Steroids DNPH deriv

The analysis of keto steroids as their 2,4-dinitrophenylhydrazone (DNPH) derivatives by TLC [30] and HPLC [31,32] is a sensitive and reliable method for the determination of these compounds in urine and in other biological fluids. The derivatives are easily separated by TLC or HPLC and can be detected in quantities as low as 1 ng. Several variations of the reaction procedure may be used. Two of these are described below. 

The use of benzoylation for the formation of UV-absorbing derivatives of non-UV absorbing hydroxy steroids has been reported for the analysis of these compounds by liquid chromatography [33]. This reaction may prove useful for the separation of steroids which cannot be separated as their DNPH derivatives. Molar absorptivities of many steroid benzoates are normally greater than 100001/mole cm at 230 nm. 

Steroidal ketones have been separated as their 2,4-DNPH derivatives [138, 1391. The neutral I7-keto steroids are of clinical interest in certain disease states the urinary 17-keto steroid level in women is indicative of adrenal activity, and that in men is a composite measure of adrenal and testicular function. Samples of urine and blood hydrolysates can be derivatized with 2,4-DNPH and analyzed by HPLC. In the work of Fitzpatrick et al. 

Sevanian et al. (1994) applied GLC and LC/TS/MS for the analysis of plasma cholesterol-7-hydroperoxides and 7-ketocholesterol. Analysis of human and rabbit plasma identified the commonly occurring oxidation products, yet dramatic increases in 7-ketocholesterol and cholesterol-5p, 6P-epoxide were observed. The study failed to reveal the presence of choles-terol-7-hydroperoxides, which were either too unstable for isolation, metabolized or further decomposed. The principal ions of cholesterol oxides monitored by LC/TS/MS were m/z 438 (cholestane triol) m/z 401 (cholesterol-7-hydroperoxide) m/z 401 (7-ketocholesterol) m/z 367 (7a-hydroxycholesterol) m/z 399 (cholesta-3,5-dien-7-one) and m/z 385 (choles-terol-5a,6a-epoxide). The major ions were supported by minor ions consistent with the steroid structure. Kamido et al. (1992a, b) synthesized the cholesteryl 5-oxovaleroyl and 9-oxononanoyl esters as stable secondary oxidation products of cholesteryl arachidonate and linoleate, respectively. These compounds were identified as the 3,5-dinitrophenylhydrazone (DNPH) derivatives by reversed-phase LC/NICI/MS. These standards were used to identify cholesteryl and 7-ketocholesteryl 5-oxovaleroyl and 9-oxononanoyl esters as major components of the cholesteryl ester core aldehydes generated by copper-catalysed peroxidation of low-density lipoprotein (LDL). In addition to 9-oxoalkanoate (major product), minor amounts of the 8, 9, 10, 11 and 12 oxo-alkanoates were also identified among the peroxidation products of cholesteryl linoleate. Peroxidation of cholesteryl arachidonate yielded the 4, 6, 7, 8, 9 and 10 oxo-alkanoates of cholesterol as minor products. The oxysterols resulting from the peroxidation of the steroid ring were mainly 7-keto, 7a-hydroxy and 7P-... 

Chromatography. DNPH-steroid derivatives may be separated on plates of silica gel G (layer thickness, 0.25 nm) with chloroform—carbon tetrachloride (2 1) or chloroform-dioxane (47 3) [30]. The spots may be quantitated directly by spectrodensitometry in situ at 367 nm. Amounts as low as 20-40 ng per spot can be detected. A chromatogram scan of 2.5 jug each of dehydroepiandrosterone, etiocholanolone and androsterone separated with chloroform—dioxane (47 3) is shown in Fig. 4.12. 

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