Chromatography as a reference technique for the determination of clinically important steroids

Abstract

Chromatographic methods (paper chromatography, thin layer chromatography, high performance liquid chromatography, gas chromatography and gas chromatography-mass spectrometry) for the determination of clinically important steroids in biological specimens are reviewed. The emphasis is on the use of gas chromatography, gas chromatography-mass spectrometry and high performance liquid chromatography as reference rather than routine techniques. Chromatographic methods are compared with colorimetric, fluorimetric and radioimmunoassay procedures in terms of simplicity of operation, cost and ability to analyse large numbers of specimens. The importance of correct specimen collection and storage are discussed. Sample preparation techniques for the various analytical methods are described. These include extraction of free and conjugated steroids from serum, plasma, urine and saliva by solvent partition, with polymer-based resins such as Amberlite XAD-2, DEAE-Sephadex and Sephadex resins bonded with various other function groups and, more recently, with chemically bonded reversed-phase silicas.

Stability studies of testosterone and epitestosterone glucuronides in urine

“…The use of mass spectrometry for estimating the T/E ratio in urine samples is mandatory in doping control 15, 17, 19, 31. To reduce the imprecision of the GC/MS assay, a sample stored under reference conditions (−80°C) analyzed at the same time as the test conditions samples has been chosen to obtain the best information on actual analyte (in)stability 21…”

“…For this reason, urine for stability testing was sterilized by filtration and stored in sterile tubes. In addition, sodium azide was added as a preservative to prevent bacterial degradation of steroids and hydrolysis of steroid conjugates 15, 34. The sterility of the urine sample was monitored by using previously described methods 20, 28, 29…”

“…Detection of doping with exogenous T has been a matter of concern for years 15–18. Estimating the T/E ratio is a critical aspect of controlling the misuse of T. This has led to discussions on the optimal method for estimating the T/E ratio, and means for ensuring that results are consistent between laboratories.…”

Testostérone libre ou biodisponible : dosages ou calculs. Comparaison critique de différents modes d'approche

“…Des procédures de référence particulièrement dédiées aux stéroïdes plasmatiques ont été publiées [27,35,39,50] et utilisées pour la validation des techniques RIA au cours des 25 dernières années. L'une d'entre elles [27] est particulière-ment adaptée au problème de l'exactitude dans les basses concentrations.…”

“…Ces niveaux de valeurs devraient pouvoir être mesurés avec une imprécision inférieure à 5 % et être bien corrélés particulièrement dans ces domaines de mesure avec les techniques de référence [27,35,39,50].…”