Formation Of Chromogen Research Articles

Novel Spectrophotometric Method Development for the Estimation of Boceprevir in Bulk and in Pharmaceutical Formulations

A New, Simple, Rapid and economical extractive spectrophotometric methods were developed for the determination of Boceprevir (anti-retroviral drug). Boceprevir is a direct acting protease inhibitor for the treatment of hepatitis C. It also has two isomers in which the S isomer is more active than the R-isomer. The methods were based on the formation of color chromogens with Bromo cresol green, Bromo thymol blue, Bromo phenol blue and Methyl orange indicator. The extractive spectrophotometry was carried out with phthalate buffer and chloroform. The absorbances of the chromogens were measured at 410 nm and 415 nm against the corresponding reagent blank. The proposed methods have been successfully applied to the bulk drug. The method has been statistically evaluated and was found to be precise and accurate.

Selective Formation of Chromogen I from N-Acetyl-d-glucosamine upon Lanthanide Coordination.

We report two nonanuclear lanthanide complexes, [Ln9(μ4-O)(μ3-OH)8(LH)4(OAc)4(H2O)12]·5ClO4·24H2O (Ln = Gd, 1; Dy, 2), where LH2- is the doubly deprotonated chiral ligand Chromogen I (2-acetamido-2,3-dideoxy-D-erythro-hex-2-enofuranose), one of the many products from the dehydration of N-acetyl-D-glucosamine (GlcNAc). Mass spectroscopic studies established the solution stability of these clusters. Through hydrogen bonding, the cluster complex self-organizes into a nanostructured 54-metal cagelike assembly featuring six of its units occupying the vertices of an octahedron. Free Chromogen I can be obtained in pure form and high yield by a straightforward workup of the cluster complex. This is the first report of dehydrating GlcNAc without the need of a catalyst or forcing conditions.

Visible Spectrophotometric Estimation of Aripiprazole

Three new, simple, sensitive and economical methods (Method I, II and III) were developed for the determination of aripiprazole in pure and pharmaceutical formulation. Method-1 is based on the formation of condensed product obtained when Aripiprazole in acidic conditions reacted with 2, 4-dinitrophenylhydrazine in methanol to form yellowish orange chromogen (hydrazone formation) exhibiting λ max at 480nm. Method-II is based on the formation of Yellow colored chromogen (formation of chalcone) by reacting with vanillin in the presence of sulphuric acid medium exhibiting λ max at 5 60nm. Method-III is based on the formation of dark yellow colored chromogen (cyclic chalcone) by reacting with Cinamaldehyde in the presence of nitric acid exhibiting λ max at 580nm. These methods obeyed Beer'slaw in the concentration range of 2.5–40μg/ml, 2–9 μg/ml and 2.5–15 μg/ml respectively. The results of analysis for the three methods were validated statistically and by recovery studies. The colored chromogens formed are stable for more than 8 hours in all the three methods. The results obtained with the proposed methods are in good agreement with labeled amounts when marketed pharmaceutical preparations are analyzed.

Determination of gallic acid with rhodanine by reverse flow injection analysis using simplex optimization

A reversed flow injection (rFI) system was designed and constructed for gallic acid determination. Gallic acid was determined based on the formation of chromogen between gallic acid and rhodanine, resulting in a colored product with a λmax at 520nm. The optimum conditions for determining gallic acid were also investigated. Optimizations of the experimental conditions were carried out based on the so-call univariate method. The conditions obtained were 0.6% (w/v) rhodanine, 70% (v/v) ethanol, 0.9molL−1 NaOH, 2.0mLmin−1 flow rate, 75μL injection loop and 600cm mixing tubing length, respectively. Comparative optimizations of the experimental conditions were also carried out by multivariate or simplex optimization method. The conditions obtained were 1.2% (w/v) rhodanine, 70% (v/v) ethanol, 1.2molL−1 NaOH, flow rate 2.5mLmin−1, 75μL injection loop and 600cm mixing tubing length, respectively. It was found that the optimum conditions obtained by the former optimization method were mostly similar to those obtained by the latter method. The linear relationship between peak height and the concentration of gallic acid was obtained over the range of 0.1–35.0mgL−1 with the detection limit 0.081mgL−1. The relative standard deviations were found to be in the ranges 0.46–1.96% for 1, 10, 30mgL−1 of gallic acid (n=11). The method has the advantages of simplicity extremely high selectivity and high precision. The proposed method was successfully applied to the determination of gallic acid in longan samples without interferent effects from other common phenolic compounds that might be present in the longan samples collected in northern Thailand.

Synthesis of New Indeno[1,2‐c]pyrazole‐Based Heterocycles and Evaluation of Their Protective Effect against DNA Damage Induced by Bleomycin–Iron

image Reaction of potassium 2‐(1‐phenyl‐1,4‐dihydroindeno[1,2‐c]pyrazole‐3‐carbonyl)hydrazine carbodithioate (2) with hydrazine hydrate, phenacyl bromides, and hydrazonoyl chlorides afforded the corresponding 1,2,4‐triazole, 1,3‐thiazole, and 1,3,4‐thiadiazole derivatives 3, 5, and 7, respectively. Reaction of 3‐mercapto‐4‐amino‐1,2,4‐triazole 3 with phenacyl bromides 4a–c and hydrazonoyl chlorides 6a,b afforded the corresponding 1,2,4‐triazolo[3,4‐b]‐1,3,4‐thiadiazine derivatives 10a–c and 13a,b, respectively, whereas its reaction with 2‐chloro‐N‐arylacetamide derivatives 11a–d afforded the corresponding 2‐(4‐amino‐5‐(1‐phenyl‐1,4‐dihydroindeno[1,2‐c]pyrazol‐3‐yl)‐4H‐1,2,4‐triazol‐3‐ylthio)‐N‐arylacetamide derivatives 12a–d. All of the new compounds were tested for their protection activity against DNA damage induced by bleomycin–iron complex. Compounds 7a and b showed the highest protection activity through diminishing chromogen formation between the damaged DNA and thiobarbituric acid.

Streptomycin interference in Jaffe reaction — Possible false positive creatinine estimation in excessive dose exposure

ObjectivesTo study the potential of commonly used aminoglycoside antibiotics to form non-creatinine chromogen with alkaline picrate reagent. Design and methodsWe studied the non-creatinine chromogen formation of various concentrations of streptomycin, amikacin, kanamycin, netilmicin, gentamicin and tobramycin added to known creatinine concentrations by the Jaffe reaction based creatinine estimation. ResultsOnly streptomycin above therapeutic concentrations of 10mg/mL interfered in the Jaffe reaction and acted as non-creatinine chromogen. ConclusionsTherapeutic doses of the aminoglycosides do not form non-creatinine chromogens.

Neuroprotection by Minocycline Caused by Direct and Specific Scavenging of Peroxynitrite

Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (•)NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 μm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.

Synthesis of New 2-Naphthyl Ethers and Their Protective Activities against DNA Damage Induced by Bleomycin-Iron

The reaction of 2-naphthaloxyacetic acid with thiosemicarbazide in the presence of phosphoryl chloride, followed by treatment with phenacylbromides, led to the formation of imidazo[2,1-b][1,3,4]thiadiazoles 3a-c. 2-(Naphthalen-3-yloxy)acetohydrazide 4 on treatment with ethyl 2-(2-arylhydrazono)-3-oxobutanoates (5a-c), 2-methoxymethylene)malononitrile, or ethyl 2-cyano-3,3-bis(methylthio)acrylate led to the formation of substituted pyrazoles 6-8. The reaction of the hydrazide 4 with hydrazonoyl chlorides 9a-c and 1,2,4,5-benzene tetracarboxylic-1,2:4,5-dianhydride produced bis-diazo compounds 10a-c and dimide 11 respectively. All new compounds were tested for their protective activity against DNA damage induced by bleomycin-iron complex. Compound 2 showed the greatest protection against DNA damage, thus diminishing chromogen formation between the damaged DNA and thiobarbituric acid.

Biological and Chemical Detections in Adsorbent Layer for Monitoring Microbial Production of Primycin

Primycin is a macrolide antibiotic complex produced in microbiological fermentation processes. The microbial production of primycin requires an in-process analytical method suitable for monitoring the level of the active agents. In this paper, a method fulfilling the described requirement is presented. This method consists of a simple, efficacious extraction step, an instrumental sample application followed by a high-performance thin-layer chromatographic separation in relatively short time and a quantitative chromatogram evaluation. A dipping technique, in a solution containing sulfuric acid followed by heating at 120 degrees C, is used for chromogen formation, resulting in an absorption maximum at 290 nm. A progress diagram of the fermentation obtained by this technique is compared with one obtained by a microbiological agar diffusion method. The bioautographic evaluation of the active spots in the chromatogram are also presented. By our TLC method, the group of the active primycin components in the fermentation broth and by-products formed during the fermentation can be well separated. The relative intensities of the different TLC spots provide some information on the formation of the active components.

Tannase Production by Aspergillus niger

A method for assay of microbial tannase (Tannin acyl hydrolase) based on the formation of chromogen between gallic acid and rhodanine is reported. Maximum Tannase production occurred in the culture broth containing 1‐2% (w/v) tannic acid and 0.05 – 0.1% (w/v) glucose. The pH, incubation period, temperature and Glucose concentration optima of Tannase production was found at 5.5, 36 h, 35°C and 0.5% respectively. These properties make the enzyme suitable for pollution control and bioprocess industry. This assay is very simple, reproducible, and very convenient, and with it Tannase activity can be measured in relation to the growth of the organism. Aspergillus niger exhibited higher enzyme activity showing about 65 mole percent conversion respectively after a 36 h incubation period. The assay is complete in a short time, very convenient and reproducible.

A Spectrophotometric Method for Assay of Tannase Using Rhodanine

A method for assay of microbial tannase (tannin acyl hydrolase) based on the formation of chromogen between gallic acid and rhodanine is reported. Unlike the previous protocols, this method is sensitive up to gallic acid concentration of 5 nmol and has a precision of 1.7% (relative standard deviation). The assay is complete in a short time, very convenient, and reproducible.

Use of deoxyribose as a probe for the determination of rate constants for reactions of hydroxyl radicals on mercury electrodes

A voltammetric procedure is described which allows determination of rate constants for reactions of hydroxyl radicals. Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H 2O 2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid under acid conditions, yield a pink chromogen with second order derivative cathodic wave at −0.88 V vs SCE. Added hydroxyl radical `scavengers' compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. The electrochemical behavior of the chromogen has been determined using voltammetry and cyclic voltammetric techniques. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of chromogen formation. For a wide range of compounds, the results fit well with rate constants previously obtained with other methods. It is suggested that voltammetric deoxyribose assay is a simple and cheap alternative for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of six kinds of active compounds extracted from rheum, a traditional Chinese herb, have been determined by this method. Reaction mechanism has also been discussed.

Effect of Acid Concentration on Chromogen Formation from Hexoses in Sulfuric Acid-Based Reactions

Effect of Acid Concentration on Chromogen Formation from Hexoses in Sulfuric Acid-Based Reactions

Attachment of an aminoglycoside, amikacin, to implantable collagen for local delivery in wounds

Cultured skin substitutes consisting of implantable collagen (COL) and cultured human skin cells often fail clinically from destruction by microbial contamination. Hypothetically, addition of selected antimicrobial drugs to the implant may control microbial contamination and increase healing of skin wounds with these materials. As a model for drug delivery, bovine skin COL (1 mg/ml) and amikacin (AM; 46 micrograms/ml) were modified by covalent addition of biotin (B-COL and B-AM, respectively) from B-N-hydroxysuccinimide and bound together noncovalently with avidin (A). B-COL was incubated with A and then with B-peroxidase (B-P) or by serial incubation with B-AM and B-P, before P-dependent chromogen formation. Colorimetric data (n = 12 per condition) from spot tests on nitrocellulose paper were collected by transmission spectrophotometry. Specificity of drug binding in spot tests was determined by (i) serial dilution of B-COL; (ii) reactions with COL, AM, or P that had no B; (iii) removal of A; or (iv) preincubation of B-COL-A with B before incubation with B-P. Binding of B-AM was (i) dependent on the concentration of B-COL; (ii) specific to B-COL, A, and B-P (P < 0.05); and (iii) not eluted by incubation in 0.15 or 1.0 M NaCl. B-AM was found to block binding of B-P to the B-COL-A complex and to retain bacteriocidal activity against 10 clinical isolates of wound bacteria in the wet disc assay. Antimicrobial activity of B-AM was removed from solution by treatment with magnetic A and a permanent magnet. These results suggest that selected antimicrobial drugs can be biotinylated for attachments to COL-cultured cell implants without loss of pharmacologic activity. Because this chemistry utilizes a common ligand, any molar ratio of agents may be administered simultaneously and localized to the site of implantation.

Malonaldehyde determination in foods by ion-pairing high-performance liquid chromatography

A direct method which involved no thiobarbiturate chromogen formation for the analysis of malonaldehyde in foods by ion-pairing high-performance liquid chromatography was developed. After deproteinization and cleaning, diluted samples (20 μl) were injected onto an octadecylsilane column (25 cm × 4·6 mm) i.d., 5 μm) and eluted with 30 m m sodium phosphate buffer, pH 6·5 containing 30% (v/v) ethanol and 0·5 m m tetradecyltrimethylammonium bromide. Detection was accomplished by monitoring absorbance at 267 nm. The lower limit for reliable quantification was 5 pmol per injection. Traces or no malonaldehyde were detected in fresh samples of selected foods. However, after a period of several weeks of refrigerator storage of opened containers, or after chemically induced lipid peroxidation, food samples contained a substantial amount of malonaldehyde.

Malonaldehyde determination in tissues and biological fluids by ion‐pairing high‐performance liquid chromatography

A method for the analysis of malonaldehyde by ion pairing high-performance liquid chromatography is described. The method is direct; no thiobarbiturate chromogen formation is required, and sample preparation is simple. After deproteinization with 50% ethanol and removal of particulate by centrifugation samples were passed through a small silica amino column to remove contaminants. Diluted samples (20 microL) were injected onto an octadecylsilane column (25 cm x 4.6 mm ID, 5 micron) which is eluted with 30 mM sodium phosphate buffer, pH 6.5 containing 30% ethanol and 1 mM tetradecyltrimethylammonium bromide. Detection was accomplished by monitoring absorbance at 267 nm. The lower limit for reliable quantification was 5 pmol per injection. The method has been successfully applied to the quantification of malonaldehyde present in plasma, urine and tissues of rats kept under different dietary conditions as well as after in vivo treatment with CCl4 and iron-dextran. The method was also applied to the quantification of malonaldehyde during liver microsomal lipid peroxidation and was compared to the thiobarbituric acid test.

Pseudo malonaldehyde activity in the thiobarbituric acid test.

In the thiobarbituric acid test for malonaldehyde, 2-hexenal, 2,4-hexadienal and 13-hydroperoxy-9Z,11E-octadecadienoic acid are found to form the characteristic 532 nm chromogen, at pH = 2.7 and at elevated iron(III) levels, faster than malonaldehyde itself. Under the same conditions, but in the presence of EDTA, the chromogen formation is dramatically suppressed. The findings show that malonaldehyde cannot be generated in situ and are interpreted in terms of chromogen formation via an iron catalysed fragmentation of TBA-aldehyde intermediates.

The deoxyribose method: A simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H 2O 2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical “scavengers” compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.

Iodination of nutrients in the presence of chlorine based disinfectants used in drinking water treatment

Under conditions simulating the gastrointestinal tract chlorine dioxide (ClO 2), HOCl, and NH 2Cl caused covalent organification of iodide to nutrient biochemicals. The extent of binding seemed to be proportional to the electromotive force (EMF) and stoichiometry of the redox couple between iodide and the oxidant. Almost half of 71 nutrients examined were found to bind reactive iodine. Iodide was found to inhibit the quinoidal chromogen formation from tyrosine and ClO 2, demonstrating the preferential generation of reactive iodine in complex organic mixtures. These findings indicate that ingestion of residual disinfectants via drinking water may pose a health risk in terms of in vivo generation of iodinated organics. Structure, formation, and biological activity of these compounds are under study.

Peroxidase associated with the root surface of Phaseolus vulgaris

The surface of bean roots demonstrates an intense peroxidase activity which was detected by hydrogen peroxide dependent formation of chromogen from chloronaphthol or dianisidine. Other peroxidase functions, oxidation of indoleacetic acid and NADPH, were catalysed by intact roots and were stimulated by Mn2+ and p-coumarate. Oxidation of NADPH involved superoxide anion [Formula: see text] and hydrogen peroxide formation. Molecular sizing chromatography of root washes demonstrated NADPH oxidase and peroxidase to be associated with higher weight components than indoleacetic acid oxidase. Root surface and root wash peroxidase displayed optimal activity between pH 7 and 8, whereas both sources of indoleacetic acid oxidase were more active at acidic pH. Native poly aery lamide gel electrophoresis of sterile root washes displayed two fast-moving anodic bands, whereas homogenates of the plant roots had several slower moving bands in addition.