Quantification of Serum Sulfamethoxazole and Trimethoprim by Ultra-Fast SPE-MS/MS.

BACKGROUND The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) is used to treat a number of bacterial infections. TMP/SMX concentrations in serum are conventionally monitored using high-performance liquid chromatography (HPLC) or liquid chromatography tandem mass spectrometry (LC-MS/MS). These methods require laborious manual extraction techniques and relatively long sample analysis times, necessitating the development of a simple, high-throughput method. A simple, high-throughput method to measure TMP/SMX using ultra-fast solid-phase extraction-tandem mass spectrometry (SPE-MS/MS) has been developed. METHODS Calibration standards, quality control materials, and patient samples were precipitated with acetonitrile containing isotopically labeled internal standards. Samples were vortexed, centrifuged for 5 minutes at 2053×g, and the resulting supernatant diluted in aqueous mobile phase and injected onto the C18 SPE cartridge. MS/MS analysis was carried out by electrospray ionization in positive ion mode at a rate of <20 seconds per sample. A five-point linear 1/x calibration curve was used to calculate sample concentrations. RESULTS The intra-assay precision coefficients of variation (PCVs) were <6% and <7% for SMX and TMP, respectively, and <10% for both inter-assay PCVs. Comparison studies using 50 patient and spiked serum samples showed r values of 0.9890 and 0.9853 and y-intercept values of -1.918 and -1.357, respectively compared to the HPLC reference method. All data points were <+/-15% of the mean. Linearity (r = 0.9952 (SMX) and 0.9954 (TMP)) was established from 12 to 400 μg/mL with a detection limit of 0.47 μg/mL, and 1.2 to 40 μg/mL with a detection limit of 0.06 μg/mL, for SMX and TMP, respectively. For either drug, no significant carryover was observed after samples at the upper limit of quantification. No interference was observed from any of the 77 drugs and respective metabolites tested. CONCLUSION A high-throughput SPE-MS/MS method for TMP/SMX quantification was developed. The <20 second analysis time is a significant improvement compared to traditional HPLC and LC-MS/MS methods, without sacrificing analytical performance.