Note

Extraction of steroid diconjugates using amberlite XAD-2 resin

This chapter discusses the newer approaches to the isolation, identification, and quantitation of steroids in biological materials. Progress in steroid analysis has been remarkable in the last 15 years. Much of present knowledge in steroid endocrinology could not have been acquired without the development of radioimmunoassay and other competitive protein-binding methods. These methods have provided the sensitivity and speed required for measurement of physiological concentrations of steroid hormones in large series of samples. Parallel with this development, both liquid and gas-liquid chromatographic methods have been markedly improved and mass spectrometers have become available for routine use. The most important advancement in this area has been the successful combination of gas chromatography and mass spectrometry (GC-MS) for analysis of molecules as large as steroids. Immunoassay and GC-MS are complementary methods. GC-MS is important as a reference method to test and validate the specificity of an immunoassay. The steroid composition of biological samples taken under different physiological and pathological conditions can be characterized by GC-MS, and immunoassays may then be developed for detailed studies of selected steroids in larger numbers of samples.

A simplified, flexible method for the analysis of metabolic profiles of steroids in urine is described. Solid extraction with Amberlite XAD-2 or Sep-Pak C_{1 g} cartridges is followed by group fractionation of unconjugated neutral and phenolic steroids, monoglucuronides, monosulphates and disulphates on the lipophilic strong anion exchanger triethylaminohydroxyproply Sephadex LH-20 (TEAP-LH-20). Following brief enzymatic hydrolysis or solvolysis the steroids are purified on TEAP-LH-20. O-Methyloxime and trimethylsilyl ether derivatives are prepared and purified by filtration through Lipidex 5000, and are then analyzed by glass capillary column gas—liquid chromatography and gas chromatography—mass spectrometry.

Between 2 and 5 ml of urine are used for a comprehensive analysis. Unconjugated neutral and phenolic steroids are isolated in half a day, corresponding steroids in the conjugate fractions in two days. No fraction containing steroids is discarded, but the analysis can be limited to a selected fraction.

Amberlite XAD-4 was shown to adsorb p-nitrophenyl glucuronide more strongly than XAD-2 does. A 10-g XAD-4 column quantitatively retained over 100 μg of p-nitrophenyl glucuronide at flow-rates up to 6 ml/min. Acetone-water (1:1) was the most suitable solvent system for elution of the glucuronides and sulfates tested on XAD-4.

A routine procedure is described for the dependable detection of various anabolic residues in tissues or urine contaminated at levels as low as 0.5–10ppb (10 parts per 10⁹). A suitable extraction and clean-up procedure was developed. permitting adequate recovery (60–80%) of various anabolics from tissue samples (50 g) or urine (50 ml). Follwong two-dimensional thin-layer chromatography, the presence of the anabolic residues are detected by sulphuric acid—induced fluorescence. The detection limit of most anabolics is of the order 1–10 ng.

[³H]androsterone was administered intraperitoneally into male Wistar rats with biliary fistulas. The major portion of the radioactivity was recovered in the bile within 24 hr. There was marked diversity in androsterone metabolism. Half of the rats excreted large amounts of steroid monoglucuronides rapidly into bile, whereas the remaining rats eliminated various steroid conjugates at a much slower rate. In both groups, androsterone was metabolized to 2- and 16-oxygenated C₁₉O₃ and C₁₉O₄ steroids and probably poly oxygenated steroids. In contrast, the injected [³H]androsterone glucuronide was recovered rapidly and quantitatively in the bile. These results indicate the extensive metabolism of androsterone prior to glucuronidation and probably the existence of variations of UDP-glucuronyltransferase toward the oxygenated metabolites of androsterone.

Coarse-grade XAD-2 resin has been micronized (medium particle size 12 μm) and used as a support for preparative high-resolution liquid chromatography. By equilibration with single-phase mixtures of water, a lower aliphatic alcohol and a hydrophobic solvent a reversed-phase partition system is formed in situ. This chromatographic technique is characterized by a large capacity, a high power of resolution and a high degree of selectivity and reproducibility, and versatile application in the isolation of polar and non-polar drug metabolites or naturally occurring compounds from complex mixtures is possible. The examples given relate particularly to the separation of water-soluble, non-extractable compounds, such as glucuronides or other conjugates, and amino acids on large-diameter columns (up to 2.5 cm).