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°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 2.5–10000 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. © 1997 John Wiley & Sons, Ltd.