Determination of a novel growth hormone secretagogue (MK-677) in human plasma at picogram levels by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.
A sensitive and specific method for the determination of N-[1(R)¿[1,2-dihydro-1-methylsulfonylspiro(3H-indole-3,4'-piper idin)-1'-yl]carbonyl¿-2-(phenylmethoxy)ethyl]-2-amino-2-meth ylpropanamide (MK-677, I), a growth hormone secretagogue, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The analyte and internal standard (II) were isolated from the basified plasma using a liquid-liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring of parent-->product ion combinations at m/z 529-->267 and 527-->267 was used to quantify I and II, respectively. The assay was validated in human plasma in the concentration range of 0.1 to 100 ng/ml, and the limit of quantification (LOQ) was 0.1 ng/ml. The precision of the assay, as expressed as coefficients of variation (C.V.,%) was less than 7% at all concentrations within the standard curve range, with adequate assay specificity and accuracy. The HPLC-MS-MS method provided sufficient sensitivity to completely map the pharmacokinetic time-course following a single 5-mg oral dose of I.
Constanzer
M L
ML
Merck Research Laboratories, West Point, PA 19486, USA.
Chavez-Eng
C M
CM
Matuszewski
B K
BK
D002853:Chromatography, Liquid; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000097:blood* / Q000493:pharmacokinetics; D013058:Mass Spectrometry; D012680:Sensitivity and Specificity; D013141:Spiro Compounds / Q000097:blood* / Q000493:pharmacokinetics
D007211:Indoles; D013141:Spiro Compounds; D019382:Human Growth Hormone; C094817:ibutamoren mesylate