Rapid method of simultaneous determination of pregnanediol and pregnanetriol in urine by glass-fiber paper chromatography

Abstract

Recently the method of hormone research has become more precisely and has been complicated. While for routine clinical use, simplicity and rapidity have been required to determine the steroid hormones.For this purpose Dingman et al. (J. clin. endocrinol., 22 : 87, 1962) have described a simple method for the determination of urinary pregnanediol and pregnanetriol by glassfiber paper chromatography. In our report, too, a more rapid method, that has been obtained from the research of simultaneous extraction of pregnanediol and pregnantriol existed in the same β-glucuronidase-hydrolysed urine and from the scrutiny of solvent system of glass-fiber paper (No. X-934-A.H.) chromatography, is established.Principal results of our investigations are summarized as follows : (1) Hydrolysis and extraction.β-glucuronidase hydrolysis is suitable for the cleavage of urinary pregnanediol and pregnanetriol. Extraction of both steroids is done simultaneously with chloroform and washing with 1.0 N NaOH is adopted. Recovery of pregnane diol is 99.0±0.7% and pregnanetriol is 87.5±6.1%.(2) Separation on simple glass-fiber paper.The Rf of pregnanediol and pregnanetriol are sufficiently separated from other steroids in benzene, benzene-isooctane or cyclohexane-acetone system of solvents by glass-fiber paper chromatography. Development is done 20cm from origin (cir. 20min.) at room temperature.(3) Recovery of glass-fiber chromatography.EtOAc is employed for extracting after separation of steroids as excellent solvent. It indicates that transferation of large dosis of steroids is not suitable in this procedure.The suitable dosis of transferated steroids are 10-20ug. Recovery of these dosis is about 90%. Absorption curves of contaminants show generally some straight line between 380 and 480mu.(4) An attempt of colorimetry.The comparison of colorimetry of steroids at room temperature and 100°C on a water bath are investigated. On pregnanediol the later procedure increases the optical sensitivity but pregnanetriol is adequated at room temperature. Absorption optima of pregnanediol is at 425m.0 and pregnanetriol is at 440mu.(5) Basing on the results described above, a new rapid method is recommended for estmation of pregnanediol and pregnanetriol in human urine. Then a few data applicated to clinical cases are demonstrated.Glass-fiber paper chromatography has a consinerable adventages, which are rapidity and accuracy of separation of steroids and simplicity of elution procedure. So these techniques are suitable for routine, simple and rapid determination of steroids.Besides, recommended method has other beneficial ideas of (a) the introduction of simultaneous hydrolysis of pregnanediol and pregnanetriol with β-glucuronidase, (b) the use of chloroform and 1.0 N NaOH for simultaneous extraction and washing of these steroids and (c) rapid colorimetry of pregnanediol.