Quantification of testosterone and epitestosterone in human urine by capillary liquid chromatography

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 column switching system with a low back pressure precolumn (PC) was used for fast loading of large sample volumes (20 μL). Chromatographic separation was carried out on a 15 cm×300 μm inner diameter (i.d.) column, packed with 3 μm Hypersil BDS-C18 at a flow rate of 4 μL/min with isocratic elution and UV absorbance detection (240 nm). Limit of detection for free testosterone was established at 0.5 ng/mL. Limits of detection were established at 1.5 and 3.2 ng/mL for testosterone and epitestosterone, respectively, after being hydrolysed from their glucuronides. Good reproducibility and robustness were observed through the entire calibration range (up to 250 ng/mL).