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Conditions for reproducible packing of fused-silica liquid chromatography capillary columns were demonstrated. The plate height vs. velocity curves were determined for successively prepared columns. In addition, the values of separation impedance were calculated. Several liquid chromatography systems were evaluated in conjunction with the slurry-packed capillaries for the retention and peak area reproducibility.

An enzymatic detection method is described for urinary conjugated steroids after chromatographic fractionation with Sephadex G-25. The principle of the method is as follows. Part of a 24-h urine sample, (1-2 ml of urine) is applied directly, to a short column of Sephadex G-25 and eluted with acetate buffer solution. Steroid conjugates in each fraction are hydrolyzed with steroid sulfatase--beta-glucuronidase. After enzymatic hydrolysis, an enzymatic color development reagent for steroids, either 3 alpha-hydroxysteroid dehydrogenase or 3 beta-hydroxysteroid oxidase, are added and the dye formed is measured spectrophotometrically. Excretion patterns of steroid-3 beta-sulfates, and steroid-3 alpha-glucuronides and steroid-3 alpha-sulfates ae shown with some patients' samples. A precision of the assay values for steroid-3 alpha-glucuronide, steroid-3 alpha-sulfate and steroid-3 beta-sulfates in urine samples and assay values for normal subjects are also studied. This simple enzymatic method for detecting the excretion patterns of urinary conjugated steroids may have a diagnostic value for clinical tests.

The combination of lipophilic gel chromatography and high-resolution glass capillary chromatography has been shown to be an important means of identification and quantification of steroid urinary metabolites. Some problems in the use of capillary columns, such as coating with polar and apolar stationary phases, column performance, thermal stability and injection systems have been investigated and this type of chromatography has been used in the analysis of 33 dihydroxysteroids. Gel chromatography is a method complementary to high-efficiency gas chromatography. The steroids are eluted according to the number of hydroxyl groups in the molecules.

Recently developed analytical procedures for the qualitative and quantitative analysis of human urine for major and minor steroid metabolites are described. Steroid profile samples were obtained by enzymic hydrolysis with beta-glucuronidase and sulfatase. Methoxime-trimethylsilyl ether derivatives of steroid metabolites were prepared; the recommended procedure converts all ketone groups (except the 11-one group) into methoxime groups and all hydroxyl groups into trimethylsily ether groups. These derivatives are thermally stable, readily volatilized, not subject to dehydration or adsorption on gas chromatographic columns, and suitable for both quanlitative and quantitative analytical studies. Thermostable glass open tubular capillary columns, coated with the non-polar phase SE-30, and containing dispersed particles of silanized silicic acid, were used for the gas chromatographic separation. Illustrations of profiles for normal female and male subjects, and patients with a testosterone-secreting ovarian tumor, congenital adrenal insufficiency and a dehydroepiandrosterone-secreting adrenal tumor are included.

The fractional assay is described of 3 beta-hydroxysteroids in various patients by thin-layer chromatography and color development using an enzyme that reacts specifically with some 3 beta-hydroxysteroids. Together with dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol and 16 alpha-hydroxydehydroepiandrosterone can be detected, but their concentrations differ with each disease. An unknown fraction, a more polar 3 beta-hydroxysteroid than 16 alpha-hydroxydehydroepiandrosterone, is also detected in moderate amounts in patients with adrenal tumor (18.3 mg/day), 21-hydroxylase deficiency (3.2 to 1.2 mg/day), and Cushing's syndrome (0.9-2.3 mg/day, as pregn-5-ene-triol).