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
Ml. Constanzer et al., 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, Journal of chromatography B. Biomedical sciences and applications, 693(1), 1997, pp. 117-129
Citations number
15
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
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.