Ethyl acetate extraction procedure and isocratic high-performance liquid chromatographic assay for testosterone metabolites in cell microsomes.
An isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for separation of testosterone and its main metabolites over the nominal range 20 to 40 microg/ml and 280 to 4600 ng/ml, respectively. Mobile phase composition (phosphate buffer-methanol-acetonitrile, 50:38.5:11.5) was optimised by studying the influence of numerous chromatographic parameters. The most critical one was the ratio CH3CN/CH3OH. Good recoveries (around 90% for all compounds) and an improved specificity were assessed by a double ethyl acetate extraction of biological samples. According to the performance criteria tested, the method could be applied to enzymatic inhibition and induction in vitro studies.
Baltes
M R
MR
Université Libre de Bruxelles, Institut de Pharmacie, Laboratoire de Chimie Bioanalytique, Toxicologie et Chimie Physique Appliquée, Brussels, Belgium.
Dubois
J G
JG
Hanocq
M
M
D000085:Acetates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D016014:Linear Models; D008861:Microsomes / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D011794:Quercetin / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013739:Testosterone / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D000085:Acetates; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D013739:Testosterone; C007650:ethyl acetate; D003577:Cytochrome P-450 Enzyme System; D011794:Quercetin