Overlay Spectra Research Articles

A validated HPTLC method for quantification of a new combination containing Lidocaine HCl and Diltiazem HCl

Regulators approve newer medications and medication combinations for the treatment of various disorders. For the treatment of anal fissures, a novel combination of currently available medications, such as lidocaine HCl and diltiazem HCl, has been approved. The current work represents high-performance thin layer chromatographic method to estimate the amounts of lidocaine hydrochloride and diltiazem hydrochloride in a combined gel dosage form called Crema-L gel (2% W/W of lidocaine and 2% W/W of diltiazem hydrochloride). This method has been validated. The mobile phase in this method consisted of a mixture of toluene, methanol, ethyl acetate (7:2: 1% v/v/v) and two drops of ammonia. The stationary phase was pre-coated aluminium sheet with silica gel G60 F254 (10 × 10 cm, 0.2 mm layer thickness). The drugs’ resolved peaks were found at the Rf values of 0.59 for Lidocaine HCl and 0.48 for Diltiazem HCl. The overlay UV spectra of the two medications were used to determine the analytical wavelength and at 220 nm, both drugs showed appreciable absorbance. The concentration range of 400–1200 ng/band was determined to be the linear approach for both lidocaine HCl and diltiazem HCl, as indicated by their respective correlation coefficients (r2) of 0.9987 and 0.9980. The technique for simultaneously estimating lidocaine hydrochloride and diltiazem hydrochloride in bulk and pharmaceutical dosage form was shown to be robust, accurate, precise, and repeatable.Graphical

DEVELOPMENT OF AN ACCURATE UV SPECTROSCOPIC METHOD FOR TINIDAZOLE IN VAGINAL BUFFERS

A precise UV spectroscopic method was developed for accurately quantifying tinidazole (TNZ) using the Shimadzu 1900i UV spectrophotometer. Different sample solutions containing TNZ were scanned across a range of concentration 6 µg mL-1 to 26 µg mL-1 between 200-400 nm, generating overlay TNZ spectra showing an absorption maximum at 318nm in vaginal buffer which is made up of phosphate buffer (PB) 4.2 pH with 2% Tween 80® LR and 4 mL of methanol. The construction of a six-point calibration curve demonstrated the linearity of TNZ within the 6-26 µg mL-1 concentration range. The regression equation derived from this calibration curve was subsequently employed to accurately determine the concentration in accuracy studies. In the analysis of bulk TNZ, a recovery rate ranging from 98.41% to 102.28% was achieved. The analysis results were validated following ICH (International Council on Harmonization) guidelines and compiled with the required criteria and standards

Simultaneous Determination of Luliconazole and Salicylic Acid by Simultaneous Equation Method and Absorbance Ratio Method

The simultaneous determination of Luliconazole and Salicylic Acid in pharmaceutical formulations holds significant importance for ensuring therapeutic efficacy and patient safety. This research focuses on developing an accurate and efficient method using UV-Visible spectrophotometry, specifically the Simultaneous Equation Method (SEM) and Absorbance Ratio Method (ARM), to quantify these active ingredients in topical creams. The study begins with the preparation of stock solutions and selection of the analytical wavelength, with 298 nm chosen in reference to the isobestic point at 230 nm. Cream samples containing 10 mg Luliconazole and 30 mg Salicylic Acid are prepared, sonicated, and filtered for analysis. The UV method is validated in accordance with ICH guidelines, encompassing linearity, precision, LOD, LOQ, specificity, robustness, and accuracy. Calibration curves show excellent linearity within the concentration range of 5-30 µg/ml for both compounds. Intra-day and inter-day precision studies exhibit low % RSD values, indicating the reliability and reproducibility of the method. LOD and LOQ values demonstrate the sensitivity of the method, enabling detection and quantification at trace levels. Specificity is confirmed through overlay spectra and comparisons of analyte responses. Robustness is assessed by varying temperature and concentration, demonstrating the method's resilience to minor changes. Accuracy is evaluated through the standard addition method, yielding recoveries within acceptable limits. The proposed UV method is successfully applied to the analysis of Luliconazole and Salicylic Acid in commercial cream samples, with assay results confirming its accuracy and suitability for routine quality control.

Development and Validation of Simultaneous Estimation of Canagliflozin and Metformin by Q Absorbance Ratio Method in its API and Tablet Formulation

A simple, precise and sensitive UV spectrophotometric method has been developed for the estimation of Canagliflozin and metformin in bulk and pharmaceutical dosage Form by Q Absorbance ratio method. Absorbance uses the ratio of absorbance at two selected wavelengths, one which is an iso absorptive point and other is the λmax of one of the two components. From the overlay spectra of two drugs, it is evident that Canagliflozin and Metformin show an isoabsorptive point at 260nm. The second wavelength used is 235nm which is λmax of Meformin HCl. Beer’s law was obeyed in range of 2-10µg/ml for both Canagliflozin and Metformin HCl. The correlation coefficient was found to be nearer to 0.9990 at both the wavelength for Canagliflozin and Metformin HCl. The result of interday and intraday precision shows standard deviation less than 2% for three concentration and three replicates. The accuracy found to be between 98% - 102%. The LOD and LOQ were found to be within the Linearity Range. The purposed method was novel and can apply for the determination of Canagliflozin and Metformin HCl in Tablet Dosage Form. The method was successfully validated as per the ICH guidelines.

Spectrophotometric simultaneous determination of efonidipine hydrochloride ethanolate and telmisartan in synthetic mixture by first order derivative method

The established method represents the development and validation of a straight forward, precise, accurate, specific and reproducible method for the simultaneous estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan in synthetic mixture. First order derivative of the overlay spectra was used for quantification of both drugs. This method involving the determination of both drugs at their respective zero crossing point working wavelength was observed 231.00 nm (Zero crossing point of Telmisartan) for Efonidipine hydrochloride ethanolate and 238.60 nm (Zero crossing point of Efonidipine Hydrochloride Ethanolate) for Telmisartan using methanol as diluent. International conference on harmonization (ICH) (Q2R1) guideline was used for method validation. The Beer-Lambert’s law obeyed in the concentration range of 2-18 μg/ml and 4-36 μg/ml for Efonidipine Hydrochloride Ethanolate and Telmisartan, respectively. The percentage recovery was found in the range of 98-101 % of Efonidipine Hydrochloride Ethanolate and 98.46-99.77% of Telmisartan. The % relative standard deviation of precision and repeatability study was found beneath 1%. This simple and precise method can be used of both drugs in quality control laboratories. Keywords: Efonidipine Hydrochloride Ethanolate, First order derivative, ICH Q2 (R1), Telmisartan, UV spectroscopy.

A VALIDATED STABILITY INDICATING UV-SPECTROPHOTOMETRIC SIMULTANEOUS ESTIMATION OF ROSUVASTATIN CALCIUM AND FENOFIBRATE IN BULK AND PHARMACEUTICAL FORMULATION

Objective: The present work deals with the development and validation of the absorbance ratio method for the estimation of rosuvastatin calcium and fenofibrate in bulk and pharmaceutical formulation. Studied forced degradation characteristics of bulk and pharmaceutical formulation as per stability guidelines. Methods: The bulk and pharmaceutical formulation studied by the absorbance ratio method. It is the ratio of absorbances at two selected wavelengths. One wavelength is the isoabsorptive point and another wavelength is λ max of one of the components. From the overlay spectra of the two drugs, ROS and FEN showed the isoabsorptive point at 249.5 nm. The second wavelength used was 287 nm, which was the λ max of FEN. Results: The drugs obeyed Beer's law and showed a good correlation. The correlation coefficient for the ROS was 0.999 and for FEN 0.999. The RSD for intraday precision was 0.57 for ROS and 0.057 for FEN. The interday precision was 0.05 for ROS and 0.03for FEN, respectively. The detection limit and quantification limit were found to be 0.048 and 0.14 μg/ml for ROS and 0.069 and 0.21μg/ml for FEN, respectively. More degradation was found in acid hydrolysis and photostability degradation. Conclusion: A simple, precise, accurate, validated, stability-indicating method for simultaneous estimation of rosuvastatin calcium and fenofibrate in bulk and pharmaceutical formulation has been developed.

SIMULTANEOUS ESTIMATION OF ASPIRIN AND OMEPRAZOLE IN LABORATORY SAMPLE BY DIFFERENT UV SPECTROPHOTOMETRIC TECHNIQUES

The aim of the present work was to develop simple, precise and economic UV- spectrophotometric methods for the simultaneous estimation of aspirin and omeprazole in a laboratory sample. The absorbance maxima (λmax) for detection of aspirin and omeprazole were selected as 274 nm and 302 nm, respectively, for simultaneous equation method while wavelength range for detection of aspirin and omeprazole were selected as 270 nm - 276 nm and 300 nm - 305 nm, respectively for area under curve method. Absorbance ratio method uses the ratio of absorbances at two selected wavelengths, one which is an isoabsorptive point and other being the λ max of one of the two components. From the overlay spectra of two drugs, it is evident that aspirin and omeprazole show an isoabsorptive point at 238.6 nm. In zero crossing first derivative spectrophotometry, aspirin showed zero crossing point at 301nm and omeprazole showed zero crossing point at 274nm. Linearity for aspirin was between 25- 125 μg/mL and omeprazole it was 3-15 μg/mL. These methods were successfully applied for estimation of aspirin and omeprazole in the laboratory sample.

Synthesis and Characterization of Homo and Mixed Ligand Complexes of Fe(III) with Hydroxypyridinone and Hydroxypyranone Type Ligands


 Hydroxypyridinone and hydroxypyranone are known to be used for the treatment of iron overload by chelation therapy for a long time. Both the ligands have their own side effects when used as medicines. In the present study homo and mixed ligand complexes of both the ligands with iron were prepared and characterized by UV-visible spectrophotometry, Potentiometric study, IR spectroscopy, SEM/EDX and XRD. Overlay spectra obtained from UV-visible spectroscopy of our studied system show the formation of different types of species and confirm that mixed ligand complex is different from the other two systems. Potentiometric titration curves of homo and mixed ligand complexes show the formation of different types of species at different pH and confirm the formation of mixed ligand complex. The comparative results of SEM images of these systems show different surface topologies and hence conform to the formation of mixed ligand complex.

TO QUANTIFY THE LUTEOLIN CONTENT FROM THE AERIAL PARTS OF HETEROPOGON CONTORTUS (L.) BEAUV. (SPEAR GRASS) THROUGH HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

Objective: The objective of this study was to quantify the luteolin content present in the extracts from different aerial plants (leaves, stem, and inflorescence) of Heteropogon contortus through high-performance thin-layer chromatography.Methods: The chromatographic fingerprint analysis of the different plant extracts has been developed using optimized mobile phase toluene: ethyl acetate: formic acid (5:5:0.7 v/v) and the developed plate is derivatized with freshly prepared anisaldehyde-sulfuric acid. Then, the plate is heated at 110–120°C. The plate is scanned for densitometry measurements and to record the overlay spectra at 366 nm absorbance/reflectance wavelength. Quantification of luteolin marker compound in different extracts of H. contortus is estimated using 2–12 ng/spot.Results: The yellow colored bands appearing on the chromatogram confirm the presence of luteolin marker compound in the different plant samples of H. contortus. Further, the presence of the luteolin marker is confirmed by comparing the Rf values (0.21) of the standard and the samples and from densitometry measurements by scanning the plate at 366 nm absorbance/reflectance. Line-to-line overlay spectra are obtained.Conclusion: From this, it is concluded that leaf sample of H. contortus contains maximum amount of luteolin, i.e., 37.13 ± 0.11 mg/g of dry wt. than inflorescence (1.60 ± 0.013 mg/g of dry wt.) and stem (0.53 ± 0.014 mg/g of dry wt.). The leaves are good source of luteolin and can be used as an alternate natural source to synthesize herbal drugs to cure cancer, hypertension, and inflammatory diseases.

TO QUANTIFY THE LUTEOLIN CONTENT FROM THE AERIAL PARTS OF HETEROPOGON CONTORTUS (L.) BEAUV. (SPEAR GRASS) THROUGH HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

Objective: The objective of this study was to quantify the luteolin content present in the extracts from different aerial plants (leaves, stem, and inflorescence) of Heteropogon contortus through high-performance thin-layer chromatography.Methods: The chromatographic fingerprint analysis of the different plant extracts has been developed using optimized mobile phase toluene: ethyl acetate: formic acid (5:5:0.7 v/v) and the developed plate is derivatized with freshly prepared anisaldehyde-sulfuric acid. Then, the plate is heated at 110–120°C. The plate is scanned for densitometry measurements and to record the overlay spectra at 366 nm absorbance/reflectance wavelength. Quantification of luteolin marker compound in different extracts of H. contortus is estimated using 2–12 ng/spot.Results: The yellow colored bands appearing on the chromatogram confirm the presence of luteolin marker compound in the different plant samples of H. contortus. Further, the presence of the luteolin marker is confirmed by comparing the Rf values (0.21) of the standard and the samples and from densitometry measurements by scanning the plate at 366 nm absorbance/reflectance. Line-to-line overlay spectra are obtained.Conclusion: From this, it is concluded that leaf sample of H. contortus contains maximum amount of luteolin, i.e., 37.13 ± 0.11 mg/g of dry wt. than inflorescence (1.60 ± 0.013 mg/g of dry wt.) and stem (0.53 ± 0.014 mg/g of dry wt.). The leaves are good source of luteolin and can be used as an alternate natural source to synthesize herbal drugs to cure cancer, hypertension, and inflammatory diseases.

Development and validation of UV spectrophotometric method for Simultaneous estimation of Empagliflozin and Metformin hydrochloride in bulk drugs

A new, simple, accurate and sensitive UV-spectrophotometric absorption correction method has been developed for simultaneous determination of Metformin HCl and Empagliflozin in bulk utilizing concept of standard addition. The method is based upon determination of Empagliflozin at 224 nm & Metformin HCl at 230 nm methanol as a solvent. Overlay spectra of both drugs shows absorbance at 227 nm. Linearity was observed in range of 10–50 μg/ml and 1–3 μg/ml for Metformin HCl and Empagliflozin respectively. The correlation coefficient value was found to near to 1. All methods were statistically validated as per ICH guidelines.

Real time band selective F1 -decoupled proton NMR for the demixing of overlay spectra of chiral molecules.

The small chemical shift dispersion and complex multiplicity pattern in proton NMR limit quantifications, for instance the determination of enantiomeric excess (ee) for an enantiomeric mixture. Herein, we present a simple proton-proton correlation experiment with band selective homonuclear (BASH) decoupling in both F1 and F2 dimensions, for the removal of scalar and residual dipolar couplings to provide collapsed singlet for each chemical site. The method has been demonstrated to separate the severely overlapped spectra of enantiomers using both chiral isotropic and anisotropic phases as well as a small biomolecule, particularly for the diastereotopic protons and also for the determination of ee. Copyright © 2016 John Wiley & Sons, Ltd.

Affordable and rapid HPTLC method for the simultaneous analysis of artemisinin and its metabolite artemisinic acid in Artemisia annua L.

Artemisinin (AN) and artemisinic acid (AA), valuable phyto-pharmaceutical molecules, are well known anti-malarials, but their activities against diseases like cancer, schistosomiasis, HIV, hepatitis-B and leishmaniasis are also being reported. For the simultaneous estimation of AN and AA in the callus and leaf extracts of A. annua L. plants, we embarked upon a simple, rapid, selective, reliable and fairly economical high performance thin layer chromatography (HPTLC) method. Experimental conditions such as band size, chamber saturation time, migration of solvent front and slit width were critically studied and the optimum conditions were selected. The separations were achieved using toluene-ethyl acetate, 9:1 (v/v) as mobile phase on pre-coated silica gel plates, G 60F254 . Good resolution was achieved with Rf values of 0.35 ± 0.02 and 0.26 ± 0.02 at 536 nm for AN and 626 nm for AA, respectively, in absorption-reflectance mode. The method displayed a linear relationship with r(2) value 0.992 and 0.994 for AN and AA, respectively, in the concentration range of 300-1500 ng for AN and 200-1000 ng for AA. The method was validated for specificity by obtaining in-situ UV overlay spectra and sensitivity by estimating limit of detection (30 ng for AN and 15 ng for AA) and limit of quantitation (80 ng for AN and 45 ng for AA) values. The accuracy was checked by the recovery studies conducted at three different levels with the known concentrations and the average percentage recovery was 101.99% for AN and 103.84% for AA. The precision was analyzed by interday and intraday precision and was 1.09 and 1.00% RSD for AN and 1.22 and 6.05% RSD for AA. The analysis of statistical data substantiates that this HPTLC method can be used for the simultaneous estimation of AN and AA in biological samples.

Influence of light on phycobiliprotein production in three marine cyanobacterial cultures

Complementary chromatic adaptation, a photomorphogenetic response, known to occur in many cyanobacteria, enables them to efficiently absorb prevalent wavelengths of light in the environment. In the present study, we have described the influence of light on phycobiliprotein production in three marine phycoerythrin producing cyanobacterial cultures, namely, Lyngbya sp. A09DM, Phormidium sp. A27DM and Halomicronema sp. A32DM. A comparative study (UV-visible overlay spectra and SDS-PAGE analyses) of phycobiliproteins purified from all the three cultures grown in white, yellow, red and green lights has been confirmed. White light was taken as control. Red and green lights were taken to check their effect on phycocyanin and phycoerythrin production, respectively. Yellow light was studied as its wavelength falls in between green and red light. Lyngbya sp. A09DM was found to be the best chromatically adapting cyanobacterium followed by Halomicronema sp. A32DM. These two cultures can be placed in group III chromatic adaptors. Phormidium sp. A27DM was the least chromatically adapting culture and can be placed in group II chromatic adaptors. The study signifies that even light plays an important role along with nutrient availability in adapting cultures to changing environmental conditions.

Magnetic Bead-Based Multiplex DNA Sequence Detection of Genetically Modified Organisms Using Quantum Dot-Encoded Silicon Dioxide Nanoparticles

In this study, we report the multiplex DNA detection of genetically modified organisms (GMOs) based on fluorescence and magnetic nanostructures built up with quantum dot-encoded silicon dioxide nanoparticles (QD-encoded SiO2 NPs) and magnetic nanoparticles (MNPs). Detection was achieved by fluorescence spectrophotometry without auxiliary instruments. GMOs, including corn, soybean, colza, and cotton, were simultaneously detected, and the quantitative detection of soybean with a limit of detection of low femtomolar concentrations was successfully achieved. SiO2 NPs were encoded by different colored QDs at various ratios through a reverse microemulsion. QD-encoded SiO2 NP-labeled probe DNAs and MNP-labeled capture DNAs were used to hybridize with the corresponding targets at the same time. After magnetic separation, the sandwich structures were measured by fluorescence spectrophotometry. According to the overlay spectra of QDs, we estimated the presence of corresponding targets by analyzing the results via X-ray photoelectron spectroscopy peak software. Multiplex label-free DNA detection of GMOs based on QD-encoded SiO2 NPs showed high sensitivity and can be applied in other samples.

A new modified ortho cleavage pathway of 3-chlorocatechol degradation by Rhodococcus opacus 1CP: genetic and biochemical evidence.

The 4-chloro- and 2,4-dichlorophenol-degrading strain Rhodococcus opacus 1CP has previously been shown to acquire, during prolonged adaptation, the ability to mineralize 2-chlorophenol. In addition, homogeneous chlorocatechol 1,2-dioxygenase from 2-chlorophenol-grown biomass has shown relatively high activity towards 3-chlorocatechol. Based on sequences of the N terminus and tryptic peptides of this enzyme, degenerate PCR primers were now designed and used for cloning of the respective gene from genomic DNA of strain 1CP. A 9.5-kb fragment containing nine open reading frames was obtained on pROP1. Besides other genes, a gene cluster consisting of four chlorocatechol catabolic genes was identified. As judged by sequence similarity and correspondence of predicted N termini with those of purified enzymes, the open reading frames correspond to genes for a second chlorocatechol 1,2-dioxygenase (ClcA2), a second chloromuconate cycloisomerase (ClcB2), a second dienelactone hydrolase (ClcD2), and a muconolactone isomerase-related enzyme (ClcF). All enzymes of this new cluster are only distantly related to the known chlorocatechol enzymes and appear to represent new evolutionary lines of these activities. UV overlay spectra as well as high-pressure liquid chromatography analyses confirmed that 2-chloro-cis,cis-muconate is transformed by ClcB2 to 5-chloromuconolactone, which during turnover by ClcF gives cis-dienelactone as the sole product. cis-Dienelactone was further hydrolyzed by ClcD2 to maleylacetate. ClcF, despite its sequence similarity to muconolactone isomerases, no longer showed muconolactone-isomerizing activity and thus represents an enzyme dedicated to its new function as a 5-chloromuconolactone dehalogenase. Thus, during 3-chlorocatechol degradation by R. opacus 1CP, dechlorination is catalyzed by a muconolactone isomerase-related enzyme rather than by a specialized chloromuconate cycloisomerase.

The role of superoxide radicals in lactoperoxidase-catalysed H 2O 2 — metabolism and in irreversible enzyme inactivation

Irreversible inactivation of lactoperoxidase in the presence of excess H2O2 has been investigated. Serial overlay absorption spectra of the Soret region show that the rate and total amount of enzyme inactivation depend on the proton concentration. Perhydroxyl or superoxide radicals (HO.2 or O-2) cannot be established as the inactivating species in this mechanism, but they influence the rate of reconversion of the intermediate lactoperoxidase-compound III back to the resting ferric form of the enzyme.