Separation of testosterone metabolites in microsomal incubates using a new capillary electrophoresis assay (original) (raw)
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Journal of Chromatography B: Biomedical Sciences and Applications, 2001
A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C analytical column (10 cm30.46 cm) and a linear mobile phase (1.25 18 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 mg / ml with a limit of detection on column of 300 ng / ml. A method for the direct HPLC analysis of liver microsomal incubates of 14 [ C]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.
Nutrition-related alterations in liver microsomal testosterone hydroxylases
International Journal of Andrology, 1988
The oxidation products of testosterone formed by liver microsomes from normal-fed and protein-energy malnourished male rats have been analysed by HPLC. Microsomes from normal-fed rats oxidized testosterone at a rate of 4.52 nmol/min/ mg protein. The major products formed were: 6β-, 7α- and 16α-hydroxytestos-terone; these three metabolites represented 65% of the total testosterone meta bolism. Microsomes from protein-energy malnourished rats oxidized testosterone at a reduced rate of 2.03 nmol/min/mg protein. The major product formed was 7α-hydroxytestosterone, which accounted for 43% of total testosterone oxidation. Microsomes from protein-energy malnourished rats showed a CO-reduced cyto-chrome P-450 spectra with a maxima at 452 nm, and a 38% decrease in the total content of cytochrome P-450. Some testosterone hydroxylases were drastically affected by protein-energy malnutrition but others, such as 7α-hydroxylase, remained unchanged. The present results suggest that nutritional status can modify the relative amounts of individual cytochrome P-450 isozymes, thus explaining the observed changes in several testosterone hydroxylases. Protein-energy malnutri tion seems to be an excellent tool with which to obtain a microsomal fraction containing predominantly P-450 isozymes, which are probably involved in key mono-oxygenations of physiological substrates.
Archives of Biochemistry and Biophysics, 1989
Hepatocytes from male or female rats were cultured for up to 2 weeks in a modified Waymouth medium supplemented with 0.1 or 1.0 I.LM dexamethasone, 10 nM insulin, and 0.1 nM glucagon with or without addition of phenobarbital, methylcholanthrene, or isoniazid. The activities of testosterone hydroxylases were measured in the intact cell monolayer and in the corresponding microsomal fraction. Aniline hydroxylase was measured in cell homogenates. In the presence of 0.1 PM dexamethasone the testosterone hydroxylase activities varied differently in hepatocytes from male and female rats during the culture period. The activities of S/3-and 15a-hydroxylases increased in female and were unchanged in male hepatocytes, while 16cy-hydroxylase activity increased in female and decreased in male, and 2a-and 7a-hydroxylase activities were unchanged in both male and female hepatocytes during the culture period. Increasing the dexamethasone concentration to 1.0 PM caused an increase in Sg-and 15cr-hydroxylase activities in cultures of hepatocytes from both sexes, whereas an increase of 2cu-and a decrease of '7~-and 17-hydroxylase activities were found only in cultures of hepatocytes from female rats. Addition of phenobarbital caused an increase in the activity of 7a-hydroxylase in both male and female hepatocytes, while the effect on the other hydroxylases differed with the sex. In hepatocytes from male rats phenobarbital addition decreased the activities of 2&-and 16a-hydroxylases, while these were increased or stable after addition of phenobarbital to hepatocytes from female rats. The activity of aniline hydroxylase was increased at Day 1 and declined afterward. The results demonstrate that the activities of different steroid hydroxylases are inducible and can be directly measured in monolayers of hepatocytes from rats. o 1989Academic PWS, IX 0003-9861/89 $3.00 C'upyrlght rc' ,989 by Academic Press. Inc.
Quantification of testosterone and epitestosterone in human urine by capillary liquid chromatography
Journal of Microcolumn Separations, 2000
A capillary-liquid chromatography LC method was developed for the quantification of the endogenous steroids testosterone and epitestosterone in human urine. One milliliter of urine was used for the overall method. Free testosterone was first separated by liquid᎐liquid extraction with n-pentane at pH 7. Glucuronides of testosterone and epitestosterone were enzymatically hydrolyzed and the free compounds were extracted with n-pentane at pH 11. A capillary Ž . column switching system with a low back pressure precolumn PC was used for Ž . fast loading of large sample volumes 20 L . Chromatographic separation was Ž . carried out on a 15 cm = 300 m inner diameter i.d. column, packed with 3 m Hypersil BDS-C at a flow rate of 4 Lrmin with isocratic elution and UV 18 Ž . absorbance detection 240 nm . Limit of detection for free testosterone was established at 0.5 ngrmL. Limits of detection were established at 1.5 and 3.2 ngrmL for testosterone and epitestosterone, respectively, after being hydrolysed from their glucuronides. Good reproducibility and robustness were observed Ž . through the entire calibration range up to 250 ngrmL . ᮊ
Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry
Scandinavian Journal of Clinical and Laboratory Investigation, 2008
Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stableisotope dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether-ethyl acetate from 250 mL or 500 mL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode after separation on a reversed-phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d 2 testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2-100 nmol/L. Interassay CVs were 4.2-7.6 % at mean concentrations of testosterone of 3.3-45 nmol/L. Total measurement uncertainty (U, k52) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal-to-noise ratio53) and the overall method recovery of testosterone was 95 %. Correlation (r) with our inhouse extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18-30, 31-50, 51-70 and over 70 years were established. Sensitivity and specificity of the LC-MS/ MS method offer advantages over immunoassay and make it suitable for use as a high-throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.
Direct microtitre plate enzyme immunoassay of testosterone in unextracted serum
Journal of Immunological Methods, 1992
A method for enzyme immunoassay of testosterone in serum has been developed which does not require extraction of the serum with organic solvents. The release of testosterone from the binding proteins is achieved by heating the serum at 70°C for 30 min in an alkaline buffer. The results correlated well (r = 0.96) with those of a radioimmunoassay using \25I-Iabelled testosterone and with enzyme immunoassay with prior extraction of samples (r = 0.98). The detection limit of the assay is 1 pg per well and the tum around time for 36 samples is 3.5 h. The procedure is simple and well suited for routine analysis.
Journal of Analytical Toxicology, 2004
The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ralio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5o~-androstanediol, and 5[~-androstanediol) together with an endogenous reference (5[3-pregnanediol) were extracted from the urines and the ~t3C/12C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum 613C-value variations (parts per thousand difference of 813C/12C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5u.-androstanediol 813C-values returned to the baseline 15 h after the second T administration, a decrease of the 5~-androstanediol ~-values could be detected for over 40 h. This suggests that measurements of 5~-androstanediol 8-values allow lhe detection of a testosterone ingestion over a longer post-administration period than other T metabolites 813C-values or than the usual T/E ratio approach.