Use of isotope-labeled Aflatoxins for LC-MS/MS stable isotope dilution analysis of foods

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods; was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins 132 and G(2), which were synthesized by catalytic deuteration of aflatoxin B, and G(1), purified, and well-characterized by NMR and MS. All four aflatoxins of interest (131, 132, G1, and G(2)) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B-2/aflatoxin B-2 and labeled aflatoxin G(2)/aflatoxin G(2). For labeled aflatoxin B-2/aflatoxin B-1, and labeled aflatoxin B-2/aflatoxin G(1) matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 mu g/kg (aflatoxin 131), 0.09 mu g/kg (aflatoxin 132), 0.38 mu g/kg (aflatoxin G(1)), and 0.32 mu g/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin 131), 3.6% (aflatoxin B-2), 14% (aflatoxin G(1)), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B, and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 mu g/kg.