Determination of 4-hydroxy-2-nonenal in serum by high-performance liquid chromatography with fluorescence detection after pre-column derivatization using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2, 1,3-benzoxadiazole.

4-Hydroxy-2-nonenal (4HNE) is a major aldehyde generated during lipid peroxidation. The clinical monitoring of 4HNE in biological fluids should be useful for the early diagnosis of several diseases involving lipid peroxidation, such as rheumatoid arthritis, Parkinson's disease and cancer. In this study, an HPLC with fluorescence detection method was developed for the determination of 4HNE in human serum. The proposed method involves the extraction of 4HNE from human serum by sub-zero temperature extraction and fluorescent labeling of 4HNE with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2, 1,3-benzoxadiazole. The lower detection limit (signal-to-noise ratio = 3) of the method was 0.06 µm in serum. The proposed method was successfully applied to the measurement of 4HNE in sera obtained from patients with rheumatoid arthritis.

Determination of 4-hydroxy-2-nonenal in serum by high-performance liquid chromatography with fluorescence detection after pre-column derivatization using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole.

4-Hydroxy-2-nonenal (4HNE) is a major aldehyde generated during lipid peroxidation. The clinical monitoring of 4HNE in biological fluids could be useful for the early diagnosis of several diseases involving lipid peroxidation, such as rheumatoid arthritis, Parkinson's disease and cancer. In this study, an HPLC with fluorescence detection method was developed for the determination of 4HNE in human serum. The proposed method involves the extraction of 4HNE from human serum by subzero temperature extraction and fluorescent labeling of 4HNE with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole. The lower detection limit (signal-to-noise ratio=3) of the method was 0.06 µm in serum. The proposed method was successfully applied to the measurement of 4HNE in sera obtained from patients with rheumatoid arthritis.

Chromatographic determination of low-molecular mass unsaturated aliphatic aldehydes with peroxyoxalate chemiluminescence detection after fluorescence labeling with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole.

A highly sensitive, selective and reproducible chromatographic method is described for determination of low-molecular mass unsaturated aliphatic aldehydes in human serum. The method combines fluorescent labeling using 4-(N,N-Dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole with peroxyoxalate chemiluminescence. The derivatives were separated on a reversed-phase column C8 isocratically using a mixture of acetonitrile and 90mM imidazole-HNO3 buffer (pH 6.4, 1:1, % v/v). The calibration ranges were: 20-420nM for methylglyoxal, 16-320nM for acrolein, 15-360nM for crotonaldehyde and 20-320nM for trans-2-hexenal. The detection limits were ranged from 4.4 to 6.5nM (88-130fmol/injection), the recovery results were within the range of 87.4-103.8% and the intra and inter-day precision results were lower than 5.5%. The proposed validated method has been successfully applied to healthy, diabetic and rheumatic arthritis patients' sera with simple pretreatment method. In conclusion, this new method is suitable for routine analysis of large numbers of clinical samples for assessment of the oxidative stress state in patients.

A novel lophine-based fluorescence probe and its binding to human serum albumin.

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510nm. The binding constant and binding number determined by Scatchard plot was 3.65×10(6)M(-1) and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.

Characterization and comparison of methacrylic acid with 2-acrylamido-2-methyl-1-propanesulfonic acid in the preparation of monolithic column for capillary electrochromatography.

Butyl methacrylate (BMA)-ethylene dimethacrylate (EDMA)-methacrylic acid (MAA) and BMA-EDMA-2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monolithic columns were prepared by varying the percentage of ionic monomers for capillary electrochromatography. Monolithic columns with a higher content of ionic monomers provided better column efficiency, and the performance of BMA-EDMA-MAA monoliths was better than BMA-EDMA-AMPS. To characterize and optimize BMA-EDMA-MAA monoliths, the effects of the content of cross-linker and the total monomer in the polymerization mixture on column performance were also studied. Plate heights of 8.2 µm for the unretained solute (thiourea) and 12.6 µm for the retained solute (naphthalene) were achieved with a monolithic column using 2.5% MAA (Column I).