Optimization and validation of a targeted high-throughput UHPLC-MS/MS method for the analysis of multiple mycotoxins...

Mycotoxins, produced by fungi, contaminate animal feed and subsequently enter food products like eggs, posing significant health risks. This study aimed to optimize and validate a sensitive, cost-efficient, high-throughput UHPLC-MS/MS method for the qualitative analysis of 38 mycotoxins, and the quantification of 30, 29 and 29 regulated and emerging mycotoxins in chicken serum, egg yolk and egg white, respectively. Sample preparation involved liquid extraction with 0.1% formic acid in acetonitrile, followed by protein and phospholipid removal using Oasis^® Ostro™. This high-throughput method processed 96 samples within 4 h. Chromatographic separation was performed on an Acquity Premier BEH C18 column using 0.1% acetic acid in both water and methanol as mobile phases, with gradient elution. The MS/MS instrument employed electrospray ionization polarity switching and operated in multiple reaction monitoring mode. To enhance performance, ¹³C-labeled internal standards were utilized. In-house method validation followed European guidelines, with procedural calibration curves constructed over a range limit quantification (LOQ) up to 200 ng/ml for serum and 20 µg/kg for egg yolk/white, demonstrating good linearity (r ≥ 0.99). LOQ values ranged between 0.05 and 1.0 ng/ml or µg/kg for serum and egg white, respectively, and 0.05-2.5 µg/kg for egg yolk. Results for within-run and between-run accuracy and precision fell within predefined ranges. The method's applicability was evaluated through the analysis of real serum and egg samples collected from 13 to 21 poultry farms in Ethiopia, respectively. Several mycotoxins were detected and quantified in all matrices, demonstrating the method's value for in-vivo monitoring of mycotoxin exposure and food safety risk assessment.