Since the isolation and identification of testosterone in normal urine by Schubert and Wehrberger (1) and Camacho and Migeon (2) a number of methods have been reported for its quantitative measurement (3-10). Some of these methods measure combined epitestosterone and testosterone while some separate out the epi form and measure only testosterone. More recently a method has been described for the separation and measurement of each in the urine of prepubertal children (11). This method however, does not employ GLC. Most of the work reported has been done on human urine using relatively large volumes of sample. The importance of epitestosterone is not fully understood at this time ( 12), but it would seem desirable to measure it as well as testosterone if this could be accomplished without additional trouble. Furthermore the analysis of monkey urine presents a particular problem due to the low volumes and low hortnone levels sometimes encountered. The methocl described in this paper was developed to meet the need for a highly sensitive, highly specific measurement of these two steroids in human and monkey urine so that the effects of stress on the androgen system could be studied. The method as described involves enzymic hydrolysis, ether extraction, thin-layer chromatography of the free (neutral) steroids, acetate formation, thin-layer chromatography of the steroid acetates, and separation and quantitation of the two steroids by gas-liquid chromatography. A flame ionization detector was used for mass measurement and the addition of a tracer amount of '%-testosterone provided t,he means for correcting for methodological losses. MATERIALS Reagents and Sohen ts 2 M acetate buffer (pH 4.8) : 272 gm NaC2H302 *3H,O (Mallinckrodt A. R.) + 120 ml glacial acetic acid diluted to 1 liter. 343 344 MOUGEY, COLLINS, ROSE, AND MASON
EDTA (Eastman Organic Chemicals) : 8 mg/ml acetate buffer. Penicillin G (Eli Lilly, buffered)-400,000 units dissolved in 14 ml acetate buffer.
NaOH (Merck pellets, low in carbonate) : 5 N solution made up and stored in a polyethylene bottle. This was filtered through a plug of glass wool and diluted to 1 N.
Ketodase (Warner-Chilcott Labs.) : beef liver /3-glucuronidase, 5000 U/ml.
Ethyl ether (Mallinckrodt A. R.) : freshly opened can used without further purification.
Benzene, acetone, ethyl acetate (Mallinckrodt "Nanograde" solvents) : used without further purification.
Pyridine, acetic anhydride (Fisher reagent) : redistilled and stored in a desiccator.
Mcthylene chloride (Merck reagent) : suitable for spectrophotometer use, purified by passing through a silica gel column (Davison Chem. activated desiccant,.
Chloroform (Merck reagent) : redistilled immediately before use.