Automated gas chromatography/tandem mass spectrometry with on-line chemical derivatization for the determination of tebufelone and two metabolites in human plasma.

An automated capillary gas chromatography/tandem mass spectrometry (GC/MS/MS) assay for the simultaneous quantitation of tebufelone (TE) and its two major metabolites (PGE-1802413 and PGE-6285825) in plasma was developed. Using a 1:1 BSTFA:pyridine derivatization cocktail to solubilize plasma extracts, trimethylsilylation (TMS) of labile alcohol and carboxylic acid functional groups occurred instantly upon introducing each sample into a 300 degrees C GC injection port. This on-line chemical derivatization process rendered these three diverse analytes equally amenable to GC analysis and circumvented laborious off-line derivatization procedures. The selectivity of MS/MS conducted on a triple-quadrupole instrument allowed minimal sample preparation and rapid analysis. Electron ionization produced molecular ions (M.+) for TMS-TE, TMS2-PGE-1802413, TMS2-PGE-6285825 and their respective stable-isotope-labeled internal standards, which were selected in Q1 to undergo collisionally activated dissociation in Q2. Quantitation was achieved through monitoring product ions in Q3 at m/z 320, 445 and 305 for respective analytes, relative to corresponding internal standard ions at m/z 323, 449 and 305. A 2.5-1000 ng per sample (approximately 25 pg to 10 ng injected) quantitation range provided access to an effective 2.5-10,000 ppb plasma concentration range (0.1-1 ml samples) for each analyte. Based on quality control data accumulated throughout 8 months of method application, the assay showed no bias and composite (N = 212) relative standard deviations of 5.6%, 7.0% and 9.5% for the respective analytes (with quality control levels typically covering a range of 10-250 ng per analyte). During this period, more than 2000 plasma study samples were analyzed, attesting to the reliability and ruggedness of this approach for routine application.