Constanzer M L, Chavez C M, Matuszewski B K, Carlin J, Graham D
Merck Research Laboratories, West Point, PA 19486, USA.
J Chromatogr B Biomed Sci Appl. 1997 May 23;693(1):117-29. doi: 10.1016/s0378-4347(97)00047-9.
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.
化合物I（4,7β-二甲基-4-氮杂胆甾烷-3-酮，MK-0386）是一种有效的1型5α-还原酶（5αR1）抑制剂。基于高效液相色谱（HPLC）与串联质谱（MS-MS）检测，已开发并验证了灵敏（0.2 ng/ml）、特异且独立的分析方法，用于分析人精液和血浆中的I及其羧酸代谢物（II）。从生物基质中液-液萃取分析物后，在分析I时，提取物在短（50 mm）分析柱上进行色谱分离，而在分析II时，在较长（150 mm）柱上用较弱流动相进行色谱分离。需要这种额外的色谱分离来将II与给药后血浆样品中存在的一种次要代谢物分离，该次要代谢物会干扰II的定量。MS-MS检测在Sciex API III Plus串联质谱仪上使用加热雾化器探头进行。在色谱分离后，以多反应监测（MRM）模式监测m/z 416→114和404→114的母离子→子离子组合，可对两种分析物进行定量。血浆中的标准曲线在I和II的浓度范围为0.2至200 ng/ml时呈线性，相关系数大于0.99，在标准曲线范围内所有浓度下重复（n = 5）分析的变异系数小于15%。对于精液分析，I的测定线性范围为0.2至50 ng/ml。这些分析方法被用于支持多项关于I的临床研究，并且在对这些研究的临床样品进行分析期间，证实了其有效性和长期性能。强调了在存在代谢物的情况下，需要仔细评估给药后生物流体样品中MS-MS分析方法的特异性。
