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

A capillary-liquid chromatography (LC) method was developed for the quantif ication of the endogenous steroids testosterone and epitestosterone in huma n urine. One milliliter of urine was used for the overall method. Free test osterone was first separated by liquid-liquid extraction with n-pentane at pH 7. Glucuronides of testosterone and epitestosterone were enzymatically h ydrolyzed and the free compounds were extracted with n-pentane at pH 11. A capillary column switching system with a low back pressure precolumn (PC) w as used for fast loading of lame sample volumes (20 muL). Chromatographic s eparation was carried out on a 15 cm x 300 mum inner diameter (i.d.) column , packed with 3 mum Hypersil BDS-C-18 at a flow rate of 4 muL/min with isoc ratic elution and UV absorbance detection (240 nm). Limit of detection for free testosterone was established at 0.5 ng/mL. Limits of detection were es tablished at 1.5 and 3.2 ng/mL for testosterone and epitestosterone, respec tively, after being hydrolysed from their glucuronides. Good reproducibilit y and robustness were observed through the entire calibration range (up to 250 ng/mL). (C) 2001 John Wiley & Sons, Inc.