Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites.
A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C18 analytical column (10 cm x 0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran-acetonitrile-water (10:10:80, v/v) changing to tetrahydrofuran-acetonitrile-water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 microg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [14C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.
Whalley
P M
PM
Division of Metabolism & Pharmacokinetics, Servier Research and Development Ltd, Slough, UK.
Bakes
D
D
Grime
K
K
Weaver
R J
RJ
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D013250:Steroid Hydroxylases / Q000378:metabolism; D013739:Testosterone / Q000302:isolation & purification* / Q000378:metabolism
D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C022724:testosterone 7-alpha-hydroxylase, hamster