Single-Laboratory Validation of Simultaneous Determination of Aflatoxins in Nutraceuticals following immune-affinity Column Cleanup and Liquid Chromatography Tandem Mass Spectrometry Analysis

BACKGROUND: Aflatoxins B1, B2, G1, and G2 are highly carcinogenic mycotoxins frequently detected in nutraceutical products. Due to their toxicological significance and the complex composition of nutraceutical matrices, sensitive and validated analytical methods are required to ensure regulatory compliance, product safety, and routine surveillance. OBJECTIVE: This study aimed to develop and perform single-laboratory validation of a reliable and sensitive method for the simultaneous determination of aflatoxins B1, B2, G1, and G2 in nutraceutical products, in accordance with AOAC Appendix F. METHOD: Samples were extracted using an organic solvent system and purified through aflatoxin-specific immunoaffinity columns to reduce matrix interference. Quantitative analysis was performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Method validation evaluated linearity, limits of detection (LOD) and quantitation (LOQ), precision (repeatability and intermediate precision), and accuracy across three representative nutraceutical matrices. RESULTS: The method showed linearity for all analytes, with coefficients of determination (R2) between 0.9984 and 0.9989. The LOD and LOQ values ranged from 0.1268 to 0.6170 µg/kg and from 0.2733 to 1.1290 µg/kg, respectively. Precision studies demonstrated acceptable repeatability and intermediate precision, with relative standard deviations ≤20%. Accuracy assessment across beverage and protein-based nutraceutical products yielded recoveries between 88% and 119% at multiple fortification levels, meeting AOAC performance requirements for mycotoxin analysis. CONCLUSIONS: The validated LC-MS/MS method is sensitive, accurate, and precise for the determination of aflatoxins B1, B2, G1, and G2 in complex nutraceutical products. The method is fit for purpose of routine quality control, regulatory testing, and surveillance of aflatoxins in nutraceutical matrices. HIGHLIGHTS: Optimized sample extraction and immunoaffinity cleanup enabled sensitive LC-MS/MS determination of aflatoxins B1, B2, G1, and G2. The method was validated for linearity, LOD, LOQ, sensitivity, precision, and accuracy, and was found to be suitable for routine aflatoxin estimation in nutraceutical products.