Differential Spectrophotometry Research Articles

Characterization of Hydrangea macrophylla Cultivars by the Anthocyanin Content in their Sepals

Abstract In order to ascertain the role of anthocyanin content on the color and brightness of Hydrangea macrophylla sepals, anthocyanin contents of different colored sepals were measured for numerous commercial cultivars. Anthocyanin contents were primarily determined by extraction of the pigment, then measurement by differential spectrophotometry. Concentrations ranged from about 25 to over 400 μg delphinidin-3-glucoside per g of fresh sepal, with the magnitude roughly proportional to the perceived intensity of sepal coloration for that Hydrangea macrophylla cultivar. However, the anthocyanin content was independent of the sepal color, being the same for red, purple, or blue sepals of the same cultivar. Even though significant sepal-to-sepal variation in color intensity existed within a single inflorescence, the extractable anthocyanin content was constant for a specific cultivar. Accordingly, Hydrangea macrophylla cultivars were classified in terms of their color brightness or anthocyanin content in sepals as blush (very light colored, 25 to 60 μg·g−1), cold-hardy (light colored, 80 to 120 μg·g−1), classic (medium colored, 140 to 190 μg·g−1), vivid (deep colored, 230 to 270 μg·g−1), or vibrant (very deep colored, >300 μg·g−1) at peak bloom. The anthocyanin content of the sepals steadily increased as the inflorescence approached peak bloom, remained constant for a week or more, then decreased. The cultivar-dependent anthocyanin contents of the sepals can be used, in part, to rationalize the relative bluing capability of the various Hydrangea macrophylla cultivars.

A method for the assay of total flavonoids in common agrimony herb

A method for assaying total flavonoids in common agrimony (Agrimonia eupatoria L.) herb based on differential spectrophotometry (analytical wavelength 403 nm) and cynaroside State Reference Compound (SRC) was developed. This method was used to analyze a series of raw material samples, which demonstrated that the flavonoid content of common agrimony herb ranged from 1.22% to 1.40% (expressed as cynaroside). Statistical analysis of these experiments showed that the error in single assays of total flavonoids in common agrimony herb was ± 4.02% at the 95% confidence level.

Cluster melting of DNA-actinomycin complexes

Sensitive methods of differential UV spectrophotometry and differential scanning microcalorimetry were used to study the interaction of small and large quantities of the natural antitumor antibiotic actinomycin D with clusters of native and fragmented calf thymus DNA during thermal melting. At micromolar (physiological) concentrations, actinomycin is incorporated in untwisted sites of DNA rather than in the double helix. Actinomycin stabilizes these sites and therefore slightly increases the overall melting temperature of DNA. The antibiotic effectively interacts with the nucleotides of native DNA at a ratio of 1 : 868, especially strongly with the clusters of satellite fractions and DNA fragments. At low concentrations, it stabilizes the "loose" clusters, i.e., those matching untwisted areas that melt first. At high concentrations, it destabilizes the double helix and causes the aggregation of DNA.

Method for Determination of Tinidazole using Direct UV-Visible Spectrophotometry and Differential Spectrophotometry in Pure and Tablet Dosage Forms

Three simple, rapid, selective, precise and accurate spectrophotometric methods for the determination of tinidazole in tablet formulation were developed. The first method was based on the direct absorbance measurements of tinidazole in 0.5 N NaOH with a λmax of 368.6 nm and linearity range of 20 – 150 µg/ml. The second method was based on direct measurement of absorbance at 279.2 nm for tinidazole in 0.5 N HCl whereby the linearity range was 50-150 µg/ml. The third method was based on the differential spectra between tinidazole solution in 0.5 N NaOH and 0.5 N HCl. The maxima recorded was 368.8 nm while the minima was 276 nm with a linearity range of 20 – 120 µg/ml. The methods were validated by determining accuracy, precision, limit of detection, limit of quantitation and performing recovery studies. The developed methods were successfully applied in the analysis of commercial samples of tinidazole and could therefore be used in the routine analysis of tinidazole formulations.

Enhanced spectrophotometric determination of two antihyperlipidemic mixtures containing ezetimibe in pharmaceutical preparations

Two spectrophotometric methods are presented for the simultaneous determination of ezetimibe/simvastatin and ezetimibe/atorvastatin binary mixtures in combined pharmaceutical dosage forms without prior separation. The first is the derivative ratio method where the amplitudes of the first derivative of the ratio spectra ((1) DD) at 299.5 and 242.5 nm were found to be linear with ezetimibe and simvastatin concentrations in the ranges 0.5-20 µgml(-1) and 1-40 µgml(-1) , respectively, whereas the amplitudes of the first derivative of the ratio spectra ((1) DD) at 289.5 and 288 nm were selected to determine ezetimibe and atorvastatin in the concentration ranges 5-50 µgml(-1) and 1-40 µgml(-1) , respectively. The second is the H-point standard additions method; absorbances at the two pairs of wavelengths, 228 and 242 nm or 238 and 248 nm, were monitored while adding standard solutions of ezetimibe or simvastatin, respectively. For the analysis of ezetimibe/atorvastatin mixture, absorbance values at 226 and 248 nm or 212 and 272 nm were monitored while adding standard solutions of ezetimibe or atorvastatin, respectively. Moreover, differential spectrophotometry was applied for the determination of ezetimibe in the two mixtures without any interference from the co-existing drug. This was performed by measurement of the difference absorptivities (ΔA) of ezetimibe in 0.07 M 30% methanolic NaOH relative to that of an equimolar solution in 0.07 M 30% methanolic HCl at 246 nm. The described methods are simple, rapid, precise and accurate for the determination of these combinations in synthetic mixtures and dosage forms.

Antioxidant and Electron Donating Function of Hypothalamic Polypeptides: Galarmin and Gx-NH2

Chemical mechanisms of antioxidant and electron donating function of the hypothalamic proline-rich polypeptides have been clarified on the molecular level. The antioxidant-chelating property of Galarmin and Gx-NH(2) was established by their capability to inhibit copper(II) dichloride catalyzed H(2)O(2) decomposition, thus preventing formation of HO(*) and HOO(*) radicals. The antiradical activity of Galarmin and Gx-NH(2) was determined by their ability to react with 2,2-diphenyl-1-picrylhydrazyl radical applying differential pulse voltammetry and UV-Vis spectrophotometry methods. Galarmin manifest antiradical activity towards 2,2-diphenyl-1-picrylhydrazyl radical, depending on the existence of phenolic OH group in tyrosine residue at the end of the molecule. The presence of antiradical activity and reduction properties of Galarmin are confirmed by the existence of an oxidation specific peak in voltammograms made by differential pulse voltammetry at E ( composite function) = 0.795 V vs. Ag/Ag(+) aq.

Spectroscopic investigations of the chiral interactions of metolachlor and its (S)-isomer with lipase and phosphatase

Metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] is a chiral acetanilide herbicide. We investigated its enantioselective interactions, and that of its (S)-isomer, with Penicillium expansum alkaline lipase and phosphatase. UV differential spectroscopy and fluorescence spectrophotometry studies were conducted in phosphate buffered solution at pH 7. Chiral differences in the UV absorption and fluorescence spectra of lipase and phosphatase with metolachlor and its (S)-isomer were detected. The results showed that the interactions of metolachlor and its (S)-isomer with lipase and phosphatase occur statically through complex formation, and enantioselectivity was clearly observed. In addition, both UV absorption and fluorescence spectrophotometry showed that the (S)-isomer interacted more strongly with lipase and phosphatase than metolachlor.

Quantitative determination of flavonoids from the aerial part of greater knapweed (Centaurea scabinosa L.)

Quantitative determination of flavonoids from the aerial part of greater knapweed (Centaurea scabiosa L.) has been carried out by differential spectrophotometry in combination with a modified method of forming flavonoid complexes with aluminum chloride in HCl. Depending on the plant collection site, the quantitative content of flavonoids in the aerial part of greater knapweed varied within 1.3 - 2.0% (calculated as rutin). The random error of measurement did not exceed 3% at a confidence probability of 0.95. The systematic error of the proposed metohd did not exceed the error of determination. The accumulation dynamics of flavonoids in the aerial part of greater knapweed during the 2006 vegetative peroid have been tracked for plants growing in Tomsk and Kemerovo regions. The maximum content of flavonoids (1.70 ± 0.04% calculated as rutin) was observed in the stage of full flowering, which is therefore the optimum period for collecting the aerial part of the plants for the maximum yield of flavonoids. The maximum amount of flavonoids was accumulated in leaves (1.96 ± 0.04%), which is somewhat greater than the content of flavonoids in the entire aerial part of greater knapweed (1.70 ± 0.04%).

Quantitative determination of the total content of flavonoids in Flavozid and Immunoflazid syrups

New syrup compositions Flavozid and Immunoflazid that contain 2% Proteflazid (ethanol extract of wild Deschampsia caespitosa L. and Calamagrostis epigeios L.) have been developed. The total content of flavonoids in both syrups is of the level of 4 – 6 gmL, which complicates the development of an analytical method. A new method for quantitative determination of flavonoids in the aforementioned syrups has been developed on the basis of differential UV spectrophotometry of their complexes with aluminum chloride. The proposed method is introduced into the Manufacturer’s Pharmacopoeic Article for the syrups. The existing formulation of Flavozid and Immunoflazid syrups is based on the active pharmaceutical ingredient Proteflazid, which was first developed and registered in Ukraine in 2001 and is the alcohol extract of wild Deschampsia caespitosa L. and Calamagrostis epigeios L. Proteflazid prepared using the original technology was recommended for use in medical practice as a composition with direct antiviral and immunomodulating action with properties of an interferon activator and DNA protease inhibitor in virus-infected cells [1 – 3]. The principal biologically active compounds of Proteflazid are flavonoids similar to quercetin (rutin), the molecules of which are based on a flavone oxygen-containing heterocycle. However, the mixture of Proteflazid flavonoids did not contain those that are frequently used as standards. Therefore, the development of a quantitative determination method for flavonoids in the syrup, where the content of Proteflazid and the corresponding flavonoids is 2% (or 4 – 6 gmL) of the starting extract, is of definite scientific interest. The goal of our work was to develop a method that would enable quantitative determination of total flavonoids, which absorb in the UV and visible spectral regions, using aluminum chloride as a complex-forming reagent. The glycoside species were converted to the aglycons by preliminary boiling of the syrup in the presence of HCl in order to increase the total amount of flavonoids in the analytes. Acid hydrolysis of the glucosides in the syrup was carried out in an excess of conc. HCl taken at 1% of the volume of the investigated syrup. The optimum conditions for acid hydrolysis with time were determined from the degree of conversion of the glycoside component into aglycons with TLC monitoring and UV spectrophotometric data. Aglycons from the syrup were extracted by ethylacetate. Solvent was vacuum distilled until production of a pasty residue that was dissolved in ethanol (96%). Studies of the optimum conditions for complexation using freshly prepared aluminum chloride (5%) in ethanol (96%) showed that increasing the volume of the solution to greater than 1 mL in the analyte did not improve the optical density parameters of the aluminum-chloride complex of the flavonoids. The maximum optical density was retained over 25 – 40 min at wavelength 422 nm. A solution with all components of the test solution with the exception of aluminum chloride was used as a reference to compensate for absorption at 420 – 424 nm of the starting extract before complex formation with aluminum chloride. Considering the complex flavonoid composition of the starting material, known standards from various flavonoid groups were used for the quantitative determination. However, the absorption maximum of the complex with aluminum chloride of none of them agreed with that for the com

Changes in electron transport pathways in endoplasmic reticulum of rapeseed in response to cold

We studied changes induced by cold on electron transfer pathways (linked to NADH or NADPH oxidation) in endoplasmic reticulum of rapeseed hypocotyls (Brassica napus L.) from a freezing-sensitive variety (ISL) and freezing-tolerant variety (Tradition). Plantlets were grown at 22 degrees C then submitted to a cold shock of 13 or 35 days at 4 degrees C. We measured the content in NADH, NADPH, NAD and NADP of the hypocotyls and the redox power was estimated by the reduced versus oxidized nucleotide ratio. The contents in cytochromes b (5) and P-450, electron acceptors of NADH and NADPH respectively, were determined by differential spectrophotometry. Finally, activity of both NADH-cytochrome b (5) reductase (E.C.1.6.2.2) and NADPH cytochrome P-450 reductase (E.C.1.6.2.4) was determined by reduction of exogenous cytochrome c. Results show that during cold shock, along with an increase of linolenic acid content, there was a general activation of the NADPH pathway which was observed more quickly in Tradition plantlets than in ISL ones. Due to transfer of electrons that can occur between NADPH reductase and cytochrome b (5), this could favor fatty acid desaturation in Tradition, explaining why linolenic acid accumulation was more pronounced in this variety. Besides, more cytochrome P-450 accumulated in ISL that could compete for electrons needed by the FAD3 desaturase, resulting in a relative slower enrichment in 18:3 fatty acid in these plantlets.

Total content of phenols and anthocyanins in leaves and flowers of some medicinal plants from Bosnia

AIM: The aim of this study was to determine the total content of phenols and anthocyanins in some medicinal plants. METHODS: Total phenols were estimated by a spectrophotometric method, and anthocyanins were measured using pH differential spectrophotometry. RESULTS: The total phenol content in rose was 106.0mg/g. In the leaves of medicinal plants the total phenol content (in mg/g) was as follows: palmarosa (Pelargonium condensatum) 66.7, yew (Taxus baccata) 31.6, lovage (Levisticum officinalis) 19.0, lemon balm (Melissa officinalis) 17.8, peppermint (Mentha piperita) 15.4, garlic (Allium sativum) 15.4, onion (Allium cepa) 14.6, wild garlic (Allium ursinum) 13.3. The total phenol content in a mixture of the leaves and flowers (in mg/g) of some medicinal plants was: yarrow (Achillea millefolium) 46.0, costmary (Chrysanthemum balsamita) 43.0, tansy (Tanacetum vulgare) 13.1. The total anthocyanin content in flowers of some medicinal plants (in mg/g) was: hippeastrum (Amaryllis) 1.04, green-winged orchid (Orchis morio) 0.95, rose (Rosa damascena) 0.047, yew 0,016. CONCLUSION: The highest content of total phenols was found in rose flowers. Also, in the leaves of Palmarosa the highest content of total phenols was found, and the lowest content of total phenols in wild garlic. The highest content of total phenols in a mixture of leaves and flowers was found in yarrow, and the lowest in tansy. The highest content of total anthocyanins was found in flowers of hippeastrum, and the lowest in yew.

Spectroscopic investigations of the chiral interactions between lipase and the herbicide dichlorprop

The enantioselective interaction between penicillium expansum alkaline lipase and chiral phenoxypropionic acid herbicide dichlorprop was studied by using UV differential spectrophotometry and fluorescence spectrophotometry in the presence of a pH 8, phosphate buffer solution. Chiral differences in the UV absorption and fluorescence spectra of lipase with dichlorprop were detected. (R)-Dichlorprop interacted the strongest with lipase as measured by both UV absorption and fluorescence spectrophotometry, followed by (Rac)-dichlorprop, while (S)-dichlorprop had the weakest interaction. The hydrophobic interaction seem to play the dominant role in the interactions and the (R)-enantiomer needed the minimum put of energy to drive the endothermic reaction, while the Rac-type and S-type compounds needed more for the reaction to take place. In the meantime, the catalytic hydrolysis of FDA with lipase show that (R)-DCPP could inhibit lipase the most strongly relatively at the same condition, perhaps because (R)-DCPP had a stronger combining effect and high enantiomeric selectivity on lipase than (Rac)-DCPP and (S)-DCPP.

New and clean strategy for the determination of Cu 2+ in electroless copper plating baths

This article concerns a new and clean strategy for the determination of Cu 2+ in electroless copper plating baths with differential spectrophotometry. With this strategy, the problem of too high absorbance of Cu 2+ under plating conditions has been solved. We investigated the influence of copper sulfate, sodium hypophosphite, nickel sulfate, sodium citrate, polyglycol, temperature and pH on the absorption spectrum of Cu 2+ in electroless copper plating baths. Five grams per litre of CuSO 4·5H 2O solution was selected as the reference solution. Experimental results prove that, this strategy has the merits of fast and high accuracy compared to the traditional techniques. Linearly dependent coefficient of the working curve is 0.9999 and the components in the formula have no obvious effect on the detection of Cu 2+ under experimental conditions. Therefore, we can directly move solutions from the EC plating baths for detection, after that the sample can still go back to the baths without any pollution from the plating process to the environment.

Studies on the oxidation reaction of tyrosine (Tyr) with H 2O 2 catalyzed by horseradish peroxidase (HRP) in alcohol–water medium by spectrofluorimetry and differential spectrophotometry

An oxidation reaction of tyrosine (Tyr) with H 2O 2 catalyzed by horseradish peroxidase (HRP) was studied by spectrofluorimetry and differential spectrophotometry in the alcohol(methanol, ethanol, 1-propanol and isopropanol)–water mutual solubility system. Compared with the enzymatic-catalyzed reaction in the water medium, the fluorescence intensities of the product weakened, even extinguished. Because the addition of alcohols made the conformation of HRP change, the catalytic reaction shifted to the side of polymerization and the polymer (A n H 2, n ≥ 3) exhibited no fluorescence. The four alcohols cannot deactivate HRP. Moreover isopropanol activated HRP remarkably.

Reactions between Human Serum γ-Globulin and Zinc Cations

Interactions of human serum gamma-globulin with zinc cations in solution were studied by differential spectrophotometry in UV light. Supraphysiological concentrations of zinc caused an increment in optical density of protein solution reflecting the effect of gamma-globulin saturation with the metal. Zinc concentrations below physiological led to hypochromism in the protein absorption spectrum. Conformation changes in gamma-globulin during interactions with zinc are analyzed for the surface and intramolecular binding sites and are compared with the effects of copper cations.

Cooperativity and Pseudo-cooperativity in the Glutathione S-Transferase from Plasmodium falciparum

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Biphasic Interaction of γ-Globulin with Copper Cations

Interactions of human serum gamma-globulin with copper cations in solution were studied by differential ultraviolet spectrophotometry. Copper in supraphysiological concentrations increases optical density of protein solution, reflecting the effect of gamma-globulin saturation with metal. In physiological and lower concentrations of copper cations we observed hypochromia in the protein absorption spectrum. Conformational changes in g-globulin molecule during interactions with copper by the surface and intramolecular binding sites and possible role of bivalent metal cations in the maintenance of certain conformations of immunoactive serum proteins are discussed.

Activation of Mucor circinelloides lipase in organic medium

An intracellular Mucor circinelloides lipase either in the form of mycelium-bound enzyme, or in the homogeneous and soluble form, was subjected to activation experiments. It was found that some compounds, such as pyridine, diethanolamine (DEtA), triethanolamine (TEtA), and cetylpyridinium bromide, either increase or decrease the synthetic lipase activity in organic solvents, dependently on their concentration. Differential spectrophotometry of the homogeneous lipase dissolved in toluene, indicate that the variation in the enzyme activity results from an interaction of these substances with the indole group(s) of the tryptophan residue(s), situated on the surface of this enzyme. Our results prove that M. circinelloides lipase is activated not only in the aqueous milieu, but also in organic systems. The molecular background of this phenomenon seems to be similar to the interfacial activation of lipases in the aqueous system (namely the ‘lid’-helix translocation), thought the reason is different.

SPECTROPHOTOMETRIC ANALYSIS OF VALSARTAN AND HYDROCHLOROTHIAZIDE

ABSTRACT Two new methods for the simultaneous determination of valsartan and hydrochlorothiazide in pharmaceutical dosage forms have been developed. The first method, based on compensation technique is presented for the derivative spectrophotometric determination of binary mixtures with overlapping spectra. By using ratios of the derivative maxima or the derivative minimum, the exact compensation of either component in the mixture can be achieved, followed by its determination. The second method, differential derivative spectrophotometry, comprised of measurement of the difference absorptivities derivatized in the first order (ΔD 1) of a tablet extract in 0.1 N NaOH relative to that of an equimolar solution in methanol at wavelengths of 227.8 and 276.5 nm, respectively. Neither sample pre-treatment nor separation were required. These methods showed good linearity, precision, and reproducibility. The results obtained were compared with the results of reversed phase HPLC. It was found that the difference was not statistically important between these methods. The proposed methods were accurate, sensitive, precise, reproducible and could be applied directly and easily to the pharmaceutical preparations.

DERIVATIVE RATIO SPECTROPHOTOMETRY AND DIFFERENTIAL DERIVATIVE SPECTROPHOTOMETRIC DETERMINATION OF ISONIAZID AND PYRIDOXINE HYDROCHLORIDE IN DOSAGE FORMS

Derivative ratio spectrophotometric and differential derivative spectrophotometric methods, for the determination of isoniazid and pyridoxine hydrochloride in pharmaceutical dosage forms (tablets) is described. The first method depends on ratio derivative spectra spectrophotometry where isoniazid and pyridoxine hydrochloride were determined by the measurement of the first derivative ratio spectra of amplitudes (Abs.) at 250.7 and 305.8 nm, respectively. The other method, is based on differential derivative spectrophotometry for the simultaneous determination of isoniazid and pyridoxine hydrochloride in binary mixture without any pre-treatment by measuring the ΔD1 values. Both methods showed good linearity, precision and reproducibility. The proposed methods were successfully applied to the pharmaceutical dosage forms containing the above-mentioned drug combination without any interference by the excipients.