Determination of a thermally labile metabolite of a novel growth hormone secretagogue in human and dog plasma by liquid chromatography with ion spray tandem mass spectrometric detection

A sensitive and selective assay for the determination of N-{1(R)-[(1,2-dihydro-1-methylsulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethoxy)-ethyl}-2-hydroxyamino-2-methylpropanamide (I), a hydroxyl amine metabolite of a novel growth hormone secretagouge (II) has been developed utilizing high-performance liquid chromatography with ion spray tandem mass spectrometric detection (HPLC–MS–MS). The analyte and an internal standard (III) were isolated from the basified biological matrix using a liquid–liquid extraction with methyl tert.-butyl ether (MTBE). The organic extract was evaporated to dryness at room temperature. The residue was reconstituted in the mobile phase and injected into the HPLC–MS–MS system. Multiple reaction monitoring using the precursor→product ion combinations of m/z 545→267 and 543 →267 was used to quantify I and III, respectively, after chromatographic separation under isocratic conditions. The assay was validated in the concentration range of 0.5 to 500 ng/0.1 ml in both human and dog plasma. The precision of the assay, expressed as relative standard deviation, was less than 10% over the entire concentration range with the exception of the low concentration of 0.5 ng/0.1 ml which was 14.0% for human plasma. The HPLC–MS–MS method provided sufficient sensitivity to completely map the pharmacokinetic time course of I following a single 5 mg dose of II to human subjects and a 0.5 mg/kg dose to beagle dogs.