AbstractThis research proposed and validated an LC-MSMS method for five reduction and hydroxylation metabolites of Mequindox (MEQ) as well as the precursor in holothurian samples. Specially, three hydroxylation metabolites (2-isoethanol-mequindox, M3, 2-isoethanol-1-desoxymequindox, M4 and 2-isoethanol-4-desoxymequindox, M5) are novel for analysis. Target compounds were extracted with methanol and ethyl acetate in turn without any complicated acidolysis, alkaline hydrolysis or enzymolysis steps. Samples were further purified with C18 solid-phase extraction cartridges for LC-MSMS analysis. Mean recoveries in spiked samples ranged from 81 to 107% with intra-day relative standard deviation (RSD) and inter-day RSD <11.2 and 9.9%, respectively. Limit of detection was determined based on signal-to-noise ratio ≥3 ranged from 0.16 to 2.11 μg kg−1 for each target. The validated protocol was successfully applied for commercial holothurian samples with a positive rate at 13.3%. And concentrations of hydroxylation metabolites were higher than reduction metabolites and precursor MEQ in positive samples.