Isocratic high-performance liquid chromatographic method for the separation of testosterone metabolites |
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| Affiliation: | 1. Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University, UCSI Heights, 56000 Cheras, Kuala Lumpur, Malaysia;2. Discipline of Chemical Engineering, School of Engineering, Monash University Malaysia, 47500 Bandar Sunway, Selangor, Malaysia;3. Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia;4. Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;5. Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;6. Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;7. School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Malaysia;8. Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia;9. Manufacturing and Industrial Processes Division, Faculty of Engineering, Centre for Food and Bioproduct Processing, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia;1. Novosibirsk State University, 630090 Novosibirsk, Russian Federation;2. V. V. Voevodsky Institute of Chemical Kinetics and Combustion, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russian Federation;3. B. I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, 220072 Minsk, Belarus |
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| Abstract: | An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-14C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsily)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver. |
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