Improved high-performance liquid chromatographic procedure for the separation and quantification of hydroxytestosterone metabolites

A reproducible, sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify hydroxytestosterone metabolites. The method features a simple one-step linear gradient of methanol in water (10%–60% methanol) for separation of the testosterone metabolites on a Supelcosil...

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Bibliographic Details
Published in:Journal of pharmacological and toxicological methods 1997-03, Vol.37 (2), p.67-73
Main Authors: Purdon, Michael P., Lehman-McKeeman, Lois D.
Format: Article
Language:English
Subjects:
Animals
Chromatography, High Pressure Liquid - methods
Cytochrome P-450
HPLC
Hydroxytestosterones - isolation & purification
Hydroxytestosterones - metabolism
Male
Mice
Microsomes, Liver - chemistry
Rats
Rats, Sprague-Dawley
Testosterone
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Summary:A reproducible, sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify hydroxytestosterone metabolites. The method features a simple one-step linear gradient of methanol in water (10%–60% methanol) for separation of the testosterone metabolites on a Supelcosil LC-18 column; metabolites are detected at 247 nm. This method provides a distinct advantage over previously developed assays in that the solvent gradient does not contribute to baseline changes throughout the chromatographic run. In this way, the flat baseline markedly improves robustness of the assay and simplifies peak integration and quantification. At the same time, resolution of 15 different steroid metabolites catalyzed by the cytochrome P-450 enzymes are readily separated in rat or mouse liver microsomes. An internal standard, cortexolone, was selected for use based on its structural and spectral similarity to the hydroxytestosterone metabolites, and quantification is based on the molar response for the testosterone/cortexolone peak area ratio. The limit of detection (LOD) is 1 pmol on-column with a limit of quantitation (LOQ) of 4 pmol on-column. The intraday repeatability is approximately 3%. This simplified procedure is straightforward and should greatly facilitate the routine use of the testosterone hydroxylation assay to measure cytochrome P-450 isozyme activity.
ISSN:1056-8719
1873-488X
DOI:10.1016/S1056-8719(97)00013-0