A new method for simple fractional simultaneous determination of 6 fractions of urinary 17-KS (androstanedione, androsterone, δ4-androstenedione, etiocholanolone, dehydroepiandrosterone and 11-oxy-17-Ketosteroids) was established and excretion values in normal adult females and several cases with ovarian dysfunction were measured by this method and several fraction ratios were evaluated as necessary informations on their androgenisity.
The new method consisted of the following procedures: 170ml of 24 hours urine was adjusted to pH 4.7 with acetic acid and then 6.8ml of 0.2M acetate buffer, 40, 800 Fishman units of β-glucuronidase (240 units/ml) and 5, 100 units of crystalline penicillin were added, then the urine was incubated in a water bath at 37°C for 24 hours. Then 20, 400 Fishman units of β-glucuronidase were added and it was successively incubated for another 24 hours. The hydrolysed urine was extracted with ethyl acetate three times, 170, 85 and 85ml of ethyl acetate were used. Then, alkali washing with 12.75ml of cold 2N-NaOH twice and water washing with 17ml of distilled water twice were treated to the organic phase, and alkali and water used for washing were reextracted with ethyl acetate. After drying with sodium sulfate anhydrous, the extract was evaporated to residue in vacuo at 60°C.
The urine layer after the extraction of β-glucuronidase hydrolysate was saturated with 34g of NaCl, and 17ml of 50% sulfuric acid, 3.4ml of 20% formalin and 170ml of ether were added, and then it was kept standing at room temperature for 4 days being shaken twice a day. After 4 days extraction, another 170ml of ether was used for the reextraction. Each 170ml of ether which extracted solvolysate was washed with 17ml of 1N-NaOH twice and then washed with 21ml of distilled water twice. Alkali and water were reextracted in the same way with the extraction of β-glucuronidase hydrolysate. The layer of ether was dried with sodium sulfate anhydrous and was evaporated to residue at 45°C.
Two step hydrolysate was chromatographed as follows;
1: Florisil column chromatography.
A glass column 28cm long, of 1cm internal diameter was packed with 4g of florisil using chloroform. Steroid residue was absorbed on the column with 3ml×4 of chloroform. The development was carried out with 10ml of chloroform and 80ml of 0.8% v/v methanol-chloroform. The chloroform fraction was discarded as prewash. The fraction of 0.8% methanol-chloroform was evaporated to residue in vacuo at 60°C. The steroid residues obtained thus contained C₁₉ steroids, and were chromatographed on the following impregnant thin layer chromatography.
2: Impregnant thin layer chromatography.
* Preparation of thin layer
20g of Kieselgel GF₂₅₄ was mixed vigorously with 40ml of 30% ethyleneglycol/methanol for 3 minutes, and the mixture was spread on a glass plate, 20×20cm, 0.45mm thick. And the plate was allowed to stand at room temperature for few minutes and then it was warmed at 37°C for 10 minutes.
* Spotting and covering
Immediately after warming the plate, each steroid residue obtained by column chromatography was spotted on the thin layer and the mixture of 5 authorized pure steroids (11-deoxy-17-KS) was paralelly spotted by 50% methanol-chloroform. After spotting, spotting areas were covered with 50% ethyleneglycol-methanol to keep those areas impregnated. Otherwise, tailing and remained steroid on the spotting area were remarkable. The plate was warmed again at 37°C for 10 minutes to evaporate methanol.
* Development and detection of spots
A chromatographic chamber was saturated with organic solvent (10% dichlormethane, 90% n-hexane). The spotted plate was developed in this chamber by the ascending method.

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