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.
Constanzer
M
M
Merck Research Laboratories, West Point, PA 19486, USA.
Chavez-Eng
C
C
Matuszewski
B
B
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D013006:Growth Hormone / Q000097:blood*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D013006:Growth Hormone