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

Compound I (4,7 beta-dimethyl-4-azacholestan-3-one, MK-0386) is a pote nt 5 alpha-reductase type 1 (5 alpha R1) inhibitor. Sensitive (0.2 ng/ ml), specific and separate assays have been developed and validated fo r the analysis of I and its carboxylic acid metabolite (II) in human s emen 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 o f I, and on a longer (150 mm) column with a weaker mobile phase during the analysis of TI. This additional chromatographic separation was re quired to separate II from a secondary metabolite present in post-dose plasma samples interfering with the quantification of II. The MS-MS d etection was performed on a Sciex API III Plus tandem mass spectromete r using the heated nebulizer probe. Monitoring the parent-->product io n combinations of m/z 416-->114 and 404-->114, in the multiple reactio n monitoring (MRM) mode, after chromatographic separation, allowed qua ntification 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 variat ion of less than 15% for replicate (n=5) analysis at all concentration s within the standard curve range. For the semen assay the linear rang e for determination of I was from 0.2 to 50 ng/ml. These assays were a pplied to support a number of clinical studies with I and their validi ty and long-term performance was confirmed during analyses of clinical samples from these studies. The need for careful assessment of the sp ecificity of MS-MS assays in post-dose biological fluid samples in the presence of metabolites was emphasized.