QUANTITATIVE SUBFEMTOMOLE ANALYSIS OF ALPHA-TOCOPHEROL AN DEUTERATED ISOTOPOMERS IN PLASMA USING TABLETOP GC MS/MS/

A rapid, high-selectivity method with subfemtomole sensitivity is repo rted for quantification of alpha-tocopherol in plasma-based gas chroma tography/tandem mass spectrometry (GC/MS/MS) using a tabletop quadrupo le ion trap mass spectrometer. Sample workup is rapid, consisting of p rotein precipitation followed by liquid/liquid extraction and O-trimet hylsilyl derivatization of alpha-tocopherol (alpha-T-TMS) and an inter nal standard, 2,2,5,7,8-pentamethyl-6-chromanol (PC-TMS). Rudimentary chromatography was carried out using an 8-m DB-5 capillary column resu lting in an analyte retention time of 7.2 min. No interferences from t he plasma matrix were observed. The assay has a detection limit of 178 amol (89.6 fg) and a lower limit of quantification of 700 amol (350 f g)of derivatized alpha-tocopherol in diluted plasma; <30 pL of plasma is estimated to yield sufficient alpha-tocopherol for quantitative ana lysis at typical concentrations found in humans. A calibration curve c onstructed from National Institute of Standards and Technology serum s tandards was linear in the working range of 1.9-1073 ng/mL (0.95-0.54 ng). Within- and between-day precision averaged 5.8% and did not excee d 11.3% for three concentrations of quality control (QC) solutions. Th e overall accuracy for the QC samples was within 7.2%. Storage studies showed that (alpha-T-TMS and PC-TMS are stable under conditions that might be encountered during analyses. In a test study, plasma kinetic curves for alpha-tocopherol-d(6) and alpha-tocopherol-d(3) were obtain ed for a catheterized pregnant ewe and her fetus who were simultaneous ly given a bolus injection of alpha-tocopherol-d(6), to the ewe and al pha-tocopherol-d(3) to the fetus. These data show that a tabletop GC i on trap can determine alpha-TT-TMS and its isotopomers quantitatively at high selectivity in a complex matrix.