The Study on the Quantitative Determination of Urinary 17-ketosteroids by Gas Liquid Chromatography

A great grogress has been made in chemical measurement of steroid hormone by the application of gas liquid chromatography, which has the advantage of high separating ability, high sensitivity, and simultaneous performance of separation and quantification in a short time. Many studies have been carried out on the determination of 17-KS through gas liquid chromatography sinoe the demonstration by Vanden Heuvel et al (1962) of the possibility of analysis with polar column on gas liquid chromatography after preparation of trimethylsilyl ether (TMSi-E) derivatives of 17-KS. However, only a few reports are available on the determination of 11-oxygenated 17-KS. Moreover, a variety of method has been carried out at the stage of purification and nothing definite has been established, and many problems still remain in the stage of TMSi-E derivative formation.<BR>The author, therefore, used a simple column chromatography in the stage of purification and studied the method of preparation of TMSi-E derivertives in an attempt to develop a method of measurement of 6 major 17-KS.<BR>After 2-step hydrolysis, the extracts were divided into two groups ; 11-deoxygenated and 11-oxygenated 17-KS, through alumina column chromatography. Each of these fractions was prepared into TMSi-E derivatives using pyridine and tetrahydrofuran as the solvent. Each group was then measured with gas liquid chromatography set at the most adequate condition for each.<BR>The instrument was a Shimazu Gas Chromatograph, Model GC-1C and GC-1B. Columns were 225 cm or 230 cm in length, 3mm-4mm i.d. packed with 80-100 mesh Chromosob W coated with 1-2% NGS and with 2% XE-60. Columns were kept at 203-230°C, while the detector cell and flash heater were kept at 240-260°C. The carrier gas the was nitrogen and flow rate of gas was 40-60-ml/min.<BR>The rate of recovery with this method averaged 95.7% except for the step of hydrolysis, and the reproducibility through this method was±1.0% (S.D.). A linearity in gas liquid chromatography was obtained above 0.06 μg.<BR>Using this method, various factors at each step of the determination of 17-KS were investigated and the following results were obtained.<BR>(1). Five hydrolytic procedures under clinical application were compared using total values of Zimmerman chromogens and values of determination by gas liquid chromato graphy. The method of 2-step hydrolysis with solvolysis after β-glucuronidase hydrolysis gave the highest value in addition to the possibility of assessing each conjugated form.<BR>(2). At the stage of purification, Florisil column chromatography was at first used to study the purification of 17-KS. Alumina column chromatography was used to study the separation and purification of 11-deoxygenated 17-KS and 11-oxygenated 17-KS. While Florisil column chromatography 'followed by alumina column chromatography achieved the best purification through measurement after division into two groups, 11-deoxygenated and 11-oxygenated 17-KS, alumina column chromatography alone without Florisil column chromatography was found to be sufficient for purification.<BR>(3). The method of preparation of TMSi-E derivatives was studied using pyridine and tetrahydrofuran as the solvent. While the method of using pyridine for 11-deoxygenated 17-KS was speedy and certain, 11-OH group in the 11-oxygenated 17-KS reacted in pyridine but did not react in tetrahydrofuran. So the tetrahydrofuran method was convenient for analysis of 11-OH group of 17-KS because of its large retention time of gas liquid chromatography.<BR>(4). With this method, urinary 17-KS values in normal female was 5.5 mg/day (total, mean). Gaschromatogram obtained in various cases were also demonstrated.<BR>With this method, however, many peaks were described on gaschromatogram when the urine from late pregnant women were determined. This indicated the need for further purification.