A quantitative method for the detection and validation of catalase activity at physiological concentration in human serum, plasma and erythrocytes.

A novel method has been proposed to develop a simple, rapid, sensitive and affordable chromogenic attempt for the quantification of catalase (CAT) activity in blood samples. The method is based on the oxidation of pyrocatechol (PC) to give quinone form which by oxidative coupling with aminyl radical of 4-aminoantipyrine (4-AAP) resulting from H2O2/CAT to produce a pink colored quinone-imine product with λmax = 530 nm in a 100 mmol/L of tris buffer of pH 9.8 at room temperature (30 °C). The linearity of CAT assay was between 0.316 and 10 U/mL. The accuracy ranges for CAT having concentrations of 1.25, 5 and 7.5 µmol/L were 89-105.52, 90-107%, and 91-104.58% respectively. Within-run and between-run precision studies showed CV's of 1.98-3.02% (n = 7) and 2.97-4.40% (n = 7), respectively. The detection and quantification limits of CAT were 0.12 and 0.225 µmol/L, respectively. The Michaelis-Menten constant and maximum velocity of the reaction was Km = 1.052 mM and Vmax = 0.168 µmol/min, respectively. The present method provides a convenient means for investigating the usefulness of CAT measurements in biological sample assessing the potential for free radical-induced pathology.

Ninhydrin-sodium molybdate chromogenic analytical probe for the assay of amino acids and proteins.

A sensitive method has been proposed for the quantification of amino acids and proteins using ninhydrin and sodium molybdate as chromogenic substrates in citrate buffer of pH5.6. A weak molybdate-hydrindantin complex plays the role in the formation of Ruhemann's purple. The linear response for the amino acid, amino acid mixture and Bovine serum albumin is between 0.999 and 66.80µM, 1.52 and 38µM and 5 and 100µg/L, respectively. The molar absorptivity of the individual amino acid by the proposed reaction extends from 0.58×104 to 2.86×104M-1cm-1. The linearity equations for the proposed ninhydrin-molybdate for amino acid mixture is Abs=0.021×Conc (µM)-0.002. The applicability of the proposed method has been justified in food and biological samples in conjunction with Kjeldahl method.

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.

Simple and sensitive method for the quantification of total bilirubin in human serum using 3-methyl-2-benzothiazolinone hydrazone hydrochloride as a chromogenic probe.

We here describe a new spectrophotometric method for measuring total bilirubin in serum. The method is based on the cleavage of bilirubin giving formaldehyde which further reacts with diazotized 3-methyl-2-benzothiazolinone hydrazone hydrochloride giving blue colored solution with maximum absorbance at 630 nm. Sensitivity of the developed method was compared with Jendrassik-Grof assay procedure and its applicability has been tested with human serum samples. Good correlation was attained between both methods giving slope of 0.994, intercept 0.015, and R(2)=0.997. Beers law obeyed in the range of 0.068-17.2 µM with good linearity, absorbance y=0.044 C(bil)+0.003. Relative standard deviation was 0.006872, within day precision ranged 0.3-1.2% and day-to-day precision ranged 1-6%. Recovery of the method varied from 97 to 102%. The proposed method has higher sensitivity with less interference. The obtained product was extracted and was spectrally characterized for structural confirmation with FT-IR, ¹H NMR.

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.

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.

Spectrophotometric method for the determination of chromium (VI) in water samples.

A simple and sensitive spectrophotometric method for the determination of chromium has been developed. The method is based on the diazotization of Dapsone in hydroxylamine hydrochloride medium and coupling with N-(1-Napthyl) Ethylene Diamine Dihydrochloride by electrophilic substitution to produce an intense pink azo-dye, which has absorption maximum at 540 nm. The Beer's law is obeyed from 0.02-1.0 microg mL(-1) and the molar absorptivity is 3.4854 L mol(-1) cm(-1). The Limits of quantification and Limit of detection of the proposed method are 0.0012 microg mL(-1) and 0.0039 microg mL(-1) respectively. The method has been successfully applied for the determination of chromium in water samples and the results were statistically evaluated with that of the reference method.

Sensitive spectrophotometric methods for the assessment of nitrite in water sample.

Two spectrophotometric methods have been developed for the determination of nitrite using dapsone (DAP) with alpha-naphthol and 4-amino-5-hydroxynapthalene-2,7-disulphonic acid monosodium salt (AHNDMS) as chromogenic reagents with maximum absorbance wavelength at 540 and 520 nm respectively. For the method that utilizes dapsone with alpha-naphthol (DAP-alpha-naphthol), the beer's law range is obeyed between 0.05-0.8 microg ml(-1) with molar absorptivity of 5.749 x 104 l mol(-1) cm(-1). The second method that uses dapsone with AHNDMS (DAP-AHNDMS), the beer's law is valid over the range 0.2-1.4 mug ml(-1) and molar absorptivity 2.44 x 104 l mol(-1) cm(-1). The common interfering ions in the analytical procedures have been studied. This proposed methods are reliable, reproducible and have been successfully applied to determine nitrite in various water sources of environmental interest.

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. Beer's law is obeyed from 0.1-7.0 microg mL-1. The limits of quantification and limits of detection were 0.11-0.18 and 0.03-0.05 microg 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.