Aflatoxins are a group of highly carcinogenic mycotoxins that contaminate a wide variety of agricultural crops and have a detrimental economic impact on industries, such as corn and ethanol production. They are regulated by the FDA, and therefore, rapid, reliable cleanup techniques with low detection limits are needed for aflatoxins in a wide array of matrices. In this study the effect of using an immunoaffinity column versus simple filtering as a cleanup was tested for afltoxins extracted from corn and Dried Distillers Grains (DDG). The aflatoxins were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The use of an immunoaffinity column resulted in greater signal-to-noise ratios (S/N), S/N of 70 vs. S/N of 5 for corn, as well as fewer non-target peaks in the analysis. Recoveries of aflatoxin using immunoaffinity ranged from 40% to 104.5% (spiked substrate) and 49% to 120% (spiked extract) while percent recoveries of filtered samples ranged from 84% to 119% (spiked substrate) and 88% to 119% (spiked extract). This comparison study showed that filtering is acceptable for small sample sets or where rapid throughput is needed. However, for larger sample sets a more stringent cleanup method is necessary to ensure instrument performance.
KeywordsAflatoxinLC-MS/MSImmunoaffinitySPE
M. A. Klich, “Aspergillus flavus: The Major Producer of Aflatoxin,” Molecular Plant Pathology, Vol. 8, No. 6, 2007, pp. 713-722. doi:10.1111/j.1364-3703.2007.00436.x
K. K. Maggon, L. Viswanathan and T. A. Venki-tasubra- manian, “The Chemistry of Aflatoxins,” The Journal of Scientific and Industrial Research, Vol. 29, 1970, pp. 8-17.
K. A. Scheidegger and G. A. Payne, “Unlocking the Secrets behind Secondary Metabolism: A Review of Aspergillus flavus from Pathogenicity to Functional Genomics,” Journal of Toxicology, Vol. 22, No. 2-3, 2003, pp. 423-459.
F. Wu, “Mycotoxin Risk Assessment for the Purpose of Setting International Regulatory Standards,” Environmental Science and Technology, Vol. 38, No. 15, 2004, pp. 4049-4055. doi:10.1021/es035353n
L. Lewis, M. Onsongo, H. Njapau, H. Schurz-Rogers, G. Luber, S. Kieszak, J. Nyamongo, L. Backer, A. M. Dahiye, A. Misore, K. DeCock and C. Rubin, “Kenya Afla- toxicosis Investigation Group. Aflatoxin Contamination of Commercial Maize Products during an Outbreak of Acute Aflatoxicosis in Eastern and Central Kenya,” Environmental Health Perspectives, Vol. 113, 2005, pp. 1763-1767. doi:10.1289/ehp.7998
Z. Liu, Z. Gao and J. Yu, “Aflatoxins in Stored Maize and Rice Grains in Liaoning Province, China,” Journal of Stored Products Research, Vol. 42, No. 4, 2006, pp. 468- 479. doi:10.1016/j.jspr.2005.09.003
F. Peers, X. Bosch, J. Kaidor, A. Linsell and M. Plujimen, “Aflatoxin Exposure, Hepatitis B Virus Infection and Liver Cancer in Swaziland,” International Journal of Cancer, Vol. 39, No. 5, 1987, pp. 545-553. doi:10.1002/ijc.2910390502
G. Wood, “Mycotoxins in Foods and Feeds in the United States,” Journal of Animal Science, Vol. 70, No. 12, 1992, pp. 3941-3949.
M. A. Klich, “Environmental and Developmental Factors Influencing Aflatoxin Production for Aspergillus flavus and Aspergillus parasiticus,” Mycoscience, Vol. 48, No. 2, 2007, pp. 71-80. doi:10.1007/s10267-006-0336-2
J. L. Firkins, L. L. Berger and G. C. Fahey, “Evaluation of Wet and Dry Distillers Grains and Wet and Dry Corn Gluten Feeds for Ruminants,” Journal of Animal Science, Vol. 60, No. 3, 1985, pp. 847-860.
E. B. Lillehoj, A. Lagoda and W. F. Maisch, “The Fate of Aflatoxins in Naturally Contaminated Corn during the Ethanol Fermentation,” Canadian Journal of Microbiology, Vol. 25, No. 8, 1979, pp. 911-914. doi:10.1139/m79-135
K. A. Nelson, P. P. Motavalli and R. L. Smoot, “Utility of Dried Distillers Grains as a Fertilizer Source for Corn,” Journal of Agricultural Science, Vol. 1, No. 1, 2009, pp. 3-12.
P. M. Scott, J. W. Lawrence and W. Van Walbeek, “Detection of Mycotoxins by Thin Layer Chromatography: Application to Screening of Fungal Extracts,” Applied Microbiology, Vol. 20, 1970, pp. 839-842.
N. A. Lee, S. W. Wang, R. D. Allan and I. R. Kennedy, “A Rapid Aflatoxin B1 ELISA: Development and Validation with Reduced Matrix Effects for Peanuts, Corn, Pistachio, and Soybeans,” Journal of Agriculture and Food Chemistry, Vol. 52, No. 10, 2004, pp. 2746-2755. doi:10.1021/jf0354038
J. Jaimex, C. A. Fente, B. I. Vazquez, A. Cepeda, G. Mahuzler and P. Prognon, “Application of the Assay of Aflatoxins by Liquid Chromatography with Fluorescence Detection in Food Analysis,” Journal of Chromatography A, Vol. 882, No. 1-2, 2000, pp. 1-10. doi:10.1016/S0021-9673(00)00212-0
M. Vahl and M. Jorgenson, “Determination of Aflatoxins in Food using LC/MS/MS,” European Food Research and Technology, Vol. 206, No. 4, 1998, pp. 243-245.
J. Stroka, E. Anklam, U. J?rissen and J. Gilbert, “Immunoaffinity Column Cleanup with Liquid Chromatography Using Post-Column Bromination for Determination of Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste, and Paprika Powder: Collaborative Study,” Journal of AOAC International, Vol. 83, No. 2, 2000, pp. 320-340.
F. Galvano, V. Galofaro and G. Galvano, “Occurrence and Stability of Aflatoxin M1 in Milk and Milk Products: A Worldwide Review,” Journal of Food Protection, Vol. 59, No. 10, 1996, pp. 1079-1090.
Code of Federal Regulations Title 40, Part 136, Appendix B, Guidelines Establishing Test Procedures for the Ana- lysis of Pollutants, U.S. Government Printing Office, Washington, DC, 2009. www.epa.gov/earth1r6/6pd/qa/qadevtools/mod4references/analytical_references/40cfr136_03.pdf.
T. M. Annesley, “Ion Suppression in Mass Spectrometry,” Clinical Chemistry, Vol. 49, No. 7, 2003, pp. 1041- 1044. doi:10.1373/49.7.1041
K. Biemann, “Contributions of Mass Spectrometry to Peptide and Protein Structure,” Biological Mass Spectrometry, Vol. 16, No. 1-12, 1988, pp. 99-111. doi:10.1002/bms.1200160119
R. Aebersold and M. Mann, “Mass Spectrometry Based Proteomics,” Nature, Vol. 422, 2003, pp. 198-207. doi:10.1038/nature01511
F. Hernandez, J. V. Sancho and O. J. Pozo, “Critical Review of the Application of Liquid Chromatography to the Determination of Pesticide Residues in Biological Samples,” Analytical Bioanalytical Chemistry, Vol. 382, No. 4, 2005, pp. 934-946. doi:10.1007/s00216-005-3185-5
H. H. Maurer, “Liquid Chromatography-Mass Spectro- metry in Forensic and Clinical Toxicology,” Journal of Chromatography B: Biomedical Sciences and Applications, Vol. 713, No. 1, 1998, pp. 3-25. doi:10.1016/S0378-4347(97)00514-8
H. S. Chun, H. J. Kim, H. E. Ok, J. Hwang and D. Chung, “Determination of Aflatoxin Levels in Nuts and Their Products Consumed in South Korea,” Food Chemistry, Vol. 102, No. 1, 2007, pp. 385-391. doi:10.1016/j.foodchem.2006.05.031
Z. Zheng, C. W. Humphrey, R. S. King and J. L. Richard, “Validation of an ELISA Test Kit for the Detection of Total Aflatoxins in Grain and Grain Products by Comparison with HPLC,” Mycopathologia, Vol. 159, No. 2, 2005, pp. 255-263. doi:10.1007/s11046-004-8666-0
H. Z. Senyuva and J. Gilbert, “Immunoaffinity Column Clean-Up Techniques in Food Analysis: A Review,” Journal of Chromatography B, Vol. 878, No. 2, 2010, pp. 115- 132. doi:10.1016/j.jchromb.2009.05.042
S. Ip and C. Che, “Determination of Aflatoxins in Chinese Medicinal Herbs by High-Performance Liquid Chro- matography Using Immunoaffinity Column Cleanup: Im- provement of Recovery,” Journal of Chromatography A, Vol. 1135, No. 2, 2006, pp. 241-244. doi:10.1016/j.chroma.2006.10.025
V. A. Vega, “Rapid Extraction of Aflatoxins from Creamy and Crunchy Peanut Butter,” Journal of AOAC International, Vol. 88, 2005, pp. 1383-1386.
M. Ofitserova, “Analysis of Aflatoxins in Ground Peanuts and Peanut Butter by HPLC with Photochemical Derivatization,” LC/GC North America Supplement, Vol. 46, 2010.
M. Muscarella, M. Iammarino, D. Nardiello, S.L. Magro and C. Palermo, “Validation of a Confirmatory Analytical Method for the Determination of Aflatoxins B1, B2, G1, and G2 in Foods and Feed Materials by HPLC with On-Line Photochemical Derivatization and Fluorescence Detection,” Food Addicitves & Contaminants: Part A, Vol. 26, No. 10, 2009, pp. 1402-1410. doi:10.1080/02652030903107906
M. Rudranhatia, “A Rapid LC/MS/MS Method for the Analysis of Aflatoxins in Complex Matrixes with Immu- noaffinity Clean-Up,” Application Note 00927, Varian, Inc. Palo Alto.