title: Low level determination of a novel 4-azasteroid and its carboxylic acid metabolite in human plasma and semen using high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.
abstract: Compound I (4.7beta-dimethyl-4-azacholestan-3-one, MK-0386) is a potent 5alpha-reductase type 1 (5alphaR1) inhibitor. Sensitive (0.2 ng/ml), specific and separate assays have been developed and validated for the analysis of I and its carboxylic acid metabolite (II) in human semen and plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. After liquid-liquid extraction of the analytes from biological matrix, the extracts were chromatographed on a short (50 mm) analytical column during analysis of I, and on a longer (150 mm) column with a weaker mobile phase during the analysis of II. This additional chromatographic separation was required to separate II from a secondary metabolite present in post-dose plasma samples interfering with the quantification of II. The MS-MS detection was performed on a Sciex API III Plus tandem mass spectrometer using the heated nebulizer probe. Monitoring the parent-->product ion combinations of m/z 416-->114 and 404-->114, in the multiple reaction monitoring (MRM) mode, after chromatographic separation, allowed quantification of both analytes. The standard curve in plasma was linear in the concentration range of 0.2 to 200 ng/ml for both I and II with correlation coefficients greater than 0.99 and coefficients of variation of less than 15% for replicate (n=5) analysis at all concentrations within the standard curve range. For the semen assay the linear range for determination of I was from 0.2 to 50 ng/ml. These assays were applied to support a number of clinical studies with I and their validity and long-term performance was confirmed during analyses of clinical samples from these studies. The need for careful assessment of the specificity of MS-MS assays in post-dose biological fluid samples in the presence of metabolites was emphasized.
authors.affiliation: Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Constanzer
authors.forename: C M
authors.initials: CM
authors.lastname: Chavez
authors.forename: B K
authors.initials: BK
authors.lastname: Matuszewski
authors.forename: J
authors.initials: J
authors.lastname: Carlin
authors.forename: D
authors.initials: D
authors.lastname: Graham
mesh_terms: D058891:5-alpha Reductase Inhibitors; D001378:Azasteroids / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D012661:Semen / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: C090665:4,7beta-dimethyl-4-azacholestan-3-one; D058891:5-alpha Reductase Inhibitors; D001378:Azasteroids; D004791:Enzyme Inhibitors