A new method for hydrolyzing sulfate and glucuronyl conjugates of steroids.

A new method for hydrolyzing steroid conjugates (both sulfates and glucuronides conjugates) that is efficient, effective, and inexpensive is described. This method comprises incubation of the conjugates--after salting-out into ethyl acetate or elution from a C18 cartridge--with anhydrous methanolic hydrogen chloride (methanolysis) for 10 min. It has been successfully applied to our routine radioimmunoassay screening and GC/MS confirmation studies of steroids in prerace and postrace equine urine samples. Comparative GC/MS studies on entire (male horse) urine samples showed that methanolysis gave amounts of free steroids (estrone, estradiols, testosterone, estrenediols, nandrolone, androstanediols) at least as large as those obtained by solvolysis. Similar studies on urine samples from a gelding that had been administered nandrolone phenylpropionate showed that methanolysis gave larger amounts of free steroids (nandrolone, estranediols) than Helix pomatia enzymatic hydrolysis or solvolysis. Also, TLC studies on methanolysis of corticosteroid conjugates such as hydrocortisone 21-sulfate and hydrocortisone 21-phosphate showed that free corticosteroid was released in 5 min.