Simultaneous determination of sulfadiazine and trimethoprim in animal feed by liquid chromatography with UV and tandem mass spectrometric detection

Abstract

A method for the simultaneous determination of sulfadiazine and trimethoprim in animal feed was developed and validated. The chromatography was performed both on a liquid chromatography–ultraviolet detection (LC–UV) and LC–tandem mass spectrometric detection (MS/MS) instrument, intended to be applied for two separate purposes. The LC–UV method was used to examine the quality requirements for medicated premixes incorporated into animal feedingstuffs. This comprised e.g. the homogeneity testing in medicated feed at the mg kg −1 level. The LC–MS/MS method was used to quantify residues of sulfadiazine and trimethoprim in unmedicated feed at the μg kg −1 level. Sulfadiazine and trimethoprim were extracted from animal feed with 50% methanol in water and with ethylacetate. Clean-up was performed on a benzenesulfonic acid SCX cartridge. The LC–UV analysis of the extracts was done on a Hypersil ODS column using a gradient of 0.5% acetic acid, 0.25% triethylamine and 5% methanol in water (A) and 5% methanol in acetonitrile (B). Detection was at 240 nm for trimethoprim and 270 nm for sulfadiazine. The same extracts were also injected for quantitation onto an ion-trap LC–MS n instrument, operating in the atmospheric pressure chemical ionization tandem MS mode (APCI–MS/MS). The elution was performed isocratically with 0.1% acetic acid in water and methanol. Both the LC–UV and LC–MS/MS methods were validated for their linearity, trueness, precision, LOD (limit of detection), LOQ (limit of quantification) and extraction recovery. All results fell within the ranges specified. Both methods were useful, but the LODs of the LC–MS/MS method were about 200-times lower than with UV detection, i.e. 0.052 mg kg −1 sulfadiazine and 0.022 mg kg −1 trimethoprim. Therefore, the LC–MS/MS method was applied for monitoring residues as a possible result from carry-over during feed production.