DNPH method HPLC analysis

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

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. 

Methods for the collection and determination of formaldehyde in water show great similarity to those methods for air described above. The methods of Tomkins et al. (1989) and EPA (1992b) for formaldehyde in drinking water and the method of Facchini et al. (1990) for formaldehyde in fog water all rely on the formation of the DNPH derivative followed by HPLC. The method of Dong and Dasgupta (1987) relies on the reaction of formaldehyde in atmospheric water with a diketone (2,4-pentanedione) and ammonimn acetate to form a ftuorescent derivative that is measured spectrophotometrically in a flow injection analysis system. 

Formaldehyde and other aldehydes are receiving increasing attention both as toxic substances and as promoters in the photochemical formation of ozone in the atmosphere. They are released into residential buildings from plywood and particle board, insulation, combustion appliances, tobacco smoke, and various consumer products. Aldehydes are released into the atmosphere in the exhaust of motor vehicles and other equipment in which hydrocarbon fuels are incompletely burned. A sensitive method for analyzing aldehydes and ketones is based on the sorption of these compounds to an SPE sorbent and their subsequent reaction with 2,4-dinitrophenylhydrazine (DNPH) on the sorbent. They are then analyzed as their hydrazones by HPLC (Fig. 7.9). A gradient analysis by HPLC may separate as many as 17 components with detection by ultraviolet (UV) light. 

There are many HPLC methods for the determination of aldehydes in ambient air. They differ in sampling technique, elution and chromatographic separation (Tuss et al., 1982). 2.4-Dinitrophenylhydrazine DNPH (LIS, 1989 VDI 3862, Part 2, 1990 Selim, 1977 Druzik et al., 1990 Lipari and Swarin, 1982 Slemr, 1991) is now widely used for derivatization because this reagent is also suitable for the simultaneous analysis of long-chain aldehydes, aromatic aldehydes and ketones. In acidic solution, DNP-hydra-zone derivatives are formed from DNPH and aldehydes or ketones as shown in Eq. (6). The reaction proceeds with nucleophilic addition to the carbonyl group followed by elimination of water. 

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