Determination of Trimetazidine and Clomiphene in Animal-Derived Foods by Mixed-Mode Reversed-Phase Strong Cation Exchange Solid Phase Extraction Cleanup Followed by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry

A method for the determination of trimetazidine and clomiphene in animal-derived foods was developed by mixed-mode reversed-phase strong cation exchange solid phase extraction cleanup followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were extracted with 0.5% (V/V) formic acid in acetonitrile, and purified on a mixed-mode reversed phase strong cation exchange column (PRiME MCX). The chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column by gradient elution using a mobile phase composed of acetonitrile and 0.1% formic acid. The analytes were detected in the positive ion mode using multiple reaction monitoring (MRM). Quantification was performed by the internal standard method. The results showed that good linearity was observed for trimetazidine and clomiphene in the concentration range of 0.3–100.0 ng/mL with determination coefficients (R²) > 0.999. The average recoveries of this method ranged from 93.4% to 112.1%, with relative standard deviations (RSDs) of 0.68%–4.81% (n = 6). The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.1 and 0.3 μg/kg, respectively. The developed method is sensitive, stable, practical, and suitable for the determination of trimetazidine and clomiphene in animal-derived foods, which can provide effective technical support for food safety assurance for sports events.