Extensive Evaluation of a Method for Quantitative Measurement of Aflatoxins B1 and M1 in Animal Urine Using High-Performance Liquid Chromatography with Fluorescence Detection

BACKGROUND: Aflatoxins (AFs) are common feed contaminants and are one of the common causes of toxin-related pet food poisoning and recalls. OBJECTIVE: Currently, there are no validated methods for the detection and quantitation of AFs in biological matrices to diagnose AF exposure in live animals. Following a successful intra-laboratory method development to quantify AFB(1) and AFM(1) in animal urine by HPLC with fluorescence detection (HPLC–FLD), the present study was conducted to extensively evaluate the method performance in an unbiased manner using blinded samples. METHODS: The evaluation included two stages. First, the performance was verified in the method-originating laboratory in a single-laboratory blinded method test (BMT-S) trial followed by a multi-laboratory blinded method test (BMT-M) trial. RESULTS: In both trials, accuracy, repeatability, and reproducibility were satisfactory confirming the relatively good ruggedness and robustness of the method and ensuring that it will perform as expected if used by other laboratories in the future. CONCLUSIONS: We extensively evaluated the performance of a quantitative method to detect AFB1 and AFM1 in animal urine by HPLC-FLD by two different laboratories in two separate BMT-S and BMT-M trials. Both BMT results demonstrated the satisfactory accuracy and precision of the method. It is now available to be adopted by other diagnostic laboratories for purposes of diagnosing AF intoxication in animals. HIGHLIGHTS: A simple urine-based diagnostic test method using HPLC–FLD that originated in a single laboratory now has passed a multi-laboratory evaluation and is now available to be shared with other diagnostic laboratories for purposes of diagnosing AF intoxication in animals so better treatment can be rendered.