Derivatization With 2,4-dinitrophenylhydrazine Research Articles

Derivatization of β‐dicarbonyl compound with 2,4‐dinitrophenylhydrazine to enhance mass spectrometric detection: application in quantitative analysis of houttuynin in human plasma

Houttuynin (decanoyl acetaldehyde), a beta-dicarbonyl compound, is the major antibacterial constituent in the volatile oil of Houttuynina cordata Thunb. In the present work, detection of houttuynin in human plasma based on the chemical derivatization with 2,4-dinitrophenylhydrazine (DNPH) coupled with liquid chromatography/tandem mass spectrometry was described. The primary reaction products between the beta-dicarbonyl compound and DNPH in aqueous phase were identified as heterocyclic structures, of which the mass spectrometric ionization and fragmentation behavior were characterized with the aid of high-resolution multistage mass spectral analysis. For quantification, houttuynin and internal standard (IS, benzophenone) in plasma were firstly converted to their DNPH derivatives without sample purification, then extracted from human plasma with n-hexane and detected by liquid chromatography tandem mass spectrometry performed in selected reaction monitoring (SRM) mode. This method allowed for a lower limit of quantification (LLOQ) of 1.0 ng/ml using 100-microl plasma. The validation results showed high accuracy (%bias < 2.1) and precision (%CV < 7.2) at broad linear dynamic range (1.0-5000 ng/ml). The simple and quantitative derivatization coupled with tandem mass spectrometric analysis facilitates a sensitive and robust method for the determination of plasma houttuynin in pharmacokinetic studies.

Determination of trace amounts of formaldehyde in acetone

A method to quantify sub-ppm levels of formaldehyde in acetone has been developed and it is reported here. In this method, the different reactivities and stabilities of sulfite with formaldehyde and acetone are used to separate the two carbonyl compounds. Sulfite reacts with formaldehyde to form hydroxymethanesulfonate (HMS), the non-volatile and stable nature of which allows its separation from bulk acetone solvent. The resulting HMS is then converted back to formaldehyde under basic conditions, and formaldehyde is derivatized with 2,4-dinitrophenylhydrazine (DNPH) and quantified in its DNP hydrazone form using high-performance liquid chromatography-UV detection. The method detection limit at the 99% confidence level was 0.051 mg L −1. A batch of samples can be processed within 4 h. The method has been applied to quantify the amount of formaldehyde in an analytical-grade acetone and in a commercial nail polish remover and the level of formaldehyde was found to be 0.175 and 0.184 mg L −1, respectively.

Determination of carbonyl compounds in the atmosphere by DNPH derivatization and LC–ESI-MS/MS detection

A method of determination of 32 carbonyl compounds by high performance liquid chromatography (HPLC) and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) after derivatization with 2,4-dinitrophenylhydrazine (DNPH) was developed and successfully applied to the atmosphere sample of a residential area of Liwan District (S1) and a research institute of Tianhe District (S2) in Guangzhou, China. Some operation conditions of ESI-MS/MS in the negative ion mode including selection of parent and daughter ions, declustering potential (DP), entrance potential (EP), collision energy (CE), collision cell exit potential (CXP) and effect of buffer in ESI-MS/MS process were optimized. The regression coefficient of the calibration curves ( R 2), recovery, reproducibility (R.S.D., n = 5) and limit of detection (LOD) were in the range of 0.9938–0.9999, 90–104%, 1.7–11% and 0.4–9.4 ng/m 3, respectively. Among most of the samples, acetone was the most abundant carbonyl in two sampling sites and formaldehyde, acetaldehyde and butyraldehyde/2-butanone were also abundant carbonyls. In contrast to LC–UV method, the LOD, the separation of some co-eluting compounds and the precision (mainly to higher molecular weight carbonyls) are all improved by LC–ESI-MS/MS.

Low Acetaldehyde Collection Efficiencies for 24-Hour Sampling with 2,4-Dinitrophenylhydrazine (DNPH)-Coated Solid Sorbents

Airborne aldehyde and ketone (carbonyl) sampling methodologies based on derivatization with 2,4-dinitrophenylhydrazine (DNPH)-coated solid sorbents could unequivocally be considered the "gold" standard. Originally developed in the late 1970s, these methods have been extensively evaluated and developed up to the present day. However, these methods have been inadequately evaluated for the long-term (i.e., 24 h or greater) sampling collection efficiency (CE) of carbonyls other than formaldehyde. The current body of literature fails to demonstrate that DNPH-coated solid sorbent sampling methods have acceptable CEs for the long-term sampling of carbonyls other than formaldehyde. Despite this, such methods are widely used to report the concentrations of multiple carbonyls from long-term sampling, assuming approximately 100% CEs. Laboratory experiments were conducted in this study to evaluate the long-term formaldehyde and acetaldehyde sampling CEs for several commonly used DNPH-coated solid sorbents. Results from sampling known concentrations of formaldehyde and acetaldehyde generated in a dynamic atmosphere generation system demonstrate that the 24-hour formaldehyde sampling CEs ranged from 83 to 133%, confirming the findings made in previous studies. However, the 24-hour acetaldehyde sampling CEs ranged from 1 to 62%. Attempts to increase the acetaldehyde CEs by adding acid to the samples post sampling were unsuccessful. These results indicate that assuming approximately 100% CEs for 24-hour acetaldehyde sampling, as commonly done with DNPH-coated solid sorbent methods, would substantially under estimate acetaldehyde concentrations.

Determination of Aldehydes and Ketones in Fuel Ethanol by High-Performance Liquid Chromatography with Electrochemical Detection

A new methodology was developed for analysis of aldehydes and ketones in fuel ethanol by high-performance liquid chromatography (HPLC) coupled to electrochemical detection. The electrochemical oxidation of 5-hydroxymethylfurfural, 2-furfuraldehyde, butyraldehyde, acetone and methyl ethyl ketone derivatized with 2,4-dinitrophenylhydrazine (DNPH) at glassy carbon electrode present a well defined wave at +0.94 V; +0.99 V; +1.29 V; +1.15 V and +1.18 V, respectively which are the basis for its determination on electrochemical detector. The carbonyl compounds derivatized were separated by a reverse-phase column under isocratic conditions with a mobile phase containing a binary mixture of methanol / LiClO4(aq) at a concentration of 1.0 × 10−3 mol L−1 (80:20 v/v) and a flow-rate of 1.1mL min−1 . The optimum potential for the electrochemical detection of aldehydes-DNPH and ketones-DNPH was +1.0 V vs. Ag/AgCl. The analytical curve of aldehydes-DNPH and ketones-DNPH presented linearity over the range 5.0 to 400.0 ng mL−1, with detection limits of 1.7 to 2.0 ng mL−1 and quantification limits from 5.0 to 6.2 ng mL−1, using injection volume of 20 μL. The proposed methodology was simple, low time-consuming (15 min/analysis) and presented analytical recovery higher than 95%.

Separation and Determination of Carbonyl Compounds in Indoor Air Using Two-Step Gradient Capillary Electrochromatography

A method of two-step gradient capillary electrochromatography (CEC) was developed to measure 12 carbonyls (aldehydes and ketones) in indoor air samples. The carbonyls were derivatized with 2,4-dinitrophenylhydrazine (DNPH) and then separated by a two-step gradient CEC on a C8 column. Effects of various instrumental conditions on the separation, including buffer concentration, organic modifiers, voltage, and cassette temperature, were investigated. The method detection limits for the 12 carbonyls ranged from 0.2 microg to 1.6 microg per sample and the recoveries were generally between 90 and 120%. A subset of 30 indoor air samples containing formaldehyde and acetaldehyde from 75 randomly selected homes in the city of Ottawa, Canada were measured using the CEC method. The concentrations of formaldehyde and acetaldehyde in these indoor air samples ranged from 5.8 microg/m3 to 85 microg/m3, and from 4.4 microg/m3 to 38 microg/m3, respectively. The comparison between CEC and the traditional HPLC method shows a good agreement in measured values.

Determination of aldehydes and ketones using derivatization with 2,4-dinitrophenylhydrazine and liquid chromatography–atmospheric pressure photoionization-mass spectrometry

Atmospheric pressure photoionization-mass spectrometry (APPI-MS) is used for the analysis of aldehydes and ketones after derivatization with 2,4-dinitrophenylhydrazine (DNPH) and liquid chromatographic separation. In the negative ion mode, the [ M − H] − pseudomolecular ions are most abundant for the carbonyls. Compared with the established atmospheric pressure chemical ionization (APCI)-MS, limits of detection are typically lower using similar conditions. Automobile exhaust and cigarette exhaust samples were analyzed with APPI-MS and APCI-MS in combination with an ion trap mass analyzer. Due to improved limits of detection, more of the less abundant long-chain carbonyls are detected with APPI-MS in real samples. While 2,4-dinitrophenylazide, a known reaction product of DNPH with nitrogen dioxide, is detected in APCI-MS due to dissociative electron capture, it is not observed at all in APPI-MS.

Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography

Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 °C for 30 min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.

Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography

Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 °C for 30 min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.

Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma.

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition.

Automated method for determination of glutardialdehyde residues in flexible endoscopes after disinfection

Glutardialdehyde (GDA) is the most commonly used disinfectant for flexible endoscopes. After inappropriate rinsing of endoscopes residual GDA in the narrow endoscope channels may lead to toxic effects in patients. Common methods for determination of aldehydes in water involve derivatization with 2,4-dinitrophenylhydrazine (DNPH), liquid–liquid or solid-phase extraction and HPLC determination. Since derivatization and extraction is both time-consuming and labor-intensive only a small number of samples can be measured. Thus, we developed a fully automated method which includes a conventional HPLC system, a programmable autosampler, and UV detection. After GDA derivatization using DNPH the samples remain in the aqueous phase and no preconcentration of the analyte is necessary. The samples are automatically derivatized through the autosampler. While derivatization in one sample takes place the previous sample is injected and measured by HPLC. Our method is well suited for screening residual GDA in endoscopes as it is both time- and labor-saving.

Determination of patterns of biologically relevant aldehydes in exhaled breath condensate of healthy subjects by liquid chromatography/atmospheric chemical ionization tandem mass spectrometry.

A method for the simultaneous determination of several classes of aldehydes in exhaled breath condensate (EBC) was developed using liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). EBC is a biological matrix obtained by a relatively new, simple and noninvasive technique and provides an indirect assessment of pulmonary status. The measurement of aldehydes in EBC represents a biomarker of the effect of oxidative stress caused by smoke, disease, or strong oxidants like ozone. Malondialdehyde (MDA), acrolein, alpha,beta-unsaturated hydroxylated aldehydes [namely 4-hydroxyhexenal (4-HHE) and 4-hydroxynonenal (4-HNE)], and saturated aldehydes (n-hexanal, n-heptanal and n-nonanal) were measured in EBC after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Atmospheric pressure chemical ionization of the analytes was obtained in positive-ion mode for MDA, and in negative-ion mode for acrolein, 4-HHE, 4-HNE, and saturated aldehydes. DNPH derivatives were separated on a C18 column using variable proportions of 20 mM aqueous acetic acid and methanol. Linearity was established over 4-5 orders of magnitude and limits of detection were in the 0.3-1.0 nM range. Intra-day and inter-day precision were in the 1.3-9.9% range for all the compounds. MDA, acrolein and n-alkanals were detectable in all EBC samples, whereas the highly reactive 4-HHE and 4-HNE were found in only a few samples. Statistically significant higher concentrations of MDA, acrolein and n-hexanal were found in EBC from smokers.

Proteomic analysis of protein oxidation in Alzheimer's disease brain.

There is a growing body of evidence that oxidative stress plays a major role in Alzheimer's disease (AD) pathogenesis. Identification of oxidatively altered proteins in AD is important for understanding the relationship between protein oxidation, protein aggregation and neurodegeneration. In this communication, we report a method that can be applied to study oxidative changes of individual proteins in brain. In order to analyze protein oxidation by detection of protein-bound carbonyls, cytosolic protein extracts were derivatized with 2,4-dinitrophenylhydrazine (DNPH) and then separated by two-dimensional (2-D) gel electrophoresis. After electrotransfer to polyvinylidene difluoride (PVDF) membranes, proteins were first stained with Sypro Ruby protein stain, and then the oxidized proteins were detected with anti-dinitrophenyl (DNP) antibody. About 150 proteins and more than 100 oxidized proteins were detected and quantified in both AD and control cases by 2-D image analysis. The amount of protein-bound carbonyls was decreased for six and increased for one protein in AD. The amount of protein was increased for three proteins in AD. Furthermore, the degree of oxidation was calculated as the ratio of protein-bound carbonyls to the total amount of an individual protein. Two proteins showed a significant decrease in the degree of oxidation in AD. Our results suggest that the balance of protein oxidation and degradation is altered in AD.

Cadmium causes the oxidative modification of proteins in pea plants

AbstractIn pea (Pisum sativum L.) leaves from plants grown in the presence of 50 µm CdCl2 the oxidative production of carbonyl groups in proteins, the rate of protein degradation and the proteolytic activity were investigated. In leaf extracts the content of carbonyl groups measured by derivatization with 2,4‐dinitrophenylhydrazine (DNPH), was two‐fold higher in plants treated with Cd than in control plants. The identification of oxidized proteins was carried out by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of proteins derivatized with DNPH and immunochemical detection with an antibody against DNPH. The intensity of the reactive bands was higher in plants exposed to Cd than in controls. By using different antibodies some of the oxidized proteins were identified as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase. The incubation of leaf crude extracts with increasing H2O2 concentrations showed a progressive enhancement in carbonyl content and the pattern of oxidized proteins was similar to that found in Cd‐treated plants. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment. In peroxisomes purified from pea leaves a rise in the carbonyl content similar to that obtained in crude extracts from Cd‐treated plants was observed, but the functionality of the peroxisomal membrane was not apparently affected by Cd. Results obtained demonstrate the participation of both oxidative stress, probably mediated by H2O2, and proteolytic degradation in the mechanism of Cd toxicity in leaves of pea plants, and they appear to be involved in the Cd‐induced senescence previously reported in these plants.

Determination of free formaldehyde in foundry resins as its 2,4-dinitrophenylhydrazone by liquid chromatography

Formaldehyde is a toxic component that is present in foundry resins. Its quantification is important to the characterisation of the resin (kind and degradation) as well as for the evaluation of free contaminants present in wastes generated by the foundry industry. The complexity of the matrices considered suggests the need for separative techniques. The method developed for the identification and quantification of formaldehyde in foundry resins is based on the determination of free carbonyl compounds by derivatization with 2,4-dinitrophenylhydrazine (DNPH), being adapted to the considered matrices using liquid chromatography (LC) with UV detection. Formaldehyde determinations in several foundry resins gave precise results. Mean recovery and R.S.D. were, respectively, >95 and 5%. Analyses by the hydroxylamine reference method gave comparable results. Results showed that hydroxylamine reference method is applicable just for a specific kind of resin, while the developed method has good performance for all studied resins.

Ozone-induced oxidation of Rubisco: from an ELISA quantification of carbonyls to putative pathways leading to oxidizing mechanisms.

In an attempt to detect a possible relationship between protein oxidation and ozone (O3) atmospheric concentration, we used a sensitive enzyme-linked immunosorbent assay (ELISA) method for measuring carbonyl formation in amino acid residues that constitute Rubisco (EC 4.1.1.39) small subunit (Rubisco-SSU). Using open-top chamber technology, bean plants (Phaseolus vulgaris L. cv. Bergamo) were exposed for 21 d (from emergence) to four different atmospheres characterized by average daylight O3 concentrations of 12, 70, 89 and 109nL L-1. Rubisco-SSU extracted from primary leaves was fixed specifically on wells coated with anti-SSU antibodies. Aldehydes and ketones, previously derivatized with 2,4-dinitrophenylhydrazine (DNP), were quantified with anti-DNP antibodies conjugated with alkaline phosphatase. A significant positive O3 effect on carbonyl formation was detected, and the number of carbonyls was found to be linearly increased (r2=0.82) when plotted against increasing external O3 dose expressed as accumulated exposure over a threshold of 40 nL L-1 h (AOT40). Furthermore, these O3-induced oxidative modifications were connected with a significant reduction in the amount of native SSU that linearly decreased (r2=0.95) as AOT40 increased from 0 to about 8295 nL L-1 h. A possible pathway leading to oxidation of Rubisco is proposed, with special reference to O3 reactivity.

Determination of 1-aminopropan-2-one, a dissolved sewage component, in water samples

A new method for the determination of 1-aminopropan-2-one (APR) in water samples was developed. APR was synthesised as its hydrochloride and derivatized with 2,4-dinitrophenylhydrazine (DNPH) for determination by high-pressure liquid chromatography with ultraviolet detection (UV-HPLC). APR was determined in water samples at pH 12 using a gas-stripping chamber, connected to a cartridge containing DNPH. Acidified water samples were injected into the gas-stripping chamber and a solution of NaOH added to bring the solution to pH 12. APR was volatilised and stripped from solution and passed onto the cartridge under a constant stream of nitrogen gas. Gas flow rates were carefully controlled to allow maximum contact of APR with DNPH on the cartridge. When the reaction time had elapsed, the cartridge was disconnected and the derivative eluted with a fixed volume of acetonitrile and injected onto the HPLC, where the APR hydrazone was resolved isocratically with a mobile phase consisting of acetonitrile and water (60 : 40). The HPLC was calibrated using standard solutions of the APR hydrazone. Recoveries of APR from standard samples were 90–100% at the 10 μM level and the detection limit for the method was calculated as 18 nM. Detection of APR in urine and primary-treated sewage samples (41 nM and 1.225 μM, respectively) confirmed the applicability of the technique to analysis of environmental samples.

Poly(allylamine) beads as selective sorbent for preconcentration of formaldehyde and acetaldehyde in high-performance liquid chromatographic analysis

Formaldehyde and acetaldehyde in water were determined by preconcentration with poly(allylamine) beads, derivatization with 2,4-dinitrophenylhydrazine (DPH) and analysis by HPLC. Poly(allylamine) beads (0.5 g) were used to adsorb formaldehyde and acetaldehyde at 1.2–150 μg l −1 and 3.5–220 μg l −1 from water (1 l). The concentration factor is 50 fold. The aldehydes were eluted and derivatized with 2 m M DPH in 0.5 M H 2SO 4 (10 ml). The time of analysis was 1 h. The detection limits ( S/N=3) for formaldehyde and acetaldehyde were 0.6 and 2 μg 1 −1, respectively.

Liquid chromatography with ultraviolet detection of lasalocid, monensin, salinomycin and narasin in poultry feeds using pre-column derivatization

A rapid and very effective analytical procedure for the simultaneous determination of four polyether antibiotics (PEs) lasalocid, monensin, salinomycin and narasin in poultry feeds was tested. PEs were extracted from samples using methanol and without any clean-up derivatized with 2,4-dinitrophenylhydrazine (DNP) in an acidic medium at 55°C. The derivatization mixture was analyzed directly on an ODS column (150×4.6 mm, 5μm) with methanol–1.5% aqueous acetic acid (90:10, v/v) as eluent and UV detection was carried out at 305/392 nm. The recoveries of the PEs from spiked samples were 85–100 % with RSDs of 4–10 % in a concentration range of 50–150 mg/kg.

Deamination of methylamine and aminoacetone increases aldehydes and oxidative stress in rats

Semicarbazide-sensitive amine oxidase (SSAO)-mediated deamination of methylamine and aminoacetone in vitro produces carbonyl compounds, such as formaldehyde and methylglyoxal, which have been proposed to be cytotoxic and may be responsible for some pathological conditions. An HPLC procedure was developed to assess different aldehydes, which were derivatized with 2,4-dinitrophenylhydrazine (DNPH). We have demonstrated in vivo deamination of methylamine and aminoacetone by examining the excretion of formaldehyde and methylglyoxal, respectively, in rats. Following chronic administration of methylamine, the urinary level of malondialdehyde (MDA), an end product of lipid peroxidation, was also found to be substantially increased. A selective SSAO inhibitor blocked the increase of MDA. The results support the idea that increased SSAO-mediated deamination of methylamine and aminoacetone can be a potential cytotoxic risk factor.