Anantharaman Shivakumar

A simple and sensitive spectrophotometric method for the determination of trace amounts of nitrite in environmental and biological samples using 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid monosodium salt

A very simple, sensitive, fairly selective and rapid spectrophotometric method for the determination of trace amounts of nitrite has been described. This method is based on the diazotized intramolecular coupling of electrophilic diazonium cation with the phenolic group of 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid monosodium salt (AHNDMS) in a phosphate buffer solution of pH 7.5. The cyclic product has a purple color with maximum absorbance at 560nm and is stable for 6h. Optimum reaction conditions and other important analytical parameters for the maximum color development were established. Beers law was found to obey for nitrite in the concentration range of 0.1-1.6microgml(-1) with molar absorptivity of 2.6x10(4)lmol(-1)cm(-1) and Sandells sensitivity of 0.0075microgml(-1). The effect of interfering ions on the determination is described. The recommended method was applied for the determination of nitrite in different water, soil and human saliva samples. The performance of the recommended method was evaluated in terms of Students t-test and variance ratio F-test, which indicated the significance of proposed method over the reference method.

A sensitive spectrophotometric method for the determination of sulfonamides in pharmaceutical preparations

A sensitive spectrophotometric method for the determination of sulfonamides in pharmaceutical preparations A new, simple and sensitive spectrophotometric method for the determination of some sulfonamide drugs has been developed. The method is based on the diazotization of sulfacetamide, sulfadiazine, sulfaguanidine, sulfamerazine, sulfamethazine, sulfamethoxazole, and their coupling with 8-hydroxyquinoline in alkaline media to yield red coloured products with absorption maxima at 500 nm. Beers law is obeyed from 0.1--7.0 μg mL-1. The limits of quantification and limits of detection were 0.11--0.18 and 0.03--0.05 μg mL-1, respectively. Intraday precision (RSD 0.1--0.5%) and accuracy (recovery 97.3--100.8%) of the developed method were evaluated. No interference was observed from common adjuvants. The method has been successfully applied to the assay of sulpha drug in pharmaceutical formulations. Osjetljiva spektrofotometrijska metoda za određivanje sulfonamida u farmaceutskim pripravcima U radu je opisana nova, jednostavna i osjetljiva spektrofotometrijska metoda za određivanje sulfonamida. Metoda se temelji na prevođenju sulfacetamida, sulfadiazina, sulfagvanidina, sulfamerazina, sulfometazina i sulfametoksazola u diazoderivate koji kondenzacijom s 8-hidroksikinolinom u alkalnom mediju daju crveno obojene produkte s maksimumom apsorpcije pri 500 nm. Beerov zakon vrijedi u koncentracijskom rasponu 0,1--7,0 μg mL-1. Granice kvantifikacije i granice detekcije su 0,11--0,18, odnosno 0,03--0,5 μg mL-1. Za predloženu metodu procijenjena je intermedijarska preciznost (RSD 0,1--0,5%) i točnost (analitički povrat 97,3--100,8). Uobičanjene pomoćne tvari u tabletama ne interferiraju tijekom određivanja. Metoda je uspješno primijenjena za analizu sulfonamida u farmaceutskim pripravcima.

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H(2)O(2)) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H(2)O(2) by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H(2)O(2) is found between 5x10(-6) and 45x10(-6) mol L(-1) at pH 4.0 and a temperature of 25 degrees C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415x10(6) M(-1) min(-1) and 9.81x10(-4) min(-1), respectively. The catalytic constant (k(cat)) and specificity constant (k(cat)/K(m)) at saturated concentration of the cosubstrates were 163.2 min(-1) and 4.156x10(6) L mol(-1) min(-1), respectively. This method can be incorporated into biochemical analysis where H(2)O(2) undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.

Use of N,N-diethyl-p-phenylenediamine sulphate for the spectrophotometric determination of some phenolic and amine drugs

Use of N, N-diethyl-p-phenylenediamine sulphate for the spectrophotometric determination of some phenolic and amine drugs Spectrophotometric methods are proposed for the determination of drugs containing a phenol group [salbutamol sulphate (SLB), ritodrine hydrochloride (RTD), isoxsuprine hydrochloride (IXP)] and drugs containing an aromatic amine group [dapsone hydrochloride (DAP), sulfamethoxazole (SFM), and sulfadiazine (SFD)] in pharmaceutical dosage forms. The methods are based on coupling of N, N-diethyl-p-phenylenediamine sulphate with the drugs in the presence of KIO4 to give a green colored product (λmax at 670 nm) and a red colored product (λmax at 550 nm), respectively. Linear relationships with good correlation coefficients (0.9986-0.9996) were found between absorbance and the corresponding concentration of drugs in the range 1-7, 2-22, 1-17, 1.5-12, 2-25, and 2-21 μg mL-1 for SLB, RTD, IXP, DAP, SFM and SFD, respectively. Variable parameters such as temperature, reaction time and concentration of the reactants have been analyzed and optimized. The RSD of intra-day and inter-day studies was in the range of 0.2-1.0 and 0.4-1.0%, respectively. No interference was observed from common pharmaceutical adjuvants. The reliability and performance of the proposed methods was validated statistically; the percentage recovery ranged from 99.5 ± 0.1 to 99.9 ± 0.3%. Limits of detection were 0.14, 0.21, 0.51, 0.44, 0.33 and 0.37 μg mL-1 for SLB, RTD, IXP, DAP, SFM, and SFD, respectively. Upotreba N, N-dietil-p-fenilenediamin sulfata za spektrofotometrijsko određivanje lijekova iz skupine fenola i amina U radu je predložena spektrofotometrijska metoda za određivanje lijekova s fenolnom skupinom [salbutamol sulfat (SLB), ritodrin hidroklorid (RTD), izoksuprin hidroklorid (IXP)] i lijekova s aromatskom amino skupinom [dapson hidroklorid (DAP), sulfametoksazol (SFM) i sulfadiazin (SFD)] u farmaceutskim dozirnim pripravcima. Metode se temelje na reakciji ljekovitih tvari s N, N-dietil-p-fenilendiamin sulfatom u prisutnosti KIO4, pri čemu nastaje zeleni (λmax pri 670 nm), odnosno crveni produkt (λmax pri 550 nm). Apsorbancije linerano ovise o koncentrancijama lijekova uz visok koeficijent korelacije (0,9986-0,9996) u koncentracijskom području 1-7, 2-22, 1-17, 1,5-12, 2-25 i 2-21 μg mL-1 za SLB, RTD, IXP, DAP, SFM i SFD. Analizirani su i optimirani promjenjivi parametri kao što su temperatura, reakcijsko vrijeme i koncentracija reaktanata. Repetibilnost i intermedijarna preciznost iznosile su 0,2-1,0, odnosno 0,4-1,0%. Nije primjećena nikakva interferencija s uobičajenim farmaceutskim pomoćnim sredstvima. Pouzdanost i izvedbene značajke predložene metode validirane su statistički. Povrat analitičke metode bio je od 99,5 ± 0,1 do 99,9 ± 0,3%. Granice detekcije bile su 0,14, 0,21, 0,51, 0,44, 0,33 i 0,37 μg mL-1 za SLB, RTD, IXP, DAP, SFM, odnosno SFD.

Development of quantitative enzymatic method and its validation for the assay of glucose in human serum.

OBJECTIVE To develop a simple, rapid, sensitive and affordable assay method for the determination of glucose in blood samples using a novel approach. DESIGN AND METHODS A spectrophotometric method for glucose quantification in human serum samples based on self-coupling of activated 2,5-dimethoxyaniline (DMA) in the presence of peroxidase (POD)/glucose oxidase (GOD) and H(2)O(2) is described. H(2)O(2) generated in situ by catalytic reaction between GOD and glucose, activates DMA in the presence of POD to form a green-colored product, which has a strong absorption at λ(max)=740 nm at room temperature (30°C) in a 100 mmol/L acetate/acetic acid buffer of pH 4.2. RESULTS The linearity ranges for the quantification of glucose by rate and one-time detection method are 0.017-0.740 and 0.017-0.478 mmol/L, respectively. Within-day and day-to-day precision were 0.98-1.4% (n=10) and 1.33-2.89% (n=15), respectively. Glucose recoveries ranged from 96.6 to 102%, indicating minimal interference by commonly present interferants in serum samples. Accuracy results were between 90 and 102%. The detection and quantification limits of glucose were 2.376 and 7.923 μmol/L, respectively. The proposed method has good correlation coefficient of 0.999 with the enzymatic kit method. CONCLUSIONS This is a rapid and convenient method to determine serum glucose using simple spectrophotometer with excellent recovery and minimal interference by interferants in serum samples with low detection limit. Therefore, this method can be considered for adoption by the clinical diagnostic laboratories.

Peroxidase-Catalyzed Oxidative Coupling of Paraphenylenediamine with 3-Dimethylaminobenzoic Acid: Application in Crude Plant Extracts

This paper presents a novel spectrophotometric method to measure peroxidase activity using paraphenylenediamine dihydrochloride (PPDD) and 3-dimethylaminobenzoic acid (DMAB). The PPDD traps free radicals and becomes oxidized to electrophillic 1,4-diimine, which couples with DMAB to give an intense green-colored chromogenic species with maximum absorbance at 710 nm. This assay was adopted for the quantification of hydrogen peroxide between 5 and 45 microM. From the kinetic data, a two-substrate ping-pong mechanism of peroxidase was established. The catalytic efficiency and catalytic constant (k(cat)) of the proposed assay were 0.54 x 10(6) M(-1) min(-1) and 0.0436 x 10(3) min(-1), respectively. As a simple, rapid, precise, and sensitive technique, PPDD-DMAB stands as a potential replacement for the traditional guaiacol method. Application of this method in plant extracts opens its relevance in the field of biochemical analysis.

Quantification of creatinine in biological samples based on the pseudoenzyme activity of copper–creatinine complex

Glomerular filtration rate (GFR), the marker of chronic kidney disease can be analyzed by the concentration of cystatin C or creatinine and its clearance in human urine and serum samples. The determination of cystatin C alone as an indicator of GFR does not provide high accuracy, and is more expensive, thus measurement of creatinine has an important role in estimating GFR. We have made an attempt to quantify creatinine based on its pseudoenzyme activity of creatinine in the presence of copper. Creatinine in the presence of copper oxidizes paraphenylenediamine dihydrochloride (PPDD) which couples with dimethylamino benzoicacid (DMAB) giving green colored chromogenic product with maximum absorbance at 710 nm. Kinetic parameters relating this reaction were evaluated. Analytical curves of creatinine by fixed time and rate methods were linear at 8.8-530 μmol L(-1) and 0.221-2.65 mmol L(-1), respectively. Recovery of creatinine varied from 97.8 to 107.8%. Limit of detection and limit of quantification were 2.55 and 8.52 μmol L(-1) respectively whereas Sandells sensitivity and molar absorption coefficient values were 0.0407 μg cm(-2) and 0.1427×10(4) L mol(-1) cm(-1) respectively. Precision studies showed that within day imprecision was 0.745-1.26% and day-to-day imprecision was 1.55-3.65%. The proposed method was applied to human urine and serum samples and results were validated in accordance with modified Jaffes procedure. Wide linearity ranges with good recovery, less tolerance from excipients and application of the method to serum and urine samples are the claims which ascertain much advantage to this method.

Determination of catalase activity using chromogenic probe involving iso-nicotinicacidhydrazide and pyrocatechol.

A biocatalatic pathway involving chromogenic probe has been proposed for the determination of catalase activity by means of iso-nicotinicacidhydrazide (INH) and pyrocatechol (PC). The assay is based on the enzymatic consumption of hydrogen peroxide using INH-PC system. The response of the catalase activity was ascertained by the rate of the reaction involving 14.10mM H(2)O(2). On addition of H(2)O(2), INH-PC indicator system formed a chromogenic product with absorbance maxima at 490 nm. Hence the activity of catalase was directly measured by the chromogenic response in the formation of the coupled product. The catalase assay was elaborated by the kinetic response of the INH-PC system. The linearity of the catalase activity and H(2)O(2) was in the range 0.2-7.0 units and 1.76-7.0mM, respectively in 3 ml solution. The catalytic efficiency and catalytic power were calculated. The Michaelis-Menten constant of INH, PC and H(2)O(2) were found to be 0.344, 0.176 and 8.82 mM, respectively. The indicator reaction was applied in the determination of catalase activity in mycelia mats and culture media.

Spectrophotometric Determination of the Trace Amount of Thallium in Water and Urine Samples by Novel Oxidative Coupling Reaction

A novel, simple, rapid, sensitive and selective method has been proposed for the trace determination of thallium by spectrophotometric detection. This method is based on the oxidation of MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) by thallium(III) to form diazonium cation, which couples with IPH (Imipramine hydrochloride) in phosphoric acid medium at room temperature giving a blue colored species having a maximum absorption at 635 nm. The reagents and manifold variables influences on the sensitivity were investigated and the optimum reaction conditions have been established. The calibration curve was found to be linear over the range 0.1-5 μg mL-1 with the molar absorptivity and Sandell’s sensitivity of 2.9x104 L mol-1 cm-1, 0.0071 μg cm-2 respectively. The tolerance limit of the method towards various ions usually associated with thallium has been detected. The relative standard deviation for five replicate determination of 2μg mL-1 thallium was 0.47%. The method has been successfully applied for the determination of thallium(ІΙΙ) and thallium(I) in synthetic, standard reference materials, water and urine samples with satisfactory results. The performance of the proposed method was evaluated in terms of student’s t-test and variance ratio F-test, to find out the significance of proposed method over the reported methods.

Amaranth dye in the evaluation of bleaching of cerium (IV) by antioxidants: Application in food and medicinal plants

A simple, low-cost, sensitive, and diversely applicable spectrophotometric method for the determination of total antioxidant capacity of several medicinal plants and food has been developed. The method is based on the bleaching of cerium (IV) by antioxidants and dye in slightly acid medium at room temperature. The unbleached dye, imparting pink color to the solution, is measured at λ(max) 530 nm which is directly proportional to the antioxidant concentration. The method is reproducible, and the trolox equivalent antioxidant capacities (TEAC coefficients) of the tested antioxidant compounds were correlated with those found by reference method such as ABTS. The recommended method was applied for the determination of total antioxidant capacity of medicinal and food samples. The performance of the recommended method was evaluated in terms of Students t-test and variance ratio F-test, which indicated the significance of proposed method over the reference method.