Determination and quantification of sulfadiazine and trimethoprim in swine tissues using liquid chromatography with ultraviolet and mass spectrometric detection

Abstract

High-performance liquid chromatographic procedures with ultraviolet detection were developed for the quantitative determination of sulfadiazine (SDA) and trimethoprim (TMP) in swine tissues (kidney, liver, muscle, fat and fat+skin). In addition, high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry was used for the confirmation of the identity of the analytes of interest. Chromatographic separation was achieved on a Spherisorb ODS-2 column (250 × 4.6 mm id, dp 5 μm). The mobile phase for SDA analysis consisted of 1% acetic acid in water–acetonitrile (85 + 15, v/v). For TMP analysis a 80 + 15 + 5 (v/v/v) mixture of 0.25% triethylammonium acetate in water, acetonitrile and methanol was used as the eluent. Sulfamerazine and ormethoprim were used as the internal standards for SDA and TMP analysis, respectively. For the isolation of the compounds of interest from biological samples, a liquid–liquid extraction with acetone and ethyl acetate, followed by a clean-up using a solid-phase extraction column (aminopropyl and benzenesulfonic acid for SDA, benzenesulfonic acid for TMP) was performed. Calibration graphs were prepared for all tissues and linearity was achieved over the concentration ranges tested (50–1000 ng g ⁻¹ for SDA, r ≥0.9979; 25–500 ng g ⁻¹ for TMP, r ≥0.9994). The method was validated at the maximum residue level (MRL, 100 ng g ⁻¹ for SDA and 50 ng g⁻¹ for TMP), at half the MRL and at double the MRL for both SDA and TMP. The accuracy and precision (expressed as the within-day repeatability) were found to be within the required ranges for each specific concentration. The quantification limits were 50 ng g ⁻¹ for SDA and 25 ng g ⁻¹ for TMP. The limits of detection were below one half the MRLs. Both methods were selective for the determination of SDA and TMP. Biological samples (kidney, liver, muscle, fat and fat + skin) from pigs that received a commercial SDA–TMP preparation with the feed for five consecutive days (dose rate: 25 mg SDA and 5 mg TMP kg body weight⁻¹ day⁻¹) were analyzed using the described methods. The quantitative results were used to calculate a withdrawal time (12 days) to reach residue levels below the respective MRLs. This calculation was performed according to the recommendations of the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/036/95).