Colorimetric determination of reactive amino groups of a solid support using Traut’s and Ellman’s reagents

A simple colorimetric method has been developed for the quantitative determination of reactive amino groups of a solid support. The method calls for the use of a reagent that reacts specifically and facilely with amino groups and concurrently introduces free sulfhydryl groups. The latter can then be titrated colorimetrically by using the well-known Ellman’s reagent. Thus, an excess of 2-iminothiolane (Traut’s reagent) was reacted with amine-carrying solid supports. After removing unreacted 2-iminothiolane and keeping the resultant solid-phase sulfhydryl groups in a reduced state by using dithiothreitol, the solid supports, after being thoroughly washed, were then reacted with 5,5′-dithiobis-(2-nitrobenzoic acid) to quantify the sulfhydryl groups that were generated from reacting solid-phase amino groups with Traut’s reagents.     

Coupling capacity of solid-phase carboxyl groups

A simple colorimetric procedure for determining the coupling capacity of solid-supported carboxyl groups has been developed. The carboxyl groups of a solid support were coupled to cystamine at pH 4–4.5, using a water soluble carbodiimide 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiime as the condensing reagent. The solid-phase coupled disulfides were then reduced to sulfhydryl groups by treating the solid phase with dithiothreitol. For every one carboxyl group coupled with cystamine, one solid-phase sulfhydryl is introduced. After removing all of the reducing reagents by extensive washing, the sulfhydryl content, which is equivalent to the carboxyl groups of the gel, was quantified by using 5,5′-dithiobis-(2-nitrobenzoic acid), the Ellman’s reagent.     

Analytical investigations on cephalosporins--IV: application of Ellman's reagent

The application of 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) for the determination of microgram quantities of various selected cephalosporins in aqueous solution is described. Cephalosporin derivatives (cephalothin sodium, cephacetrile sodium, cefamandole lithium and nafate, cefoperazone sodium and ceftizoxime sodium) have to be treated with 0.5N sodium hydroxide before determination with Ellman's reagent, which reacts with free thiol groups. An aliquot of the solution is reacted with Ellman's reagent in pH 7.2 phosphate buffer and the absorbance of the resulting yellow solution is measured at 410 nm. The method, which is simple and precise, has been applied to determination of those cephalosporins in formulations, the results being compared with those obtained by the Ni-hydroxylamine method.     

Ultrasensitive,simple and solvent-free micro-assay for determining sulphite preservatives (E220–228) in foods by HS-SDME and UV–vis micro-spectrophotometry

A new method based on headspace single-drop microextraction in combination with UV–vis micro-spectrophotometry has been developed for the ultrasensitive determination of banned sulphite preservatives (E220–228) in fruits and vegetables. Sample acidification was used for SO₂ generation, which is collected onto a 5,5′-dithiobis-(2-nitrobenzoic acid) microdrop for spectrophotometric measurement. A careful study of this reaction was necessary, including conditions for SO₂ generation from different sulphating salts, drop pH, 5,5′-dithiobis-(2-nitrobenzoic acid) concentration and potential interference effects. Variables influencing mass transfer (stirring, sample volume and addition of salt) and microextraction time were also studied. A simple sulphite extraction was carried out, and problems caused by oxidation during the extraction process were addressed. A high enrichment factor (380) allows the determination of low levels of free SO₂ in fruits and vegetables (limit of detection 0.06 μg g^?1, limit of quantification 0.2 μg g^?1) with an adequate precision (repeatability, relative standard deviation 5 %). In addition, the sulphiting process was studied through the monitoring of residual SO₂ in a vegetal sample, thus showing the importance of a sensitive tool for SO₂ detection at low levels.

The use of redox reactions in the analysis of dyes and dye intermediates: VIII. The determination of 4,4′-dinitrostilbene-2,2′-disulfonic acid by constant potential coulometry

The number of electrons exchanged in the electrochemical reduction of 4,4′-dinitrostilbene-2, 2′-disulfonic acid on a mercury cathode in Britton-Robinson buffer solution of pH 10 and in 0.1 M tetramethylammonium bromide solution was determined. A sensitive determination of the studied compound by constant potential coulometry was developed, and its accuracy and reproducibility were found.     

Differential pulse polarographic behaviors of p-nitrosodimethylaniline: Application for microdetermination of NADH

The differential pulse polarographic behavior of p-nitrosodimethylaniline has been investigated in Britton-Robinson buffer and phosphate buffer. The peak obtained at pH 8.0 is recommended for the trace determination of this compound, with an experimental detection limit of 18 ppb (1.2 × 10^?7M) in simple aqueous solution. The method is also applied for the indirect microdetermination of NADH. The experimental detection limit of NADH is shown to be 1.05 × 10^?6M.     

The determination of phenobarbital and diphenylhydantoin in blood by differential pulse polarography

A sensitive differential pulse polarographic assay was developed for the determination of phenobarbital or diphenylhydantoin in blood. The assay involves the selective extraction of the compound into chloroform from whole blood buffered to pH 7.0. After suitable “clean-up” of the sample, each compound is nitrated in 10% potassium nitrate in sulfuric acid at 25° for 1 h. The nitro-derivatives are extracted into ethyl acetate, and the residues are dissolved in 1 M phosphate buffer (pH 7.0) or 0.1 M sodium hydroxide for phenobarbital and diphenylhydantoin, respectively; the solutions are deoxygenated, and analyzed by differential pulse polarography. The overall recovery of phenobarbital and diphenylhydantoin from blood was 72.3% ±6.5 (s_r) and 76.7 ±2.3 (s_r) respectively. The sensitivity limit is 1–2 μg ml^-1 of blood for both compounds. A modified assay for the determination of both compounds in blood with t.l.c. separation was also developed.     

On-line determination of sulphite in brine by flow-injection analysis

A spectrophotometric flow-injection procedure for the determination of sulphite in aqueous media over the range 0.5–20 mg 1^?1 is described. The reagent used was the organic disulphide 5,5′-dithiobis(2-nitrobenzoic acid). Results are presented for a laboratory-based method for sulphite in water and a potential on-line method for sulphite in high ionic strength potassium chloride brine. The general attractions of flow-injection-based monitors for the on-line analysis of liquid process streams are also discussed.     

Lead ion selective PVC membrane electrode based on 5,5'-dithiobis-(2-nitrobenzoic acid)

A PVC membrane electrode for lead ions based on 5,5′-dithiobis-(2-nitrobenzoic acid) as membrane carrier was prepared. The electrode exhibits a Nernstian response for Pb²⁺ over a wide concentration range (1.0×10⁻²–4.0×10⁻⁶ M). It has a relatively fast response time and can be used for at least 3 months without any divergence in potentials. The proposed electrode revealed good selectivities for Pb²⁺ over a wide variety of other metal ions and could be used in a pH range of 2.0–7.0. It was used as an indicator electrode in potentiometric titration of lead ions and in direct determination of lead in water samples.     

Affinity chromatography of frog epidermis dopa-oxidase

Benzoic acid is a competitive inhibitor of the enzyme dopa-oxidase. p-Aminobenzoic acid was coupled by a single-step reaction of the amino group to: (1) CNBr-activated Sepharose 4B: (2) Enzacryl AA, by reaction with thiophosgene (pH 10.0); and (3) CM-Sephadex G-50, modified to azide by the Curtis procedure. These three solid supports were used as affinity adsorbents in the dopa-oxidase purification. The enzyme was obtained from frog epidermis, and was retained at pH 4.7 with 0.1 M acetate buffer. The enzyme elution was carried out using a linear pH gradient with 0.1 M phosphate buffer pH 8.0. We have examined the interaction of the enzyme with the immobilized PABA in relation to the nature of the support, the lengthening of the “arm” of the ligand, bathwise adsorption, and the enzyme activation by immobilized trypsin.     

Determination of hydroxycarboxylic acids in food products by capillary electrophoresis

A procedure for the capillary-electrophoretic determination of lactic, malic, tartaric, and citric acids in food products was developed. The use of 3-nitrobenzoic acid as a light-absorbing component of the running buffer was proposed for the indirect photometric detection of these substances. This considerably increased the determination sensitivity (up to n × 10 ??g/L), as compared with currently available analogs. The composition of the running buffer was optimized: 3-nitrobenzoic acid, 10 mM; cetyltrimethylammonium bromide, 0.5 mM; EDTA, 0.1 mM; and monoethanolamine, to pH 5.3. The procedure was tested with the samples of food products: fruits, juices, nectars, wines, beer, etc. The accuracy of the analytical results was confirmed by the standard addition method.     

Preparation and comparison of Proteus vulgaris and Proteus mirabilis bacterial electrodes for the determination of DL-phenylalanine

Bacterial electrodes for DL-phenylalanine were prepared by immobilizing the bacteria Proteus vulgaris and Proteus mirabilis on an ammonia gas-sensor. The response of the Proteus vulgaris bacterial electrode, when 10 mg of the bacteria was used, had a linear range between 3.0 × 10⁻⁴ and 1.0 × 10⁻² M DL-phenylalanine with a response slope of 43 mV/decade in pH 7.0, 0.1 M phosphate buffer solution at 30°C, while the response of the Proteus mirabilis bacterial electrode, when 3 mg of the bacteria was used, had a linear range between 3.0 × 10⁻⁴ and 3.0 × 10⁻² M DL-phenylalanine with a response slope of 49 mV/decade in pH 7.2, 0.1 M phosphate buffer solution at 30°C. The most important interferents were urea and l-asparagine, and inorganic salts reacted as an inhibitor. The Proteus vulgaris bacterial electrode could be used directly for the determination of DL-phenylalanine in nearly the same linear range during 3 days. On the other hand, the Proteus mirabilis bacterial electrode could be used continuously during 7 days in the above linear range.     

Phanquinone as a suitable derivatization reagent in micellar electrokinetic chromatography and HPLC analysis of amino acids

Phanquinone (chemically: 4,7-phenanthroline-5,6-dione) was applied as an original precolumn derivatization reagent for amino acids followed by separation using MEKC with UV detection (240 nm). The derivatization reaction was carried out at 68 degrees C in the presence of aqueous phosphate buffer (pH 8.0) and it was found to be complete after 30 min. Twelve derivatized standard amino acids were separated in about 22 min under MEKC conditions using sodium cholate (250 mM) as the surfactant in phosphate buffer (20 mM, pH 9.0). The developed method was validated for the analysis of D,L-phosphoserine (D,L-p-Ser) and L-glutamine (L-Gln); good linearity (r > 0.999) was achieved in the calibration range of 0.25-2.5 micromol/mL. The sensitivity of the MEKC method (LOD 0.1 micromol/mL; LOQ 0.25 micromol/mL, RSD% <5.0%, n = 3) was found to be adequate for quantitation of amino acids in pharmaceuticals. Quantitative applications of the validated MEKC method were carried out by the analysis of commercially available oral polyaminoacid formulations (tablets and extemporaneous solutions) containing L-Gln and D,L-p-Ser; the obtained results were found to be in agreement with those from a validated reference RP-HPLC method.     

Selective optical sensing of biothiols with Ellman's reagent: 5,5′-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles

Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB²⁻) anion as a result of reaction with biothiols was measured at 410 nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57 μM and acceptable linearity ranging from 0.4 to 29.0 μM (r = 0.998).     

The application of 2,4,6-trinitrobenzene-1-sulphonic acid in the differential pulse polarographic determination of amines

The differential pulse polarographic behaviour of 2,4,6-trinitrophenyl (TNP) derivatives of several primary amines and amino acids was investigated in the presence of sulphite ion. All the derivatives produced a polarographic peak for their complexes with sulphite (1 × 10^?2 M) in pH 8.0 phosphate buffer (0.05 M)/0.1 M potassium chloride. The derivatives of proteins and peptides did not give such a peak. A 5-min reaction time at room temperature (or 50°C for lysine) and pH 10.5 using 1 × 10^?4 M 2,4,6-trinitrobenzene-1-sulphonic acid provides the optimal conditions for the determination of 5 × 10^?6?2.5 × 10^?5 M amines. The relative standard deviation for determining 1 × 10^?5 M glycine (n = 5) was 1%.     

The Determination of Folic Acid (Pteroylmonoglutamic Acid) in Fortified Products by Reversed Phase High Pressure Liquid Chromatography

Abstract

A reversed phase HPLC method was developed for the separation and determination of pteroylglutamic acid (PGA) in fortified foods. Extraction was carried out by heating with phosphate-citrate buffer, pH 8.0 containing ascorbate, and incubation with papain at 40°C for 4 hrs. The extracts were purified and concentrated on a short DEAE column which was rinsed with phosphate buffer, pH 7.0, of increasing molarity. PGA was eluted with 0.1M phosphate buffer, pH 7.0, containing 0.5M NaCl. The eluants were chromatographed on a Spherisorb ODS 10 μm column (250 × 4.6 mm) using a 30 min linear gradient of 2% to 30% acetonitrile in 0.1M acetate buffer, pH 4.0, at 1 ml/min and an absorbance detector at 280 nm. The coefficients of variation on analysis of 8 replicate samples of a milk and soy protein based infant formulas were 5.9% (at 4.6 ng/50 μl inject) and 6.8% (at 1.8 ng/50 μl inject) respectively.     

Oxidized haematoxylin,a new reagent for the spectrophotometric determination of penicillins and cephalosporins

Haematoxylin—Chloramine-T in phosphate buffer (pH 7.0) is proposed as a new reagent for spectrophotometric determination of penicillins and cephalosporins in pure samples and pharmaceutical preparations. The method is based on acid hydrolysis of penicillins and cephalosporins with 5M HCl and subsequent treatment with oxidized haematoxylin. The resulting colour exhibits maximum absorption at 555 nm.     

Enzymatic determination of N-methylcarbamate pesticides at the nanomolar level by the stopped-flow technique

A sensitive and selective kinetic enzymatic method for the determination of N-methylcarbamate pesticides is presented. It is based on their inhibitory effect on electric eel acetylcholinesterase and the use of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as chromogenic reagent for the thiocholine released from the acetylthiocholine iodide substrate. The fast DTNB-thiocholine reaction is monitored photometrically by the stopped-flow technique. Carbaryl, propoxur and carbofuran can be determined at concentrations in the ranges 6.5-120, 2-15 and 0.1-5.0 ng/ml, respectively, by the proposed method. An interference study was also reported.     

Quantitation of sodium hydrogensulfite in parenteral samples by a flow-injection method

Sodium hydrogen sulfite reacts with 5,5′-dithiobis(2-nitrobenzoic acid) at pH 6.0 to produce a colored 2-nitrothiobenzoate ion with maximum absorbance at 410 nm. The absorbance varies linearly with concentration over the range of 1 ng-1 μg of sodium hydrogensulfite injected and the absolute detection limit is 0.3 ng. The relative standard deviation at 10 ng or greater is typically 1.5%. Matrix components in pharmaceutical parenteral solutions (e.g., amino acids, drugs, dextrose, buffers) do not interfere.     

Mediated glucose biosensor based on polyvinylferrocene

Polyvinylferrocene (PVF) was electrochemically deposited on platinum and carbon electrodes to form a stable and resilient film. During cyclic voltammetry in phosphate buffer, the PVF film deposited on carbon electrodes exhibited anodic and cathodic peaks at 214 and 68 mV, respectively. Both types of electrodes, bearing electrodeposited PVF and crosslinked glucose oxidase, were responsive to glucose, but the carbon electrode appeared to provide a faster response and could determine glucose between 0.1 and 8 mM. When protected by a layer of polymer electrochemically formed from resorcinol and phenylenediamine, the mediated biosensors based on PVF-deposited carbon electrodes were capable of determining glucose up to 25 mM with a response time of 1 min, for at least 50 repeated analyses with good reproducibility. The presence of ambient oxygen, ascorbic acid (0.1 mM), and uric acid (0.5 mM) did not affect their performance. When applied for the determination of the glucose level in reconstituted human serum, the results agreed well with those of the reference hexokinase assay.