An automated gas chromatography/tandem mass spectrometry (GC/MS/MS) based method for the rapid determination of tebufelone (TE) in animal and human plasma has been routinely applied in our laboratory to more than 3000 samples over a 2-year period. The selectivity of MS/MS conducted on a triple quadrupole instrument, combined with the use of a stable-isotope-labeled internal standard, results in excellent analytical figures of merit, as well as minimal sample preparation, rapid analysis, and high assay reliability. The work described here goes beyond initial method development and validation studies by evaluating the long-term performance of quantitative GC/MS/MS. Electron ionization produces M.+ ions for TE and the [13C, 18O]TE internal standard, which are selected in Q1 and undergo collisionally activated dissociation in Q2. Quantitation is based on monitoring daughter ions at m/z 248 and 251, respectively, in Q3. A linear range of 1-3000 ng of TE/sample (20 pg to 60 ng injected) provides access to an effective concentration range of 0.5-30,000 ppb TE in plasma (0.1-2-g samples). The assay shows no bias and less than 10% relative standard deviation over this range. In the automated mode, less than 7 min elapse from injection to report printout and more than 70 plasma samples are routinely prepared and analyzed in a day. Such performance is consistently maintained throughout long-term application.

An automated capillary gas chromatography/tandem mass spectrometry (GC/MS/MS) assay for the simultaneous quantitation of tebufelone (TE) and 13C, (18)O-labeled TE (TE-CO) in plasma was developed. This method permits the use of stable isotope coadministration (TE and TE-CO dosed concurrently via peroral and intravenous routes, respectively) for the determination of TE absolute bioavailability. 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 TE, TE-CO, and the 3-methyl-TE internal standard which were selected in Q1 to undergo collisionally activated dissociation in Q2. Quantitation was achieved through monitoring product ions at m/z 248, 251 and 248, respectively, in Q3. A 2-1000 ng per sample (40 pg to 20 ng injected) quantitation range provided access to an effective 1-5000 p.p.b. plasma concentration range (0.2-2 g samples) for both TE and TE-CO. The assay showed no bias and less than 8% relative standard deviation over the entire range. The method was used to determine plasma levels of TE and TE-CO in four dogs receiving 2.5:2.5 mg/kg TE:TE-CO, intravenously. The pharmacokinetics of both isotopomers proved to be identical, indicating no isotope effect and verifying the chemical stability of the (18)O-carbonyl label under these dosing conditions. In addition, the applicability of this analytical approach to the determination of TE peroral bioavailability was initially tested in dogs.

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