Enzymatic determination of phenols using peanut peroxidase

The influence of phenol and its derivatives on the kinetics of oxidation of aryldiamines (indicator-substrates) catalyzed by novel plant peroxidase—cationic peanut peroxidase—was studied. The character of influence of phenols on the kinetics of enzymatic oxidation of benzidine, o-dianisidine, and 3,3′,5,5′-tetramethylbenzidine (TMB) with hydrogen peroxide was found to depend on a correlation between redox properties of phenols and the indicator-substrate of peroxidase. Thus, the catalytic activity of peanut peroxidase is inhibited by phenols with redox potentials higher than that of aryldiamines mentioned above, whereas phenols with potentials below those of aryldiamines, play the role of second substrates of the enzyme. The enzymatic procedures for the determination of numerous phenols on the level of their concentrations 0.05–80 μM were developed using the reactions of benzidine, o-dianisidine, and TMB oxidation. Different analytical signals—the indicator reaction rate and the induction period duration—were used for the determination of phenols, belonging to various groups—the inhibitors and second substrates of the enzyme, respectively.     

Enzymatic reverse FIA method for total phenols determination in urine samples

A reverse flow injection spectrophotometric enzymatic method is proposed to quantify total phenols in urine samples. The polyphenol oxidase (PPO; EC 1.14.18.1) obtained as a crude extract from sweet potato root (Ipomoea batatas) was used as enzymatic catalyze. The detection limit, the sample throughput and relative standard deviation were 7.7 mg l⁻¹ of total phenols, 49 h⁻¹ and 0.9%, respectively. The method was applied to real samples and a recovery study was carried out in order to its validation.     

Enzymatic polymerization of phenols

The horseradish peroxidase-catalyzed oxidative polymerization of substituted phenols with the use of hydrogen peroxide as an oxidizer in an aqueous-organic medium has been studied. The presence of an aqueous buffer controlling activity of the catalyst is the necessary condition for this reaction. The rate of the process decreases with a reduction in the fraction of water in the reaction mixture. It has been shown that the oxidizer should be gradually added in portions to the reaction mixture. The resulting oligomers convert into high-molecular-mass crosslinked products under heating.     

Kinetic determination of phenols by a fixed-time method

The fixed-time kinetic method is used for the determination of 15 phenols in methanol and acetic acid by measurement of the yellow products from the oxidation of the phenols with sodium metaperiodate. An increase in acid and metaperiodate concentrations enhances the rate of reaction, whereas the use of 2-propanol as the solvent decreases it.     

On-line sample preparation and determination of phenols with a Flow-Analysis method

Summary A Flow-Analysis system has been developed to automate the phenol determination according to the German standard method DIN 38409-H16-2. The automation leads to a significant acceleration of the procedure. One analysis only lasts 3 min while the complete manual determination requires 3 h. Also the sample, solvent and reagent volumes are reduced to a tenth of the volumes demanded by the standard method. The described phenol determination is based on the integration of an airsegmented (Airsegmented-Flow-Analysis SFA) part in a Flow-Injection-Analysis (FIA) system. The main steps of the analytical procedure are: Reproducible inserting of the sample in a carrierstream, sample pretreatment and sample measuring. In the first step the sample is injected into the carrierstream. It transports the sample in the reactioncoil and than through the distillation unit. The steam distillation represents the sample preparation step; therefore an airsegmented stream is necessary. Afterwards the different phases (liquid and gas) were singled again and the distilled solution is fed into the FIA manifold. The determination itself takes place inside the FIA system. The limit of determination amounts to 0.01 mg l^–1 with a standard deviation of 1.5%. Different waste, surface and drinking water samples have been analyzed without any problems. The results correspond very well to those obtained by manual procedure.     

Simultaneous kinetic determination of phenols by use of the kalman filter

A multicomponent analysis method based on the Kalman filter algorithm is proposed for the determination of phenolic compounds. The method relies on the oxidative coupling of phenols (phenol, 2-chlorophenol and 3-chlorophenol) to N,N-diethyl-p-phenylenediamine in the presence of hexacyanoferrate(III), the reaction being monitored via changes in the absorbance at 660 nm of the dye formed. Phenols can be determined individually over the concentration range 1.25-25 muM with a relative standard deviation of ca 0.6-0.8%. Differences in the kinetic behaviour of the three species were exploited by using the linear Kalman filter to resolve mixtures of the phenols at the muM level in a widely variable concentration ratios with errors less than 10%.     

Enzymatic biosensor of horseradish peroxidase immobilized on Au-Pt nanotube/Au-graphene for the simultaneous determination of antioxidants

A new electrochemical method has been proposed for the simultaneous determination of butylated hydroxyanisole (BHA) and propyl gallate (PG) in food matrices based on enzymatic biosensors. Spiny Au-Pt nanotubes (SAP NTs) was first synthesized and demonstrated to exhibit intrinsic peroxidase and catalase-like activity. The structure of SAP NTs provides large surface area and favorable medium for electron transfer, on which HRP were immobilized and acted as enzymatic biosensor for the simultaneous detection of BHA and PG. The results revealed that BHA and PG both have well-defined oxidation waves with peak potentials of 624 and 655 mV, respectively. Under the optimal conditions, the method behaved satisfactory analytical performance towards BHA and PG with a wide linear range of 0.3–50 mg L⁻¹ and 0.1–100 mg L⁻¹, as well as a detection limit of 0.046 mg L⁻¹ and 0.024 mg L⁻¹ (3σ/slope), respectively. Besides, the proposed method exhibits good sensitivity, stability and reproducibility, providing an alternative to fabricate electrode and construct sensitive biosensors.     

A direct Capillary Liquid Chromatography with electrochemical detection method for determination of phenols in water samples

A fast and direct method based on the use of Capillary Liquid Chromatography (LC) with electrochemical (EC) detection has been described for phenols pollutants in water samples. Concretely, phenol, o-cresol, 2-chlorophenol and bisphenol A have been selected as target analytes. The combination of Capillary LC with EC detection avoided the necessity of preconcentration steps typically used in environmental analysis. The sample injected volume was 2μL. The achieved detection limits were between 1 and 2μg/L and the linear dynamic range was up to 50μg/L for all studied phenols. The precision and uncertainty were satisfactory. The analysis time per sample was 10min. The proposed procedure has been proved useful for treated waters.     

Semiquantitative determination of micro amounts of phenols using the ring colorimetry method

Summary The semiquantitative determination of micro amounts of pyrocatechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, andp-nitrophenol using the Weisz ring colorimetry method has been described. The results obtained deviated, on an average, from those calculated by about 4% and the amount of phenol required for one determination was about 4 g.

---

Zusammenfassung Die halbquantitative Bestimmung von Mikromengen Brenzcatechin, Resorcin, Hydrochinon, Pyrogallol, Phloroglucin und p-Nitrophenol mit Hilfe der vonWeisz angegebenen Ringkolorimetrie wurde beschrieben. Die Resultate weichen von den berechneten Werten durchschnittlich um etwa 4% ab, wobei für eine Bestimmung etwa 4 g benötigt werden.

---

Résumé On décrit le dosage semiquantitatif de microquantités de pyrocatéchol, résorcinol, hydroquinone, pyrogallol, phloroglucinol etp-nitrophénol, à l'aide de la méthode colorimétrique annulaire deWeisz. Les résultats obtenus se sont écartés, en moyenne, d'environ 4% d'avec ceux calculés et la quantité de phénol nécessaire pour un dosage s'élevait à 4 g environ.

     

Enzymatic method for the determination of inorganic and organic inhibitors of alcohol dehydrogenase

The inhibiting effect of heavy metal ions, organic nitrogen-containing heterocyclic compounds, and amino acids on the catalytic activity of yeast alcohol dehydrogenase (ADH) in the reaction of ethanol oxidation by nicotinamide adenine dinucleotide was detected. Sensitive procedures were developed for the determination of the most effective ADH inhibitors [mercury (II), silver(I), zinc(II), copper(II), 1,10-phenanthroline, 2,2′-dipyridyl, 8-hydroxyquinoline, quinoline, 3,4-dimethylimidazole, benzimidazole, 1,2,3-benzotriazole, histidine, tryptophan, proline, and histamine] with c_L = 5x 10^-10-1 x 10^-4M (RSD = 1–12%).     

Enhanced chemiluminescence determination of alpha-amylase by use of amylose labelled with horseradish peroxidase

An enhanced chemiluminescence method for determining soluble and immobilized alpha-amylase has been developed, based on use of an insoluble amylose substrate labelled with horseradish peroxidase. Soluble peroxidase-labelled fragments of the substrate, released by the action of alpha-amylase, are quantified by the peroxidase-catalyzed luminol-peroxide-p-iodophenol reaction. The detection limit for alpha-amylase was 125 fmole (12.5 nM). An insoluble amylopectin labelled with horseradish peroxidase was also effective as a substrate for this type of assay.     

Development of HPLC-MS/MS method for the simultaneous determination of environmental phenols in human urine

We present a sensitive method for simultaneous determination of bisphenol A (BPA), benzophenone-3 (BP-3), 4-tert-octylphenol (t-OP), ortho-phenylphenol (OPP), four parabens (methyl, ethyl, propyl, butyl parabens) and five chlorophenols (2,4-dichlorophenol (2,4-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4,5-trichlorophenol (2,4,5-triclorophenol), 2,4,6-trichlorophenol (2,4,6-TCP), and triclosan (TCS)), in human urine by high-pressure liquid chromatography (HPLC) mass spectrometry (MS). Samples were processed using enzymatic deconjugation of glucuronides followed by solid phase extraction (SPE) on a C18 cartridge and the eluate was concentrated. Analytes were separated by reversed-phase HPLC and then detected by atmospheric pressure chemical ionisation (APCI) MS and quantified by isotope dilution method. We describe details for optimisation of each step of the procedure. The sample treatment steps are straightforward and not labour-intensive and, therefore, permit a high sample throughput with excellent prospects for automation. This method shows low inter-day variation, and detection limits for most of the compounds are below 1 ng/mL in 1 mL of urine. The method accuracy was also verified by the analysis of proficiency testing urine samples.     

Solvent‐free method for the determination of lignin‐derived phenols in sediments

A solvent‐free method that uses headspace solid‐phase microextraction and gas chromatography with flame ionization detection is proposed for the determination of lignin‐derived phenols in sediments. The extraction and derivatization conditions for the simultaneous analysis of acetosyringone, acetovanillone, syringaldehyde, vanillin, ferulic acid, syringic acid, vanillic acid, p‐hydroxybenzoic acid, and p‐coumaric acid were optimized using a central composite design. After optimization, the best results were obtained with the following conditions: exposure of the polyacrylate fiber to the headspace with 60 μL of N ,O‐bis(trimethylsilyl)trifluoroacetamide as a derivatizing agent for 15 min and then extraction in the headspace of 100 mg of sediment (previously spiked with lignin‐derived phenols) for 35 min. The accuracy of the method was estimated based on recovery tests at two concentration levels and by comparison with a high‐performance liquid chromatography method reported in the literature. Based on the t‐test with a confidence level of 95%, no statistical differences were observed. The detection and quantification limits for the target compounds varied according to their characteristics: values at the microgram per gram level for nonacid compounds and milligram per gram level for phenolic acids, due to the lower volatility of the derivatives.     

A microanalytical method for the investigation of phenols

Summary Micro amounts of mono-, di-, and trihydric phenols were successfully identified as thep,p-nitrophenylazobenzoyl esters. A microanalytical Chromatographic procedure is described for separation of their mixtures.

---

Zusammenfassung Mikromengen ein-, zwei- und dreiwertiger Phenole lassen sich als p,p-Nitrophenylazobenzoyl-ester identifizieren. Ein mikrochromatographisches Verfahren zur Trennung ihrer Gemische wurde beschrieben.

---

---

The authors thank MadameKh. M. Badra for kind co-operation.     

Enzymatic assay technique for the determination of aspartame

An enzymatic assay technique was developed for the determination of the artificial sweetener aspartame. The peptide bond of aspartame was first cleaved by peptidase to release aspartic acid. In the presence of α-ketoglutarate, aspartic acid was then transaminated by aspartate aminotransferase to glutamate. The reaction was monitored by following the oxygen consumption during the enzymatic oxidation of glutamate by glutamate oxidase. A linear relationship between oxygen consumption and aspartame concentration up to 200 μM was obtained. The assay technique was applicable to the determination of aspartame in a variety of dietary food products. The results obtained agreed well with those determined by liquid chromatography and those reported by the product manufacturers.     

Methodologies for the practical determination and use of method detection limits

Underwater NaI(T1) and HPGe detectors are used in the environmental measurements programs at the Savannah River Site (SRS). A 22.9 cm × 10.2 cm NaI(T1) detector on the Savannah River continuously monitors effluent releases from both SRS (DOE) and Plant Vogtle (Georgia Power). Correlations with known releases indicate a sensitivity of 4 mBq/l for⁵⁸Co with 1500 min spectra; such levels are well below those of hazardous or legal concern. A 30%-efficient HPGe detector has appraised radionuclides in SRS cooling pond sediments; the dominant gamma-emitting radionuclide detected was¹³⁷Cs, at levels ranging up to 2.0 MBq/m². The pond activities were adequately quantified by 1 min counts with the HPGe detector; resulting contour maps of sediment¹³⁷Cs provided guidance for partially draining the ponds for dam repairs.     

Milligram determination of aromatic amines and phenols with N-bromosuccinimide

A simple and rapid method has been developed for the determination of milligram amounts of aromatic amines and phenols. An aliquot containing 2-4 mg of sample is dissolved in a mixture of hydrochloric and acetic acids and allowed to react with N-bromo-succinimide, the excess of which is determined iodometrically after the reaction is over. The reaction time varies from 1-10 min and the absolute error generally is negative, ranging from -1 to -4%.