Development and validation of an LC–MS method for the simultaneous determination of sulfadiazine, trimethoprim, and N4-acetyl-sulfadiazine in muscle plus skin of cultured fish

Abstract

ABSTRACTAn analytical method for the determination of both sulfadiazine (SDZ) and trimethoprim (TMP), and also N4-acetyl-sulfadiazine (AcSDZ), the main metabolite of SDZ, in fish muscle plus skin has been developed and validated. Dapsone was used as internal standard. The method involves extraction of the analytes from fish tissue by pressurized liquid extraction using water as extractant. Sample cleanup was carried out by solid phase extraction using Abselut Nexus cartridges. Target analytes were quantitatively determined by liquid–chromatography mass spectrometry using single ion monitoring. The developed method was validated according to the European Union requirements (decision 2002/657/EC). The limit of detection for SDZ and AcSDZ was 3.0 and 2.5 µg kg−1 for TMP. The limit of quantification (LOQ) was 10 µg kg−1 for SDZ and AcSDZ and 7.5 µg kg−1 for TMP. The recovery experiments carried out included the concentration levels of 0.5, 1 and 1.5 times the MRLs for SDZ and TMP. Concentration levels for AcSDZ were the same as SDZ. The values obtained were higher than 92.0% with coefficient of variation (CV, %) below 8.6%. The precision of the method, calculated as CV (%), ranged from 0.2 to 6.8% and from 0.8 to 8.9% for intra–day and inter–day analysis, respectively. Decision limit (CCα) was calculated as 104.3, 53.7 and 105.3 µg kg−1 for SDZ, TMP and AcSDZ, respectively. Detection capability (CCβ) was calculated as 110.0, 58.8 and 109.7 µg kg−1 for SDZ, TMP and AcSDZ, respectively. “Matrix effect” and “relative matrix effect” were also evaluated. The method was used for the analysis of fish samples purchased from local markets.