O-phthalaldehyde Research Articles

Simultaneous determination of o-phthalaldehyde,p-chloro-mxylenol and triclosan in compound chemical disinfectants and daily chemicals by micellar electrokinetic chromatography

A novel method for the separation and determination of o-phthalaldehyde (OPA), p-chloro-m-xylenol (PCMX) and triclosan in daily chemicals and compound chemical disinfectants in a single run by micellar electrokinetic chromatography (MEKC) was established. The factors such as the buffer concentration and pH, the concentration of sodium dodecyl sulfate (SDS), and the sample buffer, were investigated in detail. The analysis was carried out using a 50 microm uncoated capillary of 40.2 cm in total length (effective length: 30 cm). The running buffer was 20 mmol/L Na2B4O7, and 80 mmol/L SDS. The sample buffer was 2 mmol/L Na2B4O7-8 mmol/L SDS (without pH adjustment) containing 10% (v/v) methanol. The detection wavelength was 214 nm. The relative standard deviations (RSDs) of the corrected peak areas of the three components were in the range of 1.1% - 3.8%, and the RSDs of migration times were less than 0.9%. The limits of detection (LODs, S/N = 3) were 4.0, 0.4 and 0.4 mg/L, and the limits of quantification (LOQs, S/N = 10) were 12, 1.2, and 1.2 mg/L for OPA, PCMX and triclosan, respectively. The corrected peak areas and the concentrations of the three components showed good linear relationship within the ranges of 12 - 2 000 mg/L, 1.2 - 200 mg/L and 1.2 - 200 mg/L with the correlation coefficients of 0.999 4, 0.999 3 and 0.999 5 for OPA, PCMX and triclosan, respectively. The method was used for the determination of the three components in compound chemical disinfectants, hand washing liquids, soaps and a toothpaste. The results showed that the three components could be assayed in a single run with simple sample pretreatment, rapidity, accuracy and low cost, and the method is convenient for routine analysis.

Rapid Determination of Neomycin Content in Duck Egg White Using Derivative Synchronous Fluorescence-Wavelet-Subsection Genetic Algorithm-Least Squares Support Vector Regression

Derivate of neomycin and o-phthalaldehyde(OPA) has strong fluorescent characteristic under the condition of mercaptoethanol,and it can realize fluorimetric determination of neomycin content in duck egg white.In the process,synchronous fluorescence spectra from 280 nm to 390 nm were analyzed using three-dimensional synchronous fluorescence spectrometry,and the wavelength difference of 110 nm was selected to detect neomycin content in duck egg white.Then,the db10 wavelet with two levels decomposition was used to reduce the noise of first order derivative synchronous spectrum,and 14 characteristic wavelengths were obtained using subsection genetic algorithm(SGA).Lastly,least squares support vector regression(LSSVR) was applied to build the prediction model of determination of neomycin content in duck egg white,and the determination coefficient(R2) and the root mean squared error of prediction(RMSEP) were 0.9671 and 1.713 respectively.This work proved that SGA could extract effectively characteristic wavelengths of neomycin in duck egg white,and it was helpful to improve the prediction accuracy of LSSVR model.The method could achieve the rapid prediction of neomycin content in duck egg white.

Mechanistic Insights into the Inhibition of Endo-β 1,4 Xyloglucan Hydrolase by a Classical Aspartic Protease Inhibitor

This is the first report of inactivation of xyloglucanase from Thermomonospora sp by pepstatin A, a specific inhibitor towards aspartic proteases. The steady state kinetics revealed a reversible, competitive, two-step inhibition mechanism with IC 50 and K i values of 3.5 ± 0.5μM and 1.25 ± 0.5μM respectively. The rate constants determined for the isomerization of EI to EI(*) and the dissociation of EI* were 14.5 ± 1.5 × 10(-5) s(-1) and 2.85 ± 1.2 × 10(-8) s(-1) respectively, whereas the overall inhibition constant K i(*) was 27 ± 1nM. The conformational changes induced upon inhibitor binding to xyloglucanase were monitored by fluorescence analysis and the rate constants derived were in agreement with the kinetic data. The abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled xyloglucanase and far UV analysis suggested that pepstatin binds to the active site of the enzyme. Our results revealed that the inactivation of xyloglucanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis.

Comparison of four feed proteases for improvement of nutritive value of poultry feather meal

Feed industries are seeking new ways to cope with increased raw material costs, and one approach is to apply enzymatic treatment in the production of feed ingredients from animal by-products. Keratinases, a group of proteases, are capable of hydrolyzing keratin-rich material and have been applied in the production of cost-effective feather by-products for use as feed and fertilizers. The current study examined 4 commercial feed proteases from Bacillus subtilis, Bacillus licheniformis PWD-1, Aspergillus niger, and Serratia proteamaculans HY-3 used to hydrolyze chicken feather under different conditions. The degree of keratinolysis was monitored by measuring the release of NH(2) groups using o-phthalaldehyde (OPA) and by scanning electron microscopy. All 4 feed proteases were able to degrade feather at pH 5.5 and 7.0. The degree of hydrolysis was stimulated by the addition of reducing reagents such as dithiothreitol (DTT) and Na(2)SO(3). In general, the protease from B. subtilis was more efficient in degrading feather keratin compared to the other 3 feed proteases at both pH 5.5 and 7.0. For commercial production, the application of protease from B. subtilis is even more advantageous considering the lower cost-in-use.

Role of Glutamate Decarboxylase-like Protein 1 (GADL1) in Taurine Biosynthesis

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Simultaneous electrochemical measurement method of histamine and Nτ-methylhistamine by high-performance liquid chromatography–amperometry with o-phthalaldehyde–sodium sulfite derivatization

An electrochemical detection (ECD) method for analyzing sub-micro amounts of histamine (HA) and Nτ-methylhistamine (Nτ-MHA) in biological samples by high-performance liquid chromatography (HPLC)–amperometry has been developed. The method consists of a precolumn derivatization of the amines with o-phthalaldehyde (OPA) and sodium sulfite (Na2SO3) to N-alkyl-1-isoindole sulfonate and posterior separation with the HPLC system. Biological samples were pretreated by using a Vivapure sulfonic acid minifilter in which the reaction of the reagent with the amines took place during filtering. HA and Nτ-MHA retention times were 11.8±0.02 and 18.3±0.03min, respectively (means±standard deviations, n=3). The lowest limit of amperometric detection at a signal-to-noise ratio of 3:1 was 0.125pmol in both cases. HA and Nτ-MHA contents in hypothalamus, cortex, skin, and fundic gland, as well as histamine N-methyltransferase (HMT) activities of mast cell-deficient (Ws/Ws) and Wistar rats, were measured and compared with an HPLC–fluorometry system, among other experiments, in order to validate and demonstrate the usefulness of this assay system. Hence, this consequently confirms not only the sensitivity and specificity of the assay but also the potential and convenience it offers to laboratory work, especially in the analysis of the regulation of histaminergic neurons as well as enzymatic investigation of HA metabolism.

Inhibition of xyloglucanase from an alkalothermophilic Thermomonospora sp. by a peptidic aspartic protease inhibitor from Penicillium sp. VM24

A bifunctional inhibitor from Penicillium sp VM24 causing inactivation of xyloglucanase from Thermomonospora sp and an aspartic protease from Aspergillus saitoi was identified. Steady state kinetics studies of xyloglucanase and the inhibitor revealed an irreversible, non-competitive, two-step inhibition mechanism with IC50 and Ki values of 780 and 500nM respectively. The interaction of o-phthalaldehyde (OPTA)-labeled xyloglucanase with the inhibitor revealed that the inhibitor binds to the active site of the enzyme. Far- and near-UV spectrophotometric analysis suggests that the conformational changes induced in xyloglucanase by the inhibitor may be due to irreversible denaturation of enzyme. The bifunctional inhibitor may have potential as a biocontrol agent for the protection of plants against phytopathogenic fungi.

Levels of biogenic amines in retail market fermented meat products

The qualitative and quantitative profile of biogenic amines (BA) in 50 samples of dry fermented sausages sold in Greek retail markets were determined by HPLC. Putrescine, cadaverine, tryptamine, β-phenylethylamine spermidine, spermine were analysed by UV detection after pre-column derivatization with benzoyl chloride, whereas tyramine and histamine were analysed by fluorescence detection after post-column derivatization with o-phthaldialdehyde (OPA). With the exception of spermidine and spermine a wide variation of BA levels was observed among the samples. Of the BA examined, tyramine, putrescine, histamine and cadaverine showed high concentrations ranging from: 0 to 510mg/kg (median: 197.7mg/kg), 0 to 505mg/kg (median: 96.5mg/kg), 0 to 515mg/kg (median: 7.0mg/kg) and 0 to 690mg/kg (median: 3.6mg/kg), respectively. The histamine content of 28% of the samples exceeded the toxicity limit of 100mg/kg set for histamine in some fish species. Levels of tryptamine and β-phenyl-ethylamine never exceeded 50 and 29mg/kg, respectively. Results of the present study suggest that the amounts of BA in dry fermented sausages, sold in Greek retail markets, may pose a potential health risk for sensitive individuals or for those undergoing classical monoamine oxidase inhibiting (MAOI) drug therapy.

Free amino acids and biogenic amines in Alicante Monastrell wines

The simultaneous determination of 17 free amino acids and 8 biogenic amines in Alicante Monastrell wines was investigated for the first time. The quantification was carried out by using a RP-HPLC method, based on a pre-column derivatization with o-phthaldialdehyde (OPA) and fluorescence detection. From the results obtained it may be concluded that the most abundant free amino acids were Glu, Arg, Ala Asp, and Lys. None of the wine samples analysed had histamine (HIM) or Tyramine (TYM) levels above the limits considered as a possible toxic risk for healthy individuals. No measurable amounts of cadaverine (CAD) or methylamine (MEA) were found, showing no spoilage symptoms of sensory properties of the wines. Tryptamine (TRM) content was significantly higher in aged wines compared to young wines. However ethanolamine (ETA) content was lower. These data were used to make a preliminary classification of the samples using cluster analysis.

Vortex-assisted liquid–liquid microextraction coupled with derivatization for the fluorometric determination of aliphatic amines

A new one-step derivatization and microextraction technique was developed for the fluorometric determination of C1–C8 linear aliphatic primary amines in complex sample solutions containing high levels of amino acids. In this method, amines were derivatized with o-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) in aqueous solution and extracted simultaneously by vortex-assisted liquid–liquid microextraction (VALLME). Parameters affecting the extraction efficiency were investigated in detail. The optimum conditions were as follows: 50μL of isooctane as the extractant phase; 2.0mL aqueous donor samples with 12mM OPA, 24mM 2-ME, and 0.1M borate buffer at pH 10; 1min vortex extraction time; centrifugation for 4min at 6000rpm. After centrifugation, the enriched analytes in the floated extractant phase were determined by HPLC–FL in less than 14min. Under the optimum conditions, the limits of detection were of the order of 0.09–0.31nM. The calibration curves showed good linearity over the investigated concentration range between 0.4 and 40nM. The proposed method has been applied to the determination of aliphatic amines in acidophilus milk, beer, and Cu(II)/amino acid solution.

Synthesis of N4 donor macrocyclic Schiff base ligands and their Ru (II), Pd (II), Pt (II) metal complexes for biological studies and catalytic oxidation of didanosine in pharmaceuticals

A series of tetraaza (N4 donor) macrocyclic ligands (L1–L4) were derived from the condensation of o-phthalaldehyde (OPA) with some substituted aromatic amines/azide, and subsequently used to synthesize the metal complexes of Ru(II), Pd(II) and Pt(II). The structures of macrocyclic ligands and their metal complexes were characterized by elemental analyses, IR, 1H &13C NMR, mass and electronic spectroscopy, thermal, magnetic and conductance measurements. Both the ligands and their complexes were screened for their antibacterial activities against Gram positive and Gram negative bacteria by MIC method. Besides, these macrocyclic complexes were investigated as catalysts in the oxidation of pharmaceutical drug didanosine. The oxidized products were further treated with sulphanilic acid to develop the colored products to determine by spectrophotometrically. The current oxidation method is an environmentally friendly, simple to set-up, requires short reaction time, produces high yields and does not require co-oxidant.

Differentiation of pork from beef, chicken, mutton and chevon according to their primary amino acids content for halal authentication

The detection of pork in various food products has been an important subject of study in many countries. The current study was aimed to differentiate pork from selected meats of beef, mutton, chevon and chicken based on their primary amino acid contents using reverse phase-high performance liquid chromatography (RP-HPLC) with derivatization by o-phthalaldehyde (OPA) and ultraviolet (UV) detection. The results show that the most discriminative amino acids between pork and others were valine, histidine, serine, alanine and arginine. The findings here lay the ground work for the future research to develop a marker for halal meat authentication based on the amino acids content. Keywords: RP-HPLC, OPA, Amino acids, halal meat, pork

Rapid HILIC method with fluorescence detection using derivatization reaction utilizing o-phthaldialdehyde for determination of degradation product of aliskiren

A rapid procedure based on direct extraction and HILIC separation of aliskiren (ALI) degradation product - 3-amino-2,2-dimethylpropanamide (ADPA) with fluorescence detection has been developed. The formation of ADPA from ALI under different conditions was studied. The evaluation of HILIC method robustness was performed using multifactorial experiments with fixed factors (one-level Plackett-Burman design). XBridge HILIC column with isocratic elution using mobile phase 10 mM K(2)HPO(4) pH 7.2-acetonitrile (26:74; v/v) was employed. Fluorescence detection after post column derivatization using o-phthaldialdehyde (OPA) reagent was performed at excitation and emission wavelength of 345 nm and 450 nm, respectively. The reported method has an advantage of a simple sample pre-treatment and quick and very sensitive measurement. The method was successfully applied for the analysis of commercially available ALI samples.

Determination of Sulfite in Water and Dried Fruit Samples by Dispersive Liquid–Liquid Microextraction Combined with UV–Vis Fiber Optic Linear Array Spectrophotometry

A simple and rapid dispersive liquid–liquid microextraction (DLLME) method was applied to preconcentrate sulfite ions from aqueous samples as a prior step to its determination by fiber optic-linear array detection spectrophotometry. The procedure is based on the color reaction of sulfite with o-phthaldialdehyde (OPA) in the presence of ammonia to form isoindole and extraction of the formed isoindole derivative using the DLLME technique. The conditions for the microextraction performance were investigated and optimized. The calibration graph was linear in the range of 2–100 μg L−1with a detection limit of 0.2 μg L−1. The relative standard deviation for five replicate measurements of 10 and 50 μg L−1of sulfite were 2.8 and 2.0 %, respectively. Under the optimized conditions, the enrichment factor of ~133 was obtained from a sample volume of 10 mL. The proposed method was successfully applied to the sulfite determination in drinking water and in food samples.

Determination of ammonium on an integrated microchip with LED-induced fluorescence detection

A simply fabricated microfluidic device integrated with a fluorescence detection system has been developed for on-line determination of ammonium in aqueous samples. A 365-nm light-emitting diode (LED) as an excitation source and a minor band pass filter were mounted into a polydimethylsiloxane (PDMS)-based microchip for the purpose of miniaturization of the entire analytical system. The ammonium sample reacted with o-phthaldialdehyde (OPA) on-chip with sodium sulfite as reducing reagent to produce a fluorescent isoindole derivative, which can emit fluorescence signal at about 425 nm when excited at 365 nm. Effects of pH, flow rate of solutions, concentrations of OPA-reagent, phosphate and sulfite salt were investigated. The calibration curve of ammonium in the range of 0.018–1.8 μg/mL showed a good linear relationship with R2 = 0.9985, and the detection limit was (S/N = 3) 3.6 × 10−4 μg/mL. The relative standard deviation was 2.8% (n = 11) by calculating at 0.18 μg/mL ammonium for repeated detection. The system was applied to determine the ammonium concentration in rain and river waters, even extent to other analytes fluorescence detection by the presented device.

Daily intake estimates of fumonisins in corn-based food products in the population of Parana, Brazil

One hundred (100) samples of corn-based food products were collected between February 2007 and July 2010 to estimate the intake of fumonisins in the state of Paraná, Brazil. The identification of fumonisins was performed by high-performance liquid chromatography with fluorescence detection and post column o-phthaldialdehyde (OPA) derivatisation. The occurrences of fumonisins B1 (FB1) and B2 (FB2) were 82% and 51%, respectively, with concentrations ranging from 126 to 4348 μg/kg (FB1 + FB2). Among corn-based products, corn meal had a higher prevalence of fumonisin (96.6%), and corn grits had the highest level of contamination (3462 μg/kg for FB1 and 886 μg/kg for FB2). The average probable daily intake (APDI) and maximum probable daily intake (MPDI) estimated for the population of Parana, Brazil, were 120.58 and 256.07 ng/kg body weight/day, respectively, which is lower than the provisional maximum tolerable daily intake (PMTDI), 2000 ng/kg body weight/day.

The effect of zinc ion on the absorption and emission spectra of glutathione derivative: Predication by ab initio and DFT methods

Relying on the reaction of o-phthalaldehyde (OPA) with glutathione (GSH) to form a highly fluorescence derivative GSH–OPA has been widely used to measure reduced glutathione. In order to better understand spectra property of the GSH–OPA and the effect of zinc ion on it, the ground and the lowest singlet excited state properties, the electronic absorption and emission spectra are predicted by ab initio and DFT methods. The absorption spectra are simulated using time dependent DFT method (TD-DFT) whereas the emission spectra are approximated by optimizing the lowest singlet excited state by HF/CI-Singles and then subsequently using this geometry for the TD-DFT calculations. The solvent effects on transition energies have been described within the conductor-like polarizable continuum model (CPCM). The calculated transition energies (absorption and emission) are in agreement with available experimental information.

Terpene Alcohols InhibitDe NovoSphingolipid Biosynthesis

The terpene alcohols geranyllinalool, phytol (diterpene alcohol), and farnesol (sesquiterpene alcohol) were newly found to inhibit sphingolipid de novo biosynthesis in LLC-PK₁ cells (pig kidney epithelial cells). A simple chromatographic bioassay was established for the screening of inhibitory compounds able to reduce the amount of sphinganine, an intermediate metabolite of sphingolipid biosynthesis. The screening strategy was based on the degree of suppression of fumonisin B₁ (FB₁-induced sphinganine accumulation following co-treatment with selected terpene alcohols. L-cycloserine and ISP-1, specific serine palmitoyltransferase (SPT) inhibitors, were used as positive controls. Our results show that measuring reduced sphinganine levels after treatment with 2 µM FB₁ in combination with the putative inhibitory compounds provides a useful screening bioassay for evaluating compounds causing sphingolipid depletion. Intracellular sphinganine concentrations were analyzed using the fluorescent peak areas of the O-phthalaldehyde (OPA) derivatives of sphinganine eluted with 87 % acetonitrile on a reversed-phase column. Geranyllinalool, phytol, and farnesol were identified as novel SPT inhibitors that reduce FB₁-induced sphinganine accumulation and thus inhibit the first step of sphingolipid de novo synthesis.

Gold nanoparticle extraction followed by o-phthaldialdehyde derivatization for fluorescence sensing of different forms of homocysteine in plasma

This study reports a selective and sensitive method for fluorescent detection of total, protein-bound, free, and free oxidized homocysteine (HCys) using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent, fluorosurfactant-capped gold nanoparticles (FSN–AuNP) as a preconcentrating probe, and o-phthaldialdehyde (OPA) as a derivatizing agent. TCEP was used to reduce the disulfide bonds of protein-bound and free oxidized HCys. FSN–AuNPs can extract HCys from a complicated complex because the FSN capping layer stabilizes the AuNPs in a high-salt solution and inhibits non-specific adsorption. HCys was selectively derivatized with OPA in the absence of a nucleophile. By taking advantage of these features, the selectivity of the proposed system is greater than 100-fold for HCys and homocystine (HCys–HCys disulfide; diHCys) compared to any aminothiols. The limits of detection for HCys and diHCys were 4.4 and 4.6nM, respectively. Different forms of plasma HCys were determined by varying the order of disulfide reduction with TCEP.

Determination of sphingoid bases from hydrolyzed glucosylceramide in rice and wheat by online post-column high-performance liquid chromatography with O-phthalaldehyde derivatization

We developed a determination method for sphingoid bases using online post-column high-performance liquid chromatography (HPLC) with O-phthalaldehyde (OPA) derivatization. Good separation was achieved using a reversed-phase column and eluting with 50% acetonitrile containing formic acid and heptafluorobutyric acid. Using these conditions, an excellent linearity (R² > 0.999) was achieved using standard solutions of sphinganine (d18:0), sphingosine (d18:1(4t)), 4-hydroxy-sphinganine (t18:0), glucosylsphingosine (glc-d18:1(4t)), and galactosylsphingosine (gal-d18:1(4t)). Plant glucosylceramides were hydrolyzed with 1 M aqueous HCl in methanol for 18 h at 90°C, followed by extraction of sphingoid bases with diethyl ether in preparation for analysis using the proposed HPLC conditions. The glc-d18:1(4t) standard was also hydrolyzed and analyzed by HPLC using the same procedure, and the d18:1(4t) peak obtained from the hydrolyzed glc-d18:1(4t) standard was used as a reference for calculation. We also confirmed the applicability of this method to the analysis of sphingoid bases in rice and wheat, obtaining relative standard deviations of 8.0% for rice and 4.6% for wheat. The recoveries of spiked rice and wheat samples were 104% and 106%, respectively. Our proposed method enables the straightforward determination of sphingoid bases without expensive facilities, employing fluorescence detection of OPA derivatives.