Method of analysis of urinary steroids of human pregnancy by GLC and GC-MS of sephadex LH-20 chromatographic fractions

The direct analysis of pregnancy urinary steroids by GC-MS appears very difficult due to the numerous steroids and their great diversity of relative amounts, especially in overlapping peaks. Urinary steroids are hydrolyzed and extracted according to classical methods. Fractionation is carried out on Sephadex LH-20. On each fraction, separation and identification of steroids were done by GC-MS and then quantitated by GLC. We have positively identified and quantitated three 11-deoxy-17-oxosteroids, two 11-oxy-17-oxosteroids, five 16-OH-17-oxosteroids, one 18-OH-17-oxosteroid, 3 stereoisomers of 3-OH-5-pregnan-20-one, and three of pregnane-3, 20-diol, seven hydroxylated compounds of 3-OH-pregnan-20-one, the 5α-pregnane-3α, 17α, 20α-triol, four isomers of pregnane-3, 16, 20-triol, two isomers of pregnane-3, 20, 21-triol, the 5-pregnene-3β, 20α-diol and 5-pregnene-3β, 16α, 20α-triol, 17-oxo-androstane-3, 17-diol, two 5-androstenetriols, two androstanetriols and four estrogens. The two main interests of this technique rely on the module liquid fractionation which gives groups of steroids having well differentiated retention time and the quantitative analysis.