feeds– 300 ppb feeder cattle •EU legislation– 10 ppb in maize to be used as ingredient in food stuffs– 15 ppb in groundnuts before human consumption– 10 ppb in nuts and dried fruit before human consumption to be used an ingredient in food stuffs– 4 ppb in cereals intended for direct human consumption– 0.1 ppb of B1 in baby foods and processed cereal-based foods for infants and young children© 2013 Sigma-Aldrich Co. All rights reserved.12AflatoxinsStage 1 (15 minutes) Purify and Transfer (6 minutes)1. Configure manifold for waste collection 1. Configure manifold for sample collection2. Add 1 mL sample to 17 mL phosphate buffered saline, vortex 2. Mount Cartridges3. Uncap/Mount/Drain Cartridges by gravity 3. Load 2 mL sample4. Apply Reservoirs, Load Sample onto Cartridges 4. Elute and Collect Under VacuumStage 2 (15 minutes) 5. Transfer 0.2 mL Sample to Vial5. Rinse Interferences 6. Dilute Sample and Vortex6. Reconfigure Manifold for Sample Collection Analysis7. Elute/Collect SampleStage 3 (30 minutes)8. Evaporate Sample to Dryness 9. Reconstitute Sample and Vortex10. Transfer 0.2 mL Sample to Vial11. Dilute Sample and VortexAnalysisProcedure (post-extraction to analysis)© 2013 Sigma-Aldrich Co. All rights reserved.13AflatoxinsAnalysis of Aflatoxin(n = 3) Immunoaffinity Supel™ Tox Mycotoxin SPE Cartridge 60 minutes 6 minutes8 samples/day (if processing 1 at a time) 80 samples/day (if processing 1 at a time)• Large volumes of liquid •Small volumes of liquid•Controlled drop rates •Vacuum filtration used•Numerous complicated steps •Steps few and not complicated•Additional buffer salts required •No additional reagents required • Must be refrigerated, brought to room temp before use•Column does not require special storage conditions Sample Prep Time (post-extraction to pre-analysis)Ease of UseIncrease Throughput: Supel Tox can analyze10 times more samples/day than immunoaffinity© 2013 Sigma-Aldrich Co. All rights reserved.14AflatoxinsHPLC Conditionscolumn: Discovery® C18, 15 cm x 2.1 mm, 5 µm particle size (50495521)mobile phase: (A) water; (B) acetonitrile; (C) methanol; (74:13:13, A:B:C) with 0.780 g potassium bromide and 230 µL nitric acidflow rate: 0.40 mL/mincolumn temp.: 35 ºCdetector: FLD, ex 360, em 440nm FL, KOBRA cellinjection: 40 µL0 2 4 6 8 10 12 14 16 18Supeltox M Corn Matrix1020304050LU 12341. Aflatoxin G22. Aflatoxin B23. Aflatoxin G14. Aflatoxin B1Analysis of Aflatoxin in Corn MealUsing Supel Tox SPE Cartridge0 2 4 6 8 10 12 14 16 18IAC Peanut Paste2040LU 1234Analysis of Aflatoxin in Peanut Paste Using Immunoaffinity Column1. Aflatoxin G22. Aflatoxin B23. Aflatoxin G14. Aflatoxin B1© 2013 Sigma-Aldrich Co. All rights reserved.15AflatoxinsSupel™ Tox Mycotoxin G2 G1 B2 B1RSD 0.90% 1.90% 1.30% 0.90%Rcvry 107.60% 103.40% 101.10% 102.00% Immunoaffinity RSD 7.50% 14.70% 6.10% 7.60%Rcvry 92.40% 78.10% 101.10% 84.40%• Better analyte recovery than IAC• Lower %RSD than IAC• More robust method with less variability© 2013 Sigma-Aldrich Co. All rights reserved.16Aflatoxins Using Ascentis® Express0 2 4 6 8 10 12 14Time (min)20406080LU 12340 2 4 6 8 10 12 1420406080LU 12340 2 4 6 8 10 12 1420406080LU 12340 2 4 6 8 10 12 1420406080LU 1234AFLATOXIN B1&B2 (4 PPB), G1&G2( 16 PPB) STANDARDSPIKED CORN MATRIX, B1&B2 (4 PPB), G1&G2( 16 PPB) SPIKED WHEAT MATRIX, B1&B2 (4 PPB), G1&G2( 16 PPB) SPIKED PEANUT PASTE, B1&B2 (4 PPB), G1&G2( 16 PPB) 1. AFLATOXIN G22. AFLATOXIN G13. AFLATOXIN B24. AFLATOXIN B1Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 µm© 2013 Sigma-Aldrich Co. All rights reserved.17AflatoxinsAnalyte Recovery and %RSD of Aflatoxins fromWheat, Corn, and Peanut Paste (n = 3) • RECOVERY >80%• %RSD < 5%© 2013 Sigma-Aldrich Co. All rights reserved.18Deoxynivalenol (DON) and Trichothecenes, Type BOOOOH CH3OHCH3OHH HDeoxynivalenolOOOCH3OHOHOHCH3OHH HNivalenol15-Acetyldeoxynivalenol 3-Acetyldeoxynivalenol CH3OOOOOH CH3OHCH3OH HOCH3OOOCH3OHOCH3OHH H© 2013 Sigma-Aldrich Co. All rights reserved.19DON and B-TrichothecenesDON is commonly present in:• Wheat and wheat products• Corn and corn products• Barley and malted barley• Sorghum• Oats© 2013 Sigma-Aldrich Co. All rights reserved.20DON’s levels of concern:FDA advisory levels -1 ppm in finished products for human consumption1 ppm in swine feeds5 ppm in beef cattle and poultry feedsEU legislation200 ppb in processed cereal-based baby and infant food1250 ppb in unprocessed cereals (excluding durum wheat, oats and corn)1750 ppb in durum wheat, oats, corn750 ppb in cereals intended for direct human consumption, cereal flower500 ppb in bread, pastries, breakfast cerealDON and B-Trichothecenes© 2013 Sigma-Aldrich Co. All rights reserved.21DON and B-TrichothecenesExtract 25 g ground sample with 100 mL 84:16acetonitrile:water for 3 min and filter.Spiked at 2ppm with mix of B-trichothecenes.Load 2 mL of sample onto Supel Tox DON cartridgeElute with two 5 mL aliquots of extraction solventEvaporate to dryness and reconstituteProceed with LC-UV analysis© 2013 Sigma-Aldrich Co. All rights reserved.22DON0 2 4 6Time (min)020mAUDON0 2 4 6Time (min)020mAUDON0 2 4 6Time (min)020mAUDONDEOXYNIVALENOL STANDARD, 2 PPM NEAT SAMPLE, 2 PPMCORN MATRIX BLANKHPLC Conditions:column: Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 µmmobile phase: (A) water; (B) acetonitrile; (C) methanol; (92:4:4, A:B:C) flow rate: 0.400 mL/mincolumn temp.: 35 ºCdetector: UV 220 nm; 360 nminjection: 40 µL0 2 4 6Time (min)020mAUDONSPIKED CORN MATRIX, 2 PPM© 2013 Sigma-Aldrich Co. All rights reserved.23DON0 2 4 6Time (min)020mAUWHEAT MATRIX BLANK0 2 4 6Time (min)020mAU DONSPIKED WHEAT MATRIX, 2 PPM0 2 4 6Time (min) 020mAUPEANUT PASTE MATRIX BLANK0 2 4 6Time (min) 020mAU DONSPIKED PEANUT MATRIX, 2 PPM© 2013 Sigma-Aldrich Co. All rights reserved.24DONNeat Corn Wheat PeanutRecovery 79.00% 90.80% 81.30% 89.40%RSD 2.60% 2.50% 3.90% 1.80%Analyte Recovery and %RSD of DON from Wheat, Corn, and Peanut Paste (n = 3)• RECOVERY >70%• %RSD < 5%© 2013 Sigma-Aldrich Co. All rights reserved.25DON and B-TrichothecenesB-Trichothecenes 2 ppm Standard (in solvent)0 2 4 6 8 10 12Time (min)12341. Nivalenol2. DON3. 15-AcetylDON4. 3-AcetylDONcolumn: Ascentis Express C18, 10 cm x 3.0 mm, 2.7 µmmobile phase: (A) 92:4:4, water:acetonitrile:methanol; (B) acetonitrileflow rate: 0.8 mL/mintemp.: 35 ºCdet.: UV 220 nm, 16; ref 360 nm, 100injection: 40 µLgradient: Min %A %B0.0 100 05.0 100 08.0 92 810 92 8© 2013 Sigma-Aldrich Co. All rights reserved.26Conclusion• Supel Tox AflaZea sample cleanup significantly decreased sample prep time and increased reproducibility compared to traditional immunoaffinity cleanup.• Supel Tox DON provided a quick and simple method that produced sufficient and reproducible analyte recoveries for a variety of matrices.© 2013 Sigma-Aldrich Co. All rights reserved.27Future of Mycotoxins• Increasing awareness is prompting more stringent regulations• Variety of matrices and lowering detection limits pose challenges for universal methods• Supel Tox SPE addresses some of current market needs• Goal of one analytical method for all mycotoxins© 2013 Sigma-Aldrich Co. All rights reserved.28Acknowledgements• Ken Espenschied• Michael Ye• Mati Sarker• Jennifer Claus• Olga Shimelis• Marketing and R&D Staff© 2013 Sigma-Aldrich Co. All rights reserved.29