Androstanes detection

An enzyme reactor with immobilized 3 -hydroxysteroid dehydrogenase has been successfully used for the analysis of residues of 17 -methyltestosterone in trout by high-performance liquid chromatography (HPLC) (269). Following their separation by reversed-phase chromatography, the major tissue metabolites of 17 -methyltestosterone, namely 5 -androstane-17 -methyl-3, 17 -diol, and 5 -androstane-17 -methyl-3, 17 -diol, were enzymatically modified in the presence of a coreactant, nicotinamide-adenine dinucleotide (NAD), to the corresponding ketone. The position at 3 was enzymatically oxidized, and NADH, the reduced form of NAD, was produced as a coproduct and subjected to fluorescence detection. Reoxidation of NADH to NAD provides the possibility for electrochemical detection. 

Fig. 10.15. Gradient elution CEC separation of steroid hormones. Conditions capillary, 9.6 (17.6) cm x 50 pm i.d. packed with 6 pm Zorbax ODS mobile phase, A, acetonitrile-10 raM borate, pH 8 (65 35 v/v) B, acetonitrile-10 mM borate, pH 8 (85 15 v/v), 0-100% B in 5 min, 100% B for 3 min at 0.1 ml/min 14 kV 25°C detection, UV at 205 nm injection, 1 kV/0.5 s. Peak identification 1, formamide 2, corticosterone 3, testosterone 4, androsten-3,17-dione 5, androstan-3,17-dione 6, pregnan-3,20-dione. Reproduced with permission from Huber et al. [68]. Copyright 1997 American Chemical Society.

The degradation products of androgenous steroids are a further suspected cause of such febrile episodes, since severe fever could be induced by i.m. injection of aetiocholanolone (3a-hydroxy-5p-H-androstane-17-on) (A. Kappas et af, 1957). In 1958 P. K. Bondy et al. for the first time detected an increase in aetiocholanolone in the plasma of patients with unexplained fever episodes, and thus they coined the term aetiocholanolone fever. Other steroids of the 5p-H configuration also revealed this pyrogenic feature. Aetiocholanolone may be the product of disturbed hepatocellular metabolism. Further observations and examinations have meanwhile been reported. (51, 136)... 

Henbest reduction (see Chloroiridic acid). Substitution of chloroiridic acid by tris(triphenylphosphine)chlororhodium results in even greater stereospecificity thus 5/3-androstane-3,17-dione is reduced to the 3/3-alcohol and 3 -alcohol in the ratio 50 1, with no detectable reduction of the 17-keto group. The reaction is preferably run in a sealed tube t 82°. 2-Ketosteroids are reduced entirely to the axial 2/3-alcohol (Henbest s conditions give 1% of the 2a-alcohol).1... 

Many of the organic contaminants which were found in Lippe river water were also present in the source samples (see Table 3). The sewage effluent sample and the Seseke river showed the best accordance with the compound spectrum of the Lippe river. However, also in the two tributaries from the rural upper reaches of the river, numerous specific contaminants like 9-methylacridine (No. 8), alkyl phosphates (Nos. 31, 32) and chlorinated alkyl phosphates (Nos. 34, 36) appeared. In the effluent of a pharmaceutical plant, only a few Lippe river contaminants like n-alkanes (No. 1), naphthalene (No. 3), TXIB (No. 21) and caffeine (No. 67) were detected (see Table 3). Therein, mainly structural relatives of androstanone like 3p-hydroxy-5p-androstan-17-one, 3a-hydroxy-5p-androstan-17-one and androstan-50-3,17-dione were present. These compounds are probably by-products of the synthesis of hormone preparations. Some polycyclic aromatic compounds, halogenated compounds and terpenoids were not detected in the source samples (see the underlined compounds in Table 3) and probably have another origin. Representative sampling of various input sources have to be carried out to prove the origin of these compounds. Hexachlorobutadiene (No. 38) and bis(chloropropyl)ethers (No. 44) appear exclusively at the lower reaches of the Lippe river (see Table 1), downstream the chemical plants in Marl. They are attributed to inputs of the chlorochemical industry (see section 3.1). Hence, this suggests their input by an industrial point source. 

Several groups are included in this class of steroids including corticosteroids, pregnanes, androstanes and oestranes. Extensive reviews on the chromatography of these compounds have been previously published (Heftman, 1983 Heftman and Lin, 1982 Purdy et al, 1982 O Hare and Nice, 1982 Schmidt, 1982). It is not the purpose of this section to provide a comprehensive review of the Uterature but to provide the potential chromatographer with a number of alternative strategies for the separation and detection of the various steroid hormones. For ease of reference the individual classes of steroid hormones have been separated into individual sub-sections. 

The most convenient method of detection of the androstanes is to utilise their characteristic UV absorbance. The d -3-ketones (including androstenedione and testosterone) have a strong absorption at 254 nm such that as little as 30 ng of testosterone can be detected. The... 

Androstane is converted in glacial acetic acid (CH3CO2H/CH2CI2 = 3 2, i = 40mAcm", T = 20°C, Pt electrode) to 17% androstanyl acetate (19) and 38% androstane is recovered. Remarkable is the selectivity of the oxidation It was found that the 6-, 7- and 12-acetates are formed in the ratio 35 1 2.5, and no other acetates could be detected. Cholestane affords 15% cholestanyl acetates the 6-, 7- and 12-acetates are formed in the ratio 40 1 2. 

Bjorkhem, 1., and Ek, H. (1983) Detection and quantitation of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major urinary metabolite of methenolone acetate (Primobolan) by isotope dilution—Mass spectrometry. Journal of Steroid Biochemistry, 18,481-487. 

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