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Mycotoxins negatively impact animal health. Aflatoxins (AFs) are the most common mycotoxins affecting both large and small animals and are a common cause of toxin-related pet food recalls. Definitive diagnosis of aflatoxicosis is constrained by a lack of validated ante-mortem analytical methods for detection and quantitation of AFs and their metabolites in biological specimens. Herein, we developed and evaluated a urine-based quantitative method for measurement of aflatoxin B1 (AFB1) and its metabolites aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1) in animal urine. (Some of the results have been presented at 59th AAVLD conference, Greensboro, North Carolina, October 13-19th, 2016.) This method uses an immuno-affinity column for clean-up and pre-column derivatization followed by high performance liquid chromatography analysis with fluorescence detection. The method has high selectivity, recovery (>81%) and sensitivity with an instrument limit of detection of 0.20-1.02 pg; instrument limit of quantitation of 0.77-4.46 pg; and a method lower limit of quantitation of 0.30-2.5 ng/mL. The method has high accuracy, repeatability, and is rugged against minor changes. However, because of poor sensitivity of AFQ1 at low concentrations we recommend this method for quantitative determination of AFB1 and AFM1, and for qualitative measurement of AFQ1 in animal urine for diagnosis of aflatoxicosis., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Automated immunoaffinity solid-phase extraction followed by liquid chromatography-tandem mass spectrometry and chemical analogue internal standardization is employed to detect and quantify the aflatoxins AFB(1), AFB(2), AFG(1), AFG(2), and the metabolites AFM(1) and AFP(1) in urine. The dynamic range of the method is nearly three orders of magnitude with limits of detection in the low femtogram on column range. The method was validated over a 12-day period by eight analysts. This method is suitable for agricultural, forensic, and public health laboratories during an accidental outbreak or a chemical terrorism event where a rapid and accurate diagnosis of aflatoxicosis is needed.