Separation and Identification of Human Urinary Dihydroxysteroids By High Resolution Glass Capillary Columns

Packed gas chromatographic columns are used at the present time in many steroid laboratories. The separations allowed by these columns are limited, especially for the study of complex biological mixtures. Vastly improved efficiency can be obtained by using capillary columns. High resolution glass capillary columns allow the successful specific analysis of C19 and C21 urinary human dihydroxysteroids. This report deals with some problems in the use of capillary columns: coating of stationary phase, injector systems either in solid phase or in liquid phase, columns performances. The resolution of stereoisomers depends on the derivatives used (trimethylsilyl ethers, trifluoroacetates and heptafluorobutyrates). The high resolution glass open tubular columns allow separation and specific measurement of seven dihydroxysteroids: 5α-androstane-3α:,17β/3β,17β-diols, androst-5-ene-3β, 17β/3β,17α-diols, 5α/5β-pregnane-3α,20α-diols and 5β-androstane-3α,17β-diol. These dihydroxysteroids are identified by their retention index and by their mass spectra. A coupling device of capillary columns to a mass spectrometer is described. It is shown that this coupling fully maintains the gas chromatographic resolution. The fragmentation of heptafluorobutyrates is presented. The scheme of fragmentation of androstane- and pregnane-diheptafluorobutyrates is characteristic and can be a supplementary element in difficult identification. The rapid approach to the simultaneous determination of seven neutral steroids in urine is particularly interesting in the study of pathological cases.