Dinitrophenylhydrazone derivative

The metabolism of NPYR is summarized in Figure 1. a-Hy-droxylation (2 or 5.position) leads to the unstable intermediates and decomposition of gives 4-hydroxybutyraldehyde [ ]. The latter, which exists predominantly as the cyclic hemiacetal 1, has been detected as a hepatic microsomal metabolite in rats, hamsters, and humans and from lung microsomes in rats (9-13). The role of 1 and as intermediates in the formation of 6 and 7 is supported by studies of the hydrolysis of 2-acetoxyNPYR and 4-(N-carbethoxy-N-nitrosamino)butanal, which both gave high yields of 7 (9,14). In microsomal incubations, can be readily quantified as its 2,4-dinitrophenylhydrazone derivative (15). The latter has also been detected in the urine of rats treated with NPYR ( ). 

Unfortunately, there are no universal methods to detect all types of protein oxidation, because the products formed can be so diverse in nature. However, some forms of protein oxidation can be assayed using chemical modification (Davies et al., 1999 Shacter, 2000). In particular, the formation of carbonyl groups on proteins can be targeted using the reagent 2,4-dinitrophenyl-hydrazine (DNPH). This compound reacts with aldehydes to form 2,4-dinitrophenylhydrazone derivatives, which create chromogenic modifications that can be detected at high sensitivity in microplate assays or Western blot analysis (Buss et al., 1997 Winterbourn et al., 1999). 

Cyclobutanecarboxaldehyde has been prepared in very low yield by the Rosenmund reduction procedure.6 A 46% yield of the 2,4-dinitrophenylhydrazone derivative has also been reported, with the aldehyde formed as an intermediate, in the reaction of the acid chloride and lithium tri-Mmtoxyaluminum hydride at —78° in diglyme.7... 

Fraction 1 was found to have the same retention time as that of authentic cyclohexanone, and the infrared spectrum showed a strong absorption band at 1720 cm."1 (C=0). The 2,4-dinitrophenylhydrazone derivative was prepared, and a mixed melting point with an authentic sample was not depressed, m.p., 156°-157°C. [literature value 160.2°C. 

Derivatization is a procedure in which analyte is chemically modified to make it easier to detect or separate. For example, formaldehyde and other aldehydes and ketones in air, breath, or cigarette smoke25 can be trapped and derivatized by passing air through a tiny cartridge containing 0.35 g of silica coated with 0.3 wt% 2,4-dinitrophenylhydrazine. Carbonyls are converted into the 2,4-dinitrophenylhydrazone derivative, which is eluted with 5 mL of acetonitrile and analyzed by HPLC. The products are readily detected by their strong ultraviolet absorbance near 360 nm. 

The reaction of an aldehyde or a ketone with 2,4-dinitrophenylhydrazine produces a 2,4-dinitrophenylhydrazone derivative. This reaction is also used as a test for the presence of an aldehyde or ketone. A drop or two of a suspected aldehyde or ketone is added to a solution of 2,4-dinitrophenylhydrazine in ethanol and water. The formation of a precipitate, usually orange or red, of the derivative indicates that the unknown is an aldehyde or ketone ... 

Analysis of aldehydic products of lipid peroxidation as 2,4-dinitrophenylhydrazone derivatives In order to enhance the detectability of saturated aliphatic carbonyls... 

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. 

Although alcaptonuria is a relatively harmless condition, such is not the ca.se with other errors in amino acid metabolism. In maple syrup urine dis-eim, the oxidative decarboxylation of cy-kctoacids derived from valine, isoleucinc, and leucine is blocked because the branched-chain dehydrogenase is missing or defective. Hence, the levels of these cx-ketoacids and the branched-chain amino acids that give rise to them are markedly elevated in both blood and urine. The urine of patients has the odor of maple syrup-hence the name of the disease (also called branched-chain ketoaciduria). Maple syrup urine disease usually leads to mental and physical retardation ss the patient is placed on a diet low in valine, isoleucinc, and leucine early in life. The disease can be readily detected in newborns by screening urine samples with 2,4-dinitrophenylhydrazine, which reacts with a-ketoacids to form 2,4-dinitrophenylhydrazone derivatives, A definitive sis can be made by mass spectrometry. 

FIGURE 16.4 High-pressure liquid chromatogram of a mixture of the 2,4-dinitrophenylhydrazone derivatives of a standard carbonyl mixture. 

Detection systems for GC are chosen for their sensitivity and selectivity for a particular class of VOCs. Detectors for GC include FID, the BCD, the photoionization detector (PID), the pulsed discharge detector (PDD), and the reduction gas detector (RGD). A variety of mass spectrometers can also be interfaced with a GC for confirmation of molecular structure and quantitation. Singlewavelength ultraviolet-visible detectors (190 to 600 nm) and diode array detectors are used to detect carbonyls as their 2,4-dinitrophenylhydrazone derivatives. The absorption maxima for aliphatic carbonyls, aromatic carbonyls, and dicarbonyls are near 360 nm, 385 to 390 nm, and 415 to 430 nm, respectively. Formic, acetic, and pyruvic acid are detected by ion conductivity. 

Bell et al. (243) used the formation of the 2,4-dinitrophenylhydrazone derivative of formaldehyde in SSS as the means to quantitate the level of formaldehyde in SSS. 

Seligman, R.B. and M.D. Edmonds Chromatography of 2,4-dinitrophenylhydrazone derivatives 9th Tobacco Chemists Research Conference, Program Booklet and Abstracts, Vol. 9, Paper No. 8, 1955, pp. 4—5. 

Kolliker, S. and Oehme, M., Structure elucidation of 2,4-dinitrophenylhydrazone derivatives of carbonyl compounds in ambient air by HPLC/MS and multiple MS/MS using atmospheric chemical ionization in the negative ion mode. Anal. Chem., 70, 1979, 1998. 

Two new alkaloids of the flindersine group, zanthophylline (13 R = Me) and desmethylzanthophylline (13 R = H), were obtained from Zanthoxylum mono-phyllum by chromatography of the chloroform extract of the plant. The structure of zanthophylline was indicated by and n.m.r. spectroscopy and by hydrolysis to 8-methoxyflindersine (2 R = OMe) and formaldehyde (identified as its 2,4-dinitrophenylhydrazone derivative). When aqueous acid was used in the isolation procedure, the only alkaloid obtained was 8-methoxyflindersine, and this is apparently an artefact formed by hydrolysis of zanthophylline. Desmethylzanthophylline is only a minor constituent of Z. monophyllum although the alkaloid has not been fully investigated, its mass spectrum is in accord with structure (13 R = H), and the presence of N-acetoxymethyl and hydroxy-groups is confirmed by the appropriate i.r. absorptions. 

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