Spectrophotometric determination of folic acid in pharmaceutical preparations by coupling reactions with iminodibenzyl or 3-aminophenol or sodium molybdate-pyrocatechol

Novel coupling reagents are used for the simple and sensitive spectrophotometric determination of folic acid either in pure form or in its pharmaceutical preparations. The methods are based on the probable diazotization of the p-aminobenzoylglutamic acid obtained after reductive clevage of folic acid, followed by either coupling with iminodibenzyl to give a violet product with lambda(max) of 580nm or coupling with 3-aminophenol to produce an orange yellow-colored product with lambda(max) of 460nm. Sodium molybdate and pyrocatechol are used in the third method and the pale red-colored product formed has a lambda(max) of 490nm. The methods are highly reproducible and have been applied to the determination of folic acid in tablets and the results compare favorably with the official method. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods.     

Spectrophotometric determination of caffeine and theophylline in pure alkaloids and its application in pharmaceutical formulations

A novel-coupling reagent is used for the simple and sensitive spectrophotometric determination of caffeine (CF) and theophylline (TP) in pure or pharmaceutical formulations. The method is based on the oxidation of CF and TP with sodium metaperiodate in the presence of acetic acid, followed by coupling with 3-methyl 2-benzo thiazolinone hydrazone hydrochloride (MBTH), to produce a blue-colored product with a lambda(max) of 630 nm. The method is reproducible and has been applied for the determination of CF and TP in the tablets. The results are comparable to those obtained with the British pharmacopoeia (BP) method. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed method.     

Spectrophotometric determination of acid mucopolysaccharides

The present report describes a novel spectrophotometric method for the quantitative determination of acid mucopolysaccharides based on the interaction of these macromolecules with the zirconyl ion. The method is simple, accurate, and involves the determination of the acid mucopolysaccharide molecules rather than their hydrolytic components (as in the case of existing methods of analysis). Substances, which are normally present in the acid mucopolysaccharide preparations (such as protein, glycoprotein, and nucleic acid), do not interfere with the determination.     

The adenosine triphosphate-pyrophosphate isotopic exchange reaction: a tool for determination of tryptophan

Quantitative determination of tryptophan at the picomole level is described, using the ATP-[32P]PPi isotopic exchange reaction catalyzed by tryptophanyl-tRNA synthetase. Sensitivity limits of 500 fmol were obtained. The presence of other amino acids at a 1000-fold excess over tryptophan did not interfere significantly with the quantitative determination of tryptophan. The specificity of the reaction was checked using five tryptophan analogs. These analogs did not prevent the determination of tryptophan when present in the same concentration range as tryptophan. When sensitive determination of a single amino acid is needed, the ATP-[32P]PPi exchange reaction catalyzed by aminoacyl-tRNA synthetases is suggested as a general method and as an alternative to HPLC procedures.     

Direct determination of tryptophan using high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Here we present a new method to rapidly quantify tryptophan (Trp) in proteins, animal feed (Mehaden fishmeal), cell cultures, and fermentation broths. Trp is separated from common amino acids by anion-exchange chromatography in 12min and directly detected by integrated pulsed amperometry. The estimated lower detection limit for this method is 1pmol. Alkaline (4M NaOH) hydrolysates can be directly injected, and therefore we used this method to determine the optimum alkaline hydrolysis conditions for the release of Trp from a model protein, bovine serum albumin (BSA). This method accurately determined the Trp content of BSA and fishmeal. High levels of glucose (2%, w/w) do not interfere with the chromatography or decrease recovery of Trp. We used this method to monitor free Trp during an Escherichia coli fermentation.     

Fluorometric determination of trioses with o-phenylphenol.

A spectrophotometric method for determining ascorbic acid based on the redox reaction between a copper (II)-2,2′-biquinoline solution and ascorbic acid was developed. The purple color of the copper (I)-2,2′-biquinoline complex formed in a buffered acetone-water solution was measured at 540 nm. Minerals, sugars, and other vitamins do not interfere when present in quantities usually found in pharmaceutical preparations. Ferrous interference was eliminated by treating the sample solution with the cation-exchange resin Dowex 50W-X12. Several single component and multivitamin formulations were satisfactorily analyzed by this method. Its high sensitivity permits measurements of quantities of ascorbic acid to 3.4 μg/ml of sample solution. The procedure is simple, rapid, and suitable for routine control.     

A colorimetric method for the determination of indole, and its application to assay of tryptophanase.

Indole reacts with sodium nitrite and glycine-HCl buffer, pH 2.6, to form a red color that is stable for more than 1 week. The reaction is reproducible and is linear over a wide range of indole concentrations (0.05–1.00 μmol). Twelve indole derivatives, including tryptophan, and 17 protein amine acids do not interfere. Indole-3-acetic acid, indole-3-acrylic acid, indole-3-pyruvic acid, 5-indole carboxylic acid, and 5-hydroxyindole-3-acetic acid interfere to varying extents (16–27%). Free indole was determined in biological material containing tryptophan by the present method. The method is also applicable to the assay of tryptophanase activity without prior indole extraction.     

Selective spectrofluorimetric method for determination of Lisinopril in pharmaceutical preparations and in presence of hydrochlorothiazide: Application to content uniformity testing

A novel sensitive and cost‐effective spectrofluorimetric method has been developed and validated for determination of lisinopril (an angiotensin converting enzyme inhibitor) in its pure form and pharmaceutical preparations. The method is based on the reaction of the drug with ninhydrin and phenylacetaldehyde in buffered medium (pH 7.0) to form a highly fluorescent product measured at 460 nm after excitation at 390 nm. Different experimental parameters were optimized and calibration curve was constructed. The fluorescence‐concentration relationship was linear in the range of 0.15–4.0 μg mL^?1. The calculated Limit of detection (LOD) and Limit of quantitation (LOQ) were 0.04 and 0.12 μg mL^?1, respectively. The method was successfully applied for the analysis of pharmaceutical preparations containing the studied drug either alone or co‐formulated with hydrochlorothiazide. The obtained results were in agreement with those of the reported method in respect to accuracy and precession. Moreover, the method was applied content uniformity testing according to United States Pharmacopeia (USP) guidelines.     

L-arginine assay with the use of arginase I

A highly selective and sensitive method for the quantitative determination of L-arginine (Arg) with a fluorescent detection of the reaction product has been developed. The method is based on the use of human liver arginase I isolated from a recombinant producer strain, yeast Hansenula polymorpha, and 2,3-butanedione monoxime, which is used to detect carbamide—the product of enzymatic reactions. The linear concentration range for determining Arg in the final reaction mixture varies from 0.2 to 250 μM, and the detection limit is 0.16 μM. Tests of the new method using commercial Arg-containing pharmaceutical preparations showed a high correlation (R = 1.0) of the results with the manufacturer’s data and the results of other methods for Arg detection.     

Highly sensitive spectrofluorimetric method for the determination of the genotoxic methylglyoxal in glycerol-containing pharmaceuticals and dietary supplements

Methylglyoxal (MGO) is a genotoxic α-dicarbonyl compound. Recently, it was found to be formed in glycerol preparations during storage through auto-oxidation. A simple fluorimetric determination of the carcinogenic degradation product of glycerol, MGO, was developed and validated. The proposed method is based on the derivatization of MGO with 4-carbomethoxybenzaldehyde (CMBA) and ammonium acetate to yield a fluorescent imidazole derivative that can be measured at 415 nm after excitation at 322 nm. The optimized conditions were determined to be 0.2 M CMBA, 1.0 M ammonium acetate and a reaction time of 40 min at 90°C using ethanol as diluting solvent. The linear range was 10.0–200.0 ng/ml. Detection and quantification limits were 2.22 and 6.72 ng/ml, respectively. The proposed method was validated according to International Council for Harmonisation (ICH) guidelines and compared with the reported method and no significant difference was found. It was successfully applied for the determination of MGO in six different glycerol-containing pharmaceutical preparations and dietary supplements.     

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.     

A validated silver‐nanoparticle‐enhanced chemiluminescence method for the determination of citalopram in pharmaceutical preparations and human plasma

A simple and sensitive chemiluminescence (CL) method was developed for the determination of citalopram in pharmaceutical preparations and human plasma. The method is based on the enhancement of the weak CL signal of the luminol–H₂O₂ system. It was found that the CL signal arising from the reaction between alkaline luminol and H₂O₂ was greatly increased by the addition of silver nanoparticles in the presence of citalopram. Prepared silver nanoparticles (AgNPs) were characterized by UV–visible spectroscopy and transmission electron microscopy (TEM). Various experimental parameters affecting CL intensity were studied and optimized for the determination of citalopram. Under optimized experimental conditions, CL intensity was found to be proportional to the concentration of citalopram in the range 40–2500 ng/mL, with a correlation coefficient of 0.9997. The limit of detection (LOD) and limit of quantification (LOQ) of the devised method were 3.78 and 12.62 ng/mL, respectively. Furthermore, the developed method was found to have excellent reproducibility with a relative standard deviation (RSD) of 3.65% (n = 7). Potential interference by common excipients was also studied. The method was validated statistically using recovery studies and was successfully applied to the determination of citalopram in the pure form, in pharmaceutical preparations and in spiked human plasma samples. Percentage recoveries were found to range from 97.71 to 101.99% for the pure form, from 97.84 to 102.78% for pharmaceutical preparations and from 95.65 to 100.35% for spiked human plasma. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple colorimetric method for the determination of tryptophan

A simple, sensitive, and reproducible colorimetric method for the determination of tryptophan in amounts as low as 2 μg is described. It is based on the oxidation of tryptophan by sodium nitrite and the coupling of the oxidized product to the leucodye N-1-(naphthyl)ethylenediamine dihydrochloride. The purple-pink product has an absorption maximum at 550 nm. There is no interference by carbohydrates, other amino acids, neutral salts, or a number of other compounds likely to be found in tissue hydrolysates. A number of indole derivatives including indole-3-acetic acid also react to give a colored product. Dipeptides containing tryptophan are much less reactive than free tryptophan; hence proteins must be hydrolyzed completely for the method to be useful. The assay is carried out at room temperature and can be modified easily to increase or decrease its sensitivity. It has been employed to determine the tryptophan content of a number of proteins following alkaline hydrolysis. Generally, values obtained were in close agreement with values reported in the literature.     

A rapid and direct method for the quantitative determination of tryptophan in the intact protein

1. A method is given for the quantitative determination of free tryptophan or tryptophan in the intact protein by treating with ninhydrin in a mixture of formic acid and hydrochloric acid (reagent b), for 10min at 100 degrees C. Glycyltryptophan was used as a standard for the determination of tryptophan in the intact protein. The extinction at 390nm was linear in the range 0.05-0.5mumol for free tryptophan (in7120) and 0.05-0.30mumol for glycyltryptophan (in15400). 2. Free tryptophan in the presence of protein may be determined by treating with ninhydrin in a mixture of acetic acid and 0.6m-phosphoric acid (reagent a) for 10min at 100 degrees C, the extinction being linear for tryptophan in the range 0.05-0.9mumol. N-Terminal tryptophan peptides also give the typical yellow product on treatment with reagent a. 3. Tryptophan content of several pure intact proteins when treated with the above method gave values in good agreement with those reported by others. A mean tryptophan content of 11.25 (s.e.m. +/-0.08) mumol/100mg of protein was found in rat brain during development from 1 to 82 days after birth.     

Microbiological method for the determination of L-tryptophan

Sebek, Oldrich K. (The Upjohn Company, Kalamazoo, Mich.). Microbiological method for the determination of l-tryptophan. J. Bacteriol. 90:1026-1031. 1965.-The ability of Chrombacterium violaceum to utilize l-tryptophan for the synthesis of a purple pigment, violacein, served as a basis for the development of a quantitative estimation of this amino acid. The method consists of suspending washed colorless cells of the organism in an agar layer, placing a paper disc impregnated with a tryptophan solution on top of the layer, and allowing the system to incubate. As tryptophan diffuses into the agar, it is converted into violacein, and appears as a zone of striking purple color. Since the diameter of the zone is a function of the amount of tryptophan applied, the amino acid can be quantitatively estimated within the range of 10 to 320 mug per sample with 5.6% standard deviation. The method is fairly specific for free tryptophan, since only indole, indole-3-pyruvic acid, and, to a small degree, anthranilic acid interfere. Other amino acids, tissue homogenates, tryptophan in peptide linkage, or compounds related to this amino acid do not affect its determination. The bacterium does not utilize tryptophan for the synthesis of cellular material unless its growth has been initiated by another substrate.     

Spectrofluorimetric method for the determination of sulpiride in pharmaceutical preparations and human plasma through derivatization with 2‐cyanoacetamide

A sensitive and accurate spectrofluorimetric method has been developed for the determination of sulpiride in pharmaceutical preparations and human plasma. The developed method is based on the derivatization reaction of 2‐cyanoacetamide with sulpiride in 30% ammonical solution. The fluorescent derivatized reaction product exhibited maximum fluorescence intensity at 379 nm after excitation at 330 nm. The optimum conditions for derivatization reactions were studied and the fluorescence intensity versus concentration plot was found to be linear over the concentration range 0.2–20.0 µg/mL with a correlation coefficient of 0.9985. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.82 and 2.73 ng/mL, respectively. The proposed method was validated according to ICH guidelines. The effects of common excipients and co‐administered drugs were also studied. The accuracy of the method was checked using the standard addition method and percent recoveries were found to be in the range of 99.00–101.25% for pharmaceutical preparations and 97.00–97.80% for spiked human plasma. The method was successfully applied to commercial formulations and the results obtained for the proposed method were compared with a high‐performance liquid chromatography reference method and statistically evaluated using the Student's t‐test for accuracy and the variance ratio F‐test for precision. A reaction pathway was also proposed. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide

A convenient method for postcolumn carbohydrate labeling has been developed. Eluates of borate complex anion-exchange columns are mixed with a reagent solution prepared from an aqueous solution of 2-cyanoacetamide and a borate buffer (pH 10.5), and the mixture is heated in a 10-m reaction coil at 100°C. Measurement of the absorbance of the product at 276 nm permits high reproducibility determination of 5 to 500 nmol of aldoses. Some carbonyl compounds are positive to this reaction, but most do not interfere with the analysis because their peaks do not appear in the aldose region. Ascorbate gives a small peak between those of mannose and fucose, but interference is negligible for equimolar amounts of ascorbate and these aldoses. This method is applied to and gives satisfactory results in the analysis of monosaccharides from various types of glycoconjugates.     

A simple and sensitive spectrofluorimetric method for the determination of furosemide using zinc(II)–1,4‐bis(imidazol‐1‐ylmethyl)benzene complexes

A novel spectrofluorometric method for the determination of furosemide (FUR) is described. The method is based on enhancement of fluorescence emission of FUR in the presence of zinc (II) complexes of 1,4‐bis(imidazol‐1‐ylmethyl)benzene. Under optimum conditions, the enhanced fluorescence intensity is linearly related to the concentration of FUR. The proposed method has been successfully applied to the determination of FUR in pharmaceutical preparations. The possible mechanism of this reaction is discussed briefly based on data from fluorescence spectroscopy, UV–vis absorption and infrared spectroscopy. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive spectrofluorimetric determination of tizanidine in pharmaceutical preparations,human plasma and urine through derivatization with dansyl chloride

A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1‐dimethylaminonaphthalene‐5‐sulphonyl chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511 nm after excitation at 383 nm. The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescence–concentration plots were rectilinear over the ranges 50–500 and 20–300 ng/mL for plasma and urine, respectively, detection limits of 1.81 and 0.54 ng/mL and quantification limits of 5.43 and 1.62 ng/mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t‐ and F‐tests. Copyright © 2011 John Wiley & Sons, Ltd.     

Flow injection chemiluminescence determination of dihydralazine sulphate based on permanganate oxidation sensitized by rhodamine B.

A novel flow injection chemiluminescence (CL) method for the determination of dihydralazine sulphate (DHZS) is described. The method is based on the CL produced during the oxidation of DHZS by acidic permanganate solution in the presence of rhodamine B. Rhodamine B is suggested as a fluorescing compound for the energy-transferred excitation. The CL emission allows quantitation of DHZS concentration in the range 5-800 ng/mL, with a detection limit of 1.9 ng/mL (3sigma). The experimental conditions for the CL reaction are optimized and the possible reaction mechanism is discussed. The method has been applied to the determination of DHZS in pharmaceutical preparations and compares well with the high performance liquid chromatography (HPLC) method.