Comparison of Four Methods of Determining Urinary Pregnanediol.

Abstract

0b Four methods for determining urinary pregnanediol are compared. The procedures involve several hydrolysis, extraction, and purification methods, and utilization of gas chromatography and spectrophotometry for quantitation. Enzymatic hydrolysis of pregnanediol glucosiduronate, preliminary purification using silica gel column chromatography, and quantitation by means of gas chromatography was found to be the preferred method.HE DEVELOPMENT of methods for T determining urinary pregnanediol (5p-pregnane-3a,20a-diol) originated with Venning (1 6, 17) and involved the gravimetric assay of pregnanediol glucosiduronate. In this procedure sodium pregnanediol glucosiduronate was extracted with butanol and purified by an acetone precipitation technique. Among the disadvantages of this method were the occurrence of emulsions during the butanol extraction and the difficulty of purifying the extract (14). Astwood and Jones (1) attempted to overcome the difficulties of the Venning method by hydrolyzing the conjugate with mineral acid in the presence of toluene. This method has gained wide acceptance, although many modifications have been reported.The advent of gas chromatography of steroids prompted many investigators to apply this technique to the determination of pregnanediol in biological extracts. In most cases the toluene-acid hydrolysis procedure has been utilized (2, 3, 7 , I S , 16,18) and the extract submitted to analysis either directly or following prepurification and acetylation. Enzyme hydrolysis has been reported by some investigators and the extract is either purified by means of thin layer or column chromatography and acetylated, converted to the trimethylsilyl ether, or analyzed directly (9)(10)(11)(12). Unfortunately, very little information has been given concerning the reliability of these methods.The present investigation was undertaken to evaluate these possible methods for determining urinary pregnanediol using gas chromatography. The methods were : toluene-acid hydrolysis and extraction ( I S , 15); extraction of the conjugate with ether-ethanol (8) and hydrolysis with perchloric acid in anhydrous tetrahydrofuran (6) ; and enzyme hydrolysis, chloroform extraction, and silica gel column purification. These methods were compared with the spectrophotometric method of Goldzieher and Nakamura (4) used in these laboratories for 2 years.The methods were evaluated by study ing the recovery of free pregnanediol added to urine and by analyzing a serially diluted pregnancy urine pool. EXPERIMENTALApparatus. Model 15 and Series 5000 Barber-Colman gas chromatography units equipped with strontium-90 detectors (A-4150) and a Packard Model 7503 dual column gas chromatograph equipped with tritium detectors were used in this study. The stationary phase consisted of a mixture of 1% XE-60 and lyO SE-30 coated by the filtration technique (6) on 100-1 10 mesh Anakroxy ABS (Analabs, Inc., Hamden Conn.) previously washed with methanol and methylene chloride. Glass columns 1.8 meters X 6 mm. were used, with a column temperatu...