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

摘要

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.