Abstract
A major problem in the analysis of steroids by gas chromatography-mass spectrometry or other ultramicro methods is the preliminary purification and fractionation. Conventional methodology involves the use of thin-layer chromatography, but poor recoveries, chemical transformations and introduction of interfering materials are common problems with this method. For this reason work has been directed towards development of column systems employing inert synthetic gels as the stationary phase (see Sjövall J., Nyström E. & Haahti E.: Advances in Chromatography 6 (1968) 119). The most versatile phase so far obtained is a hydroxyalkoxypropyl derivative of Sephadex (Elling-boe J., Nyström E. & Sjövall J.: J. Lipid Res. 11 (1970) 266). This material gives a stationary phase which is less polar than the solvent when it is used with solvent mixtures such as methanol/water/chloroform, 8:2:1. When columns of the same Sephadex derivative are prepared in a mixture of hexane and chloroform, 9:1, a straight phase system is obtained which is suitable for separation of very non-polar steroids. The effect of different substituents on the mobilities in different solvent system will be described. Quantitative studies indicate no loss of picogram quantities of steroids, and the method has been used in combination with competitive proteinbinding to give a simple rapid method for determination of 17α-hydroxyprogesterone in plasma (Holmdahl & Sjovall, to be published).
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