Development and validation of a simple solid-liquid extraction protocol coupled with LC-ESI-MS/MS for the determination of aflatoxin M1 in products of colostrum-based supplements and whey protein-based sports food

Abstract

Special categories, such as infants and athletes, rely heavily on milk-based products. However, aflatoxin M1 (AFM1) contamination is a prevalent risk. This study aims to develop and validate a simple solid-liquid extraction protocol coupled with LC-ESI-MS/MS for AFM1 determination in colostrum-based supplements and whey protein-based sports foods. Extracts of acetonitrile-water (3:2, v/v) were directly injected into LC-ESI-MS/MS in a positive-ion mode. Separation was performed on the Agilent Poroshell 120 EC-C18 column with core-shell properties in a total analysis run time of 7.5 min. Among the tested extraction solvents, acetonitrile-water (3:2, v/v) provided an efficient direct extraction without any further treatments. AFM1 was eluted at a tR of 4.20 ± 0.05 min using the Agilent Poroshell column, and a tolerable ME% was successfully achieved. In both commodities studied, the recovery percentage, repeatability, intermediate precision, LOD, LOQ, linearity range, and linear regression coefficient (R2) results were 94.3–104.1% with RSDs of 4.70–8.40%, 4.49–9.7%, 7.89–10.6%, 0.0015 µg/kg, 0.005 µg/kg, 0.005–1.000 ng/mL, and 0.9999, respectively. Applicability was demonstrated on two proficiency testing (PT) samples and 60 domestic samples. Results have confirmed the practicality of the proposed assay protocol, with concentrations ranging from 0.010 to 4.423 µg/kg for positive samples. Out of the tested samples, 4.8% of the whey protein-based sports foods violated the EU's established limits. This validated assay protocol would help increase sample processing capacity while also ensuring effective and regular oversight by national regulatory authorities.