The analysis of urinary steroids using gas-liquid chromatography

A method is described using gas-liquid chromatography for the routine determination of urinary C₁₉O₂ and C₁₉O₃-17-Ketosteroids, pregnanediol, and pregnanetriol in the urine of normal subjects. Following hydrolysis, the extract is applied to thin-layer silica gel and developed in a system which allows separation of steroid metabolites into 3 zones. The steroids in each zone are measured using conditions that provide optimal separation from other metabolites and impurities. Pregnanediol is analyzed as its diacetate after preliminary thin-layer chromatography. These methods have high accuracy, speed and precision, and may be used in normal as well as abnormal states.     

Measurement of urinary testosterone by gas-liquid chromatography

A method has been developed which employs gas-liquid chromatography for the assay of testosterone from extracts of human urine prepared according to an earlier method described by Camacho & Migeon in 1963. The trimethyl silyl ethers of testosterone and an internal standard, cistestosterone, were separated on the nitrile silicone rubber stationary phase XE-60. This method has given results which correspond satisfactorily with those obtained with UV spectrophotometry or measurement of sulfuric acid chromogens after paper chromatography.     

Quantitative analysis of carbohydrates using gas-liquid chromatography

Sugars can be analyzed quantitatively as their trimethylsilyl derivatives using gas-liquid chromatography. Standardization of polyethylene glycol succinate columns with α-methylgalactopyranoside as the standard gave a quantitative correlation between GLC relative peak areas and relative molar compositions of sugar standard solutions for: the pentose, ribose; the deoxyhexose, fucose; and the hexoses, glucose, galactose, and mannose. Quantitative data were also obtained for N-acetylamino sugars. Mixtures of sugars were analyzed by using the standardization graphs, and the amounts determined were accurate within 10%. This use of GLC will make it easier for the biochemist to attack problems in carbohydrate chemistry by enabling quantitative work to be done readily on mixtures heretofore difficult to analyze.     

Thin-layer chromatography of urinary 17-oxosteroids using dansylhydrazine as a prelabelling reagent

This paper describes a modification of a high-performance liquid chromatographic method for measurement of 17-oxosteroids in biological fluids for use with thin-layer chromatography and fluorometric scanning detection. After extraction from urine samples with Separon-C₁₈ microcolumns, free oxosteroids were labelled with dansylhydrazine in acetonitrile-acetic acid and chromatographed on silica gel F-254 plates with the solvent system chloroform—methanol (97:3). Linearity of fluorescence detection (Shimadzu CS-9000 densitometer) was obtained between 30 and 1000 ng.     

Semi-automated fluorimetric method for the estimation of urinary catecholamines using high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of adrenaline and noradranaline in urine is described, using fluorescence detection. The effluent from the liquid chromatograph is led directly into an analyser to produce the fluorescent trihydroxyindoles from the catecholamines. The method is more reliable and specific than conventional fluorescence techniques. Both catecholamines can be detected at levels of 0.5 ng on the column.     

A modified method for the estimation of pregnanediol, pregnanetriol and seven common 17-ketosteroids in urine by gas-liquid chromatography

A method is described for simultaneous determination of seven common urinary 17-ketosteroids, pregnanediol (5β-pregnane- 3α, 20α-diol) and pregnanetriol (5β-pregnane-3α,17α,20α-triol) by gas-liquid chromatography. The essential steps include hydrolysis of conjugates by an extract from $\text{Helix}$$\text{pomatia}$, use of 5β-cholestan-3α-ol as an internal standard, silylation and gas chromatography using 3% XE-60 as a stationary phase. The chromatograms were clear and the peaks easily identifiable. Laborious purification procedures were not necessary. 5β-pregnane-3α,20α-diol and 5β-pregnane-3α, 17α,20α-triol were measured in 22 adult males and 17 adult females. The subjects were trained hospital personnel, healthy and without medications. The results, in general, were in agreement with those reported by others.     

The determination of urinary testosterone using thin-layer chromatography and gas chromatography

A new method has been devised for the relatively rapid and sensitive determination of testosterone in human urine. One fourth of a 24 hour urine sample was incubated with β-glucuronidase after addition of a tracer dose of 1,2–3H-testosterone for the calculation of recovery and extracted with ethyl ether. The crude extract was subjected to thin-layer chromatography (TLC), eluted, acetylated, subjected to TLC again and analyzed by gas chromatography. This method detected as little as 5 μg of testosterone per 24 hour and the rate of recovery was 54.8±0.4% (Mean±S.E.). The value for normal adult men ranged from 41 to 200 μg/day, showing definite decline with advancing age. While the urinary testosterone exhibits a very low level (5>-8 μg/day) in normal adult women, hirsute women excreted almost as much testosterone as normal adult men.     

Simultaneous estimation of pregnenolone, 17-hydroxypregnenolone and dehydroepiandrosterone by gas-liquid radiochromatography

Mixtures of the 5-ene-3β-hydroxysteroids pregnenolone, 17-hydroxypregnenolone and DNA have been simultaneously separated and quantitated by gas-liquid radiochromatography. Over 98% of the injected radioactivity was accounted for using an improved trapping technique. The identity of the recovered steroids was confirmed by mass spectrometry.The analytical method has been applied successfully to the analysis of the in vitro metabolism of pregnenolone by the microsomes of the human adrenal gland.