Development and Validation of a High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method to Determine...

This study aimed to determine promethazine (PMZ) and its metabolites, promethazine sulfoxide (PMZSO) and monodesmethyl-promethazine (Nor1PMZ), in swine muscle, liver, kidney, and fat. A sample preparation method and high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were established and validated. The samples were extracted using 0.1% formic acid-acetonitrile and purified with acetonitrile-saturated n-hexane. After concentration by rotary evaporation, the extract was re-dissolved in a mixture of 0.1% formic acid-water and acetonitrile (80:20, v/v). Analysis was performed using a Waters Symmetry C18 column (100 mm × 2.1 mm i.d., 3.5 μm) with 0.1% formic acid-water and acetonitrile as the mobile phase. The target compounds were determined using positive ion scan and multiple reaction monitoring. PMZ and Nor1PMZ were quantified with deuterated promethazine (PMZ-d6) as the internal standard, while PMZSO was quantified using the external standard method. In spiked muscle, liver, and kidney samples, the limits of detection (LOD) and limits of quantification (LOQ) for PMZ and PMZSO were 0.05 μg/kg and 0.1 μg/kg, respectively, while for Nor1PMZ, these values were 0.1 μg/kg and 0.5 μg/kg, respectively. For spiked fat samples, the LOD and LOQ for all three analytes were found to be 0.05 μg/kg and 0.1 μg/kg, respectively. The sensitivity of this proposed method reaches or exceeds that presented in previous reports. The analytes PMZ and PMZSO exhibited good linearity within the range of 0.1 μg/kg to 50 μg/kg, while Nor1PMZ showed good linearity within the range of 0.5 μg/kg to 50 μg/kg, with correlation coefficients (r) greater than 0.99. The average recoveries of the target analytes in the samples varied from 77% to 111%, with the precision fluctuating between 1.8% and 11%. This study developed, for the first time, an HPLC-MS/MS method for the determination of PMZ, PMZSO, and Nor1PMZ in four swine edible tissues, comprehensively covering the target tissues of monitoring object. The method is applicable for monitoring veterinary drug residues in animal-derived foods, ensuring food safety.