DNPH method

A number of intercomparison studies of the various methods of measurement of HCHO have been carried out. As might be expected given the specificity of spectroscopic methods, the results of FTIR, DOAS, and TDLS are generally in good agreement, within 15% of their mean value in one study in a polluted atmosphere (Lawson et al., 1990). During the same study, the diacetyldihydrolutidine derivative method and the DNPH method were lower by 15-25% than the spectroscopic mean, whereas the enzymatic method was higher by 25%. 

In another intercomparison using TDLS, several DNPH methods, the 1,3-cyclohexanedione diffusion scrubber, and the enzymatic method were compared using both spiked samples and ambient air. The TDLS was used as a standard for comparison. For ambient air measurements, results obtained with the 1,3-cyclohexanedione diffusion scrubber were about 30% higher than those obtained with TDLS, whereas results for the enzymatic method were about 35% lower. The DNPH cartridge measurements were quite variable, which may... 

Multifunctional acids containing a carbonyl group such as pyruvic acid [CH3C(0)C00H] are typically measured using the derivatization techniques used for aldehydes and ketones, such as the DNPH method (e.g., see Lee et al., 1995). 

The fact that DHA possesses vitamin C activity was recognized early in studies of AA and was studied by several groups (13,14), Enzymes that catalyze the reduction of DHA to AA have been demonstrated in many systems and are discussed later in this chapter. Dimeric DHA, or bisdehydroascorbic acid (BDHA), and DHA both are important in nutrition this importance has been taken for granted in that the common assay of ascorbic acid, the dinitrophenylhydrazine (DNPH) method, does not distinguish between these forms. 

A diflFerent method for determination of RAA, DHA, and DKG, based on the work of Roe et al. (27), has been used by some investigators (12,13,28). Total AA is determined by the DNPH method previously described, where the RAA is oxidized with Norit or bromine to DHA. DHA and DKG react with the DNPH to form the red osazones. To determine DHA and DKG, the extract is not oxidized, but is reacted directly with the DNPH. The RAA is determined by difference. Total DKG is measured by reducing the DHA in the extract to RAA, prior to osazone formation. In cases where it is suspected that a relatively large proportion of the RAA has been oxidized, either to DHA or DKG, as in the case of frozen fruits and vegetables (12,29), the differential assay may be of use. However, for routine assays, the DCIP or micro-fluorometric assays, or a combination of the two will yield adequate information. 

Table 1.2-2. HPLC-paramctcrs for the separation and deteetion of aldehydes and ketones by use of the DNPH-method.

K perchlorate, etc) form easily detonatable expls. Colver (Ref 5, 291) describes preparative methods and gives mp s of some of the additive compds. For example, 2,4-DNPh (mp 112-14°) combines with aniline to form an additive compd of mp 82° [See also GerP 72945 (1893) 73205... 

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). 

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). 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

Carbonyl compounds for instance can be trapped by absorption in a reagent solution containing 2, 4-dinitrophenylhydrazine and hydrogen chloride. Details of this method are extensively described elsewhere (8). The principle of the method is that the carbonyl compounds, in case of rendering plant emission the aldehydes, react with the 2,4-dinitrophenylhydrazine and form 2,4-dinitrophenylhydrazones (2,4-DNPH s) according to the scheme. 

HPLC methods with fluorescence detection have also been developed for the determination of nitro-policyclic aromatic hydrocarbons (PAHs) (among which 9-nitroanthracene and 1-nitronaph-thalene) [238] in atmosphere. Samples have been collected in a standard high-volume sampler with a Teflon-coated glass fiber filter, and the Soxhlet extraction was performed with dichloromethane as the solvent. RP HPLC/UV techniques are used for the determination of aldehydes, ketones, and carbonylic compounds after derivatization with DNPH [239],... 

In addition to the spectroscopic methods, there are a number of derivatization methods, in which a derivative of the carbonyl compound that can be easily separated and measured is formed. The most common of these is the use of 2,4-dinitrophenylhydrazine (DNPH), which reacts to form the hydrazone ... 

In order to express the absorbances obtained by this method in terms of moles of carbonyl, the spectral absorbances of DNPH derivatives of several purified carbonyl compounds were measured. At 430 nm, molar extinction coefficients of 16,000 and 21,350 were determined for the saturated and unsaturated carbonyl compounds, respectively, whereas at 460 nm, the values were 12,450 and 28,100, respectively. Based on the present methodology and use of 1-cm cuvettes, the above equations were derived (Henick et al., 1954). 

Describes a simple and sensitive spectrophotometric method to estimate the content of total carbonyl compounds in rancid fats and foods by trapping them with 2,4-DNPH the technique determines total carbonyls, including those that are nonvolatile, decreasing the ability of the assay to correlate well with sensory data. Although gas chromatographic techniques are better suited for determining volatile carbonyl compounds from lipid oxidation, this is still the classical colorimetric assay. 

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. 

Alternatively, acidified DNPH coated silica gel or florisil use as adsorbent derivative analyzed by HPLC (U.S. EPA Method TO 11) recommended flow rate 500 mL/min sample volume 100 L. 

Trace acetone in water may be determined by a fast HPLC method (Takami et al., 1985) aqueous sample passed through a cartridge packed with a moderately sulfonated cation-exchange resin charged with 2,4-dinitrophenylhydrazine (DNPH) DNPH derivative eluted with acetonitrile and analyzed by HPLC with a 3-mm ODS column. 

Air drawn through a midget impinger containing 10 mL of 2 N I ICl/0.5% DNPH and 10 mL isooctane the stable DNPH derivative formed partitions into isooctane layer, isooctane layer separated aqueous layer further extracted with 10 mL 70/30 hexane/methylene chloride the latter combined with isooctane the combined organic layer evaporated under a steam of N2 residue dissolved in methanol the DNPH derivative determined by reversed phase HPLC using UV detector at 370 nm (U.S. EPA Method TO-5, 1988). 

Alternatively, a measured volume of air drawn through a prepacked cartridge coated with acidified DNPH the DNPH derivative of acetone eluted with acetonitrile and measured by HPLC-UV as above (U.S. EPA Method TO-11, 1988). 

Air drawn through a cartridge containing silica gel coated with acidified DNPH derivative eluted with acetonitrile and determined using isocratic reverse phase HPLC with UV detection at 360 nm (U.S. EPA 1988, Method TOll) recommended flow rate 1 L/min sample volume 100 L. 

Zwiener, C., T. Glauner, and F.H. Frimmel. 2002. Method optimization for the determination of carbonyl compounds in disinfected water by DNPH derivatization and LC-ESI-MS-MS. Anal. Bioanal. Chem. 372 615-621. 

Colorometric procedures involving reaction of aldehydes with hydrazines, semicarbazide, or piperidine/nitroprusside solutions are also non-specific and lack sensitivity (15, 35, 36). Schmidt et al. (33) have proposed an HPLC method for analyzing the 2,4-dinitrophenylhydrazone (DNPH) derivatives of specific aldehydes. This procedure allows for a number of ddehydes to be separated and measured simultaneously, however, HPLC methods in general suffer from poor resolving power and may have low sensitivity (37). In addition, hydrazine derivatizations are often performed under acidic conditions for maximal reactivity these conditions would not provide quantitative information on total aldehyde content. 

---