An improved method for the simultaneous determination of individual 17-oxosteroids and of pregnanediol in urine by gas-liquid chromatography.

An improved method for the simultaneous gas-chromatographic determination of individual 17-oxosteroids and of pregnanediol in the same sample of urine is described. The method involves the following steps. The urinary sample is chromatographed on Amberlite XAD-2. The fraction containing the steroid conjugates is evaporated; the residue is dissolved in n-heptane and the organic phase extracted with water. The steroid glucuronides are hydrolysed by a bacterial beta-glucuronidase; subsequently, the steroid sulphates are solvolysed in ethyl acetate. Free 17-oxosteroids and pregnanediol are separated by two-dimensional thin-layer chromatography. The zone containing the steroids to be investigated is eluted; after evaporation, the residue is treat with N-methyl N-trimethyl silyl trifluoroacetamide. The reaction mixture is injected into a gas chromatograph; the trimethyl silyl ethers of the individual 17-oxosteroids and of pregnanediol are quantitated by measuring the peak heights.