High-throughput assay for quantification of the plasma concentrations of thiopental using automated solid phase extraction (SPE) directly coupled to LC-MS/MS instrumentation.

Most previous assays for thiopental are time-consuming due to laborious sample extraction steps prior to analysis using gas chromatography or high pressure liquid chromatography. Here, we describe the first high-throughput liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for quantification of thiopental concentrations in samples of human plasma. Robotic on-line solid phase extraction (SPE) was used to elute the analytes of interest from samples of human plasma (50μL) loaded onto C18 SPE cartridges to which were added aliquots (50μL) of internal standard solution (thiopental-d5 100ng/mL) and 0.5% formic acid in water (100μL). Cartridges were washed using 10% methanol in ammonium acetate buffer (50mM, pH 7) before elution with mobile phase comprising 0.1% formic acid in water and acetonitrile with a flow rate of 0.55mL/min using a 7.2min run time. The analytes were separated on a C18 XTerra(®) analytical column. Mass spectrometry detection was performed using a QTrap 5500 mass spectrometer (AB Sciex) with negative ionisation. The multiple reaction monitoring (MRM) transitions for thiopental and the internal standard were 241→58, and 246→58, respectively. The calibration curve was linear over a range of 6-600ng/mL. Thiopental was stable in human plasma samples for at least 36h in the autosampler, as well as after three cycles of freeze and thaw, and after 3h storage at room temperature. The absolute recovery and matrix effect were 102% and 6.9%, respectively, and the within-run and between-run precision and accuracy were ≤15%. Our method is fully-validated and satisfies the requirements of the 2012 European Medicines Agency (EMEA) guideline for Bioanalytical Method Validation.