Keto 2,4-dinitrophenylhydrazones

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. 

Dinitrophenylhydrazones of carbonyl compounds may be formed as described for keto steroids (Section 4.1.1.4.1) by precipitation or extraction from aqueous perchlorate as described by Neuberg et al. [65], by the method of Houben-Weyl [66] or by the method of Shriner et al. [67] using sulfuric acid—water-ethanol as the reaction medium. 

Menkes (1959) reported data collected from six more children. All showed symptoms similar to those described above, and died within 15 days to 20 months of birth. In one case, Menkes was able to obtain urine samples during the last months of the infant s life. When he treated the urine with 2,4-dinitrophenylhydrazone, which forms colored precipitates with keto compounds, he found three a-keto acids in unusually large amounts ... 

Dinitrophenylhydrazones (DNPHs) were applied to the GC analysis of keto acids. As with carbonyl compounds, they are prepared by reaction with 2,4-dinitrophenylhydrazine and are also used mainly for the preliminary isolation of keto acids. They can be isolated from a dilute aqueous sample by adsorption on activated carbon and selective desorption [178] hydrazones of aldehydes with a methyl formate-dichloromethane mixture and hydrazones of keto acids with a pyridine-water azeotropic mixture. Hydrazones of acids are released from their pyridine salts with methanol containing hydrogen chloride. After... 

The 2,4-dinitrophenylhydrazone derivatives of a-ketoisovaleric acid and a-keto-jS-methylvaleric acid are separated from 2,4-dinitrophenylhydrazine by chromatography at room temperature on a Zorbax Cjg column (4.6 mm x 250 mm). Solvent A was 25% acetonitrile in water containing 0.1% triethyla-mine (v/v) and adjusted to pH 4.5 with acetic acid. Solvent B was acetonitrile. A linear gradient from 20 to 50% B was made within 20 minutes. The effluent was monitored at 254 nm. 

It also has been used as a spray reagent and for quantitation in paper and thin layer chromatography Section 6.5). It should be noted that A - 4-3-keto steroids react much less readily with isonicotinic acid hydrazide than do A4-3-keto steroidsThe 2,4-dinitrophenylhydrazone has also been described4. 

Carbonyl compounds (after ref. 5]. McMurry58 desired to selectively reduce an olefinic double bond with diborane in the presence of a keto group. This was successfully achieved by conversion to the dinitrophenylhydrazone, hydroboration and removal of the protective group by ozonolysis in ethyl acetate at — 78°. Oximes and oxime acetates are reduced by BH3. Attempted protection by ketalization in this case was unsuccessful because of simultaneous migration of the double bond. 

Other workers separate by paper chromatography the amino acids obtained by catalytic hydrogenolysis of the 2,4-dinitrophenylhydrazones of the a-keto acids (H19, K19, T24). 

N6. Neish, W. J. P., On the separation of u-keto acid dinitrophenylhydrazones by ionophoresis and their polarographic estimation. Rec. trav. chim. 72, 105 (1953). 

T2. Tauber, H., Separation of a-keto acid dinitrophenylhydrazones by paper electrophoresis and their colorimetric determination. Anal, Chem. 27, 287 (1955). 

Neubauer B) and Knoop (Ba) demonstrated in the early part of this century the metabolic interconversion of a-amino and a-keto acids. In 1920, Thunberg (S) observed that glutamate was oxidized in the presence of frog muscle, and, in 1936, Weil-Malherbe (4) prepared an extract from brain tissue which catalyzed glutamate oxidation and identified the product, a-ketoglutarate, as its 2,4-dinitrophenylhydrazone. 

The oxidation product has been isolated as its 2,4-dinitrophenylhydrazone and corresponds with -keto-6-aminovaleric acid. Putrescine, arginine, Manske s 1 (+) acetylornithine (118) and other related amino acids are oxidized much less readily if at all. The same authors have extracted an ornithine dehydrogenase from the young roots of Datura tatula. It requires the cooperation of a coenzyme not yet identified, and appears unable to oxidize putrescine and amino acids other than ornithine and to a lesser extent glutamic. Either of these systems, or the two linked into a H-transfer chain, would seem able to catalyze the oxidation of ornithine in the living tissues. No carbon dioxide was released from ornithine by the poly-phenolase system but on addition of an unwashed belladonna tissue-suspension carbon dioxide was liberated, presumably by decarboxylation of the a-keto-5-aminovaleric acid formed by the oxidation. 

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-... 

Apart from citrus oils, other essential oils have also been analyzed by means of LC, such as the blackcurrant bud essential oil [100]. The latter was fractionated into hydrocarbons and oxygenated compounds, and the two fractions were submitted for RP-HPLC analysis. Volatile carbonyls consist of some of the most important compounds for the blackcurrant avor and, hence, were analyzed in detail. The carbonyls were converted into 2,4-dinitrophenylhydrazones and the mixture of 2,4-dini-trophenylhydrazones was separated into derivatives of keto acids and monocarbonyl and dicarbonyl compounds. Each fraction was submitted to chromatographic investigation. 

For the determination of keto-acids, e.g. pyruvic, a-ketoglutaric and oxalacetic acids in mixtures and in biological materials, a method has been proposed employing paper electrophoretic separations of the 2,4-dinitrophenylhydrazones of these acids.< ) After elution, the polarographic wave corresponding to the reduction of the nitro-groups (in the 2,4-dinitrophenylhydrazone formed) was followed in 0-1 N HCl and the first wave at —0T4 V measured. 

The IR (1690 cm ) and UV (257 nm) spectra, and the formation of a 2,4-dinitrophenylhydrazone supported the presence of an a,p-unsaturated carbonyl group in tomentellin (581). Hydrogenation of (581) which gave methyl 3-methoxy-4-hydroxybenzoate and tetrahydroderivative, and analysis of the H-NMR and mass spectra [m/z 181 +H (65%) and 151 (100%)] led to the conclusion that tomentellin was 3,7-dimethyl-5-keto-2,6-octadienyl 3,4-dimethoxybenzoate (581). The position of the carbonyl group was further confirmed by the bathochromic shift of the UV absorption band to 257 nm after addition of aq. NaOH. The geometry of the enone system was presumed to be s-cis from the observation that the IR absorption band of the double bond was more intense than that of a carbonyl group. 

Kallio, H. and Linko, R.R. (1973), Gas-liquid chromatographic analysis of 2,4-dinitrophenylhydrazones of keto acid methyl esters. J. Chromatogr., 76,229. 

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