Running Title: Steroid Analysis by LC/MS/MS *Corresponding Author:

The use of anabolic steroids in sports is prohibited by World Anti-Doping Agency. Most of the methods for the detection of steroids are currently based on GC/MS analysis. These methods involve lengthy extraction procedures followed by derivatization of steroid metabolites. Additionally, GC/MS methods are used to detect steroid metabolites instead of parent compounds due to their lower sensitivity. We have recently developed an LC/MS/MS method for the detection, identification and quantitation of anabolic steroids in urine samples. Using an Applied Biosystems LC/MS/MS 3200 QTRAP system, we were able to identify several anabolic steroids of abuse (boldenone, methenolone, methandienone, nandrolone, stanozol, mesterolone, norethandrolone and androstenedione) with simultaneous quantitation of testosterone and epitestosterone levels to determine the T/E ratios. Using this method, we were able to detect and quantitate parent compounds of boldenone, nandrolone and stanozol in urine samples of actual steroid users. Briefly, the method involved hydrolysis of glucuronated steroids from urine samples with β-glucuronidase (from H. pomatia) followed by liquid/liquid extraction. The extracts were injected on to a column (C18 reverse phase, 50 mm x 4.6 mm, 3μ) using a Shimadzu autosampler and the anabolic steroids were separated by gradient elution (ammonium acetate / methanol/aceti