Selective liquid chromatographic isolation procedure for gas chromatographic-mass spectrometric analysis of 3-ketosteroids in biological materials.
A method for the isolation of 3-ketosteroids based on the positive charge of their oximes is described. The biological extract is filtered through a column of sulphoethyl Sephadex LH-20 (H+). Steroids in the filtrate are converted into oximes and the dried reaction mixture is filtered through a column of diethylaminohydroxypropyl Sephadex LH-20 (OH-) in methanol. After evaporation of the solvent and hydroxylamine, the oximes are taken up by and separated on a column of sulphoethyl Sephadex LH-20 (H+) in  methanol. Following elution of other steroids, the oximes of 3-ketosteroids are eluted as a group with methanol-pyridine (20:1, v/v), and are converted into trimethylsilyl ethers. Removal of reagents and further purification of the sample is achieved by rapid filtration through Lipidex 5000 in n-hexane-pyridine-hexamethyldisilazane-dimethoxypropane (97:1:2:10). The derivatives are then analyzed by computerized gas chromatography-mass spectrometry using open-tubular glass capillary columns. Recoveries of picogram amounts of 3H-labelled steroids carried through the entire isolation procedure are 80-90%. The purification achieved in analyses of plasma permits solid injection of the equivalent of 1-2 ml of plasma without overloading of the capillary column. The principle of the isolation procedure might be applicable to other groups of compounds that possess keto or aldehydo groups.
D002849:Chromatography, Gas; D002853:Chromatography, Liquid; D005260:Female; D005898:Glass; D006801:Humans; D007664:Ketosteroids / Q000097:blood*; D013058:Mass Spectrometry; D008722:Methods; D008832:Microchemistry; D010091:Oximes / Q000097:blood; D011247:Pregnancy; D012997:Solvents; D014297:Trimethylsilyl Compounds / Q000097:blood
D007664:Ketosteroids; D010091:Oximes; D012997:Solvents; D014297:Trimethylsilyl Compounds