Abstract
The conditions for optimal determinations from urine of 17-ketosteroids were investigated by various means of each steps of hydrolysis, removal of impurities, extractions and Zimmermann reaction colometry.
1. Hydrolysis.
The optimal method for hydrolysis of total contents of 17-ketosteroid as simplified clinical test was acid hot hydrolysis; urine was heated at 100°C for 15 min. with conc·H2SO4 of 1/10 vol. of urine.
While optimal hydrolysis method for chromatographic separation was acid hot hydrolysis after acid room temperature hydrolysis; urine was shaken at room temperature for 6 days with 50% H2SO4 of 1/10 vol. of urine and equal vol, of ether, the urine was then extracted with ether and the residual urine was heated at 100°C for 15 min. with conc·H2SO4 1/20 vol, of urine.
2. Removal of impurities.
The removal impurities in 17-ketosteroid were better Girard keto separation and method by addition of formaldehyde than the other methods.
The best condition for the two methods were established, and the removal of impurities by addition of formaldehyde could carried out easily as clinical method than Girard keto separation, and it was purified completely as well as Girard.
3. Extraction.
The ether was good solvent to extract of 17-ketosteroids in hydrolyzed urine, an concentration of NaOH to removed of impurities was 4 % best.
4. Colormetry
The optimal method of colormetry of extracted 17-ketosteroid as followed; 17-ketosteroids in each tubes were added 0.4 ml of 1 % m-dinitrobenzene in ethanol and 0.2 ml of 8 N-KOH aqueous solution, and they were allowed to stand for 30-60 min. at room temperature in dark place and were then added 70% ethanol. The resultant chromogens were readed with the Beckman DU spectrophotometer at 460, 520 and 580 mμ.
The results were calculated by application of a correction factor according to the principles of Allen.
