Determination of 8α-hydroxymutilin as a Marker Residue for Tiamulin in Swine Tissue by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Tiamulin is a semi-synthetic derivative of the natural antibiotic pleuromutilin and is widely used as a veterinary drug for swine. Herein, we report the development of a sensitive and reliable method for determining 8α-hydroxymutilin as a marker residue for tiamulin in swine tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method consists of sample extraction with acetone, defatting by acetonitrile/hexane partitioning, hydrolysis of the tiamulin metabolites to 8α-hydroxymutilin under alkaline conditions, liquid-liquid extraction with ethyl acetate, cleanup using a primary secondary amine cartridge, and LC-MS/MS analysis. The developed method was validated for 8α-hydroxymutilin in swine muscle, fat, and liver at two levels, namely 0.01 mg/kg and the maximum residue limits established in Japan (i.e., 0.1 mg/kg for swine muscle and fat, and 0.6 mg/kg for liver). The trueness ranged from 82 to 89%, and the relative standard deviations ranged from 1 to 3%. No chromatographic interference was observed near the retention time of 8α-hydroxymutilin, and matrix effects were negligible for all matrices, suggesting that the cleanup protocol was effective. The calibration curve was linear in the 0.005–0.5 μg/mL range, with a coefficient of determination greater than 0.997. The developed method enabled accurate quantification using solvent-based calibration without compensating for matrix effects and losses during sample preparation. The limit of detection of the method was 0.0005 mg/kg for each matrix. The developed method is suitable for regulatory-purpose analysis of 8α-hydroxymutilin as a marker residue for tiamulin as defined by the European Union and several other countries.