Efficient chromatographic fractionation of steroids in human serum through regulation of liquid--liquid distribution ratios.
To improve the clean-up process in the analysis of biological fluid constituents, an efficient liquid--liquid distribution system was developed. Closed-bed columns containing fine diatomaceous earth granules were prepared by slurry packing for the fractionation of steroid hormones in human serum before quantitative assay by liquid chromatography or radioimmunoassay. Four columns were connected to construct the aqueous liquid--liquid chromatography--fractionation system. The first was coated with neutral water for distribution of serum, the second was weakly alkaline with sodium hydrogen carbonate for extrusion of strong acidic components, and the third was strongly alkaline with sodium hydroxide to capture oestrogens. The final column was acidic with sulphuric acid to remove basic components. Optimization of the stepwise gradient solvents was achieved on the basis of the results of a linear relationship between the logarithms of the capacity ratios and solvent composition determined from an analytical run. Neutral steroid hormones added to serum were eluted from the column system by a stepwise gradient elution technique to obtain first very non-polar materials, then progesterone and testosterone, and finally to extract the corticosteroids. Phenolic oestrogens were recovered from the strong alkaline column with a mobile phase solvent after the pH of the stationary phase had been adjusted with a phase transfer neutralizer. The fractional constituents were purified and enriched. This procedure was used to determine Solu-medrol, an acidic corticosteroid drug, in human serum.
D002853:Chromatography, Liquid; D003345:Corticosterone / Q000097:blood; D004958:Estradiol / Q000097:blood; D004970:Estrone / Q000097:blood; D006801:Humans; D006854:Hydrocortisone / Q000097:blood; D007202:Indicators and Reagents; D008776:Methylprednisolone Hemisuccinate / Q000097:blood; D011374:Progesterone / Q000097:blood; D011863:Radioimmunoassay; D013256:Steroids / Q000097:blood*; D013739:Testosterone / Q000097:blood
D007202:Indicators and Reagents; D013256:Steroids; D004970:Estrone; D013739:Testosterone; D011374:Progesterone; D004958:Estradiol; D008776:Methylprednisolone Hemisuccinate; D003345:Corticosterone; D006854:Hydrocortisone