Densitometric Measurements Research Articles

Novel Validated HPTLC Method for the Analysis of Two Binary Mixtures Containing Tamsulosin Hydrochloride with Antimuscarinic Agents.

A validated and selective high-performance thin-layer chromatography (HPTLC) method was developed for the analysis of mixures of tamsulosin hydrochloride (TAM) with either tolterodine tartrate (TOL) or solifenacin succinate (SOL) in bulk drug and in combined dosage forms. The proposed method is based on HPTLC separation of the three drugs followed by densitometric measurements of their spots at 224 nm. Separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using ethyl acetate-methanol-ammonia (6:4:0.05, v/v) as mobile phase. The linear regression analysis data were used for the regression line in the range of 0.1-0.7, 0.4-4 and 1-6 μg band-1 for TAM, TOL and SOL, respectively. The proposed method was validated and successfully applied for the analysis of their pharmaceutical formulations and laboratory-prepared mixtures containing the two bicomponent combinations. The method was validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability.

697 Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts

Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. The present study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA). The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA, furthermore, the DNA synthetic activity also rose in a concentration-dependent manenr in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.

Validated spectrophotometric and chromatographic methods for analysis of the recently approved hepatitis C antiviral combination ledipasvir and sofosbuvir

This work describes five simple and reliable spectrophotometric and chromatographic methods for analysis of hepatitis C antiviral binary mixture of ledipasvir (LPV) and sofosbuvir (SBV). Method I is based on the use of Amax and derivative spectrophotometry with the zero-crossing technique where LPV was determined using its Amax and 1D amplitudes at 324 and 338nm respectively, while SBV was determined by measuring the 1D amplitudes at 276nm. Method II involves the application of the ratio spectra derivative spectrophotometry. For LPV, 12μg/mL SBV was used as divisor and the 1DD amplitudes at 239.8nm were plotted against LPV concentrations; while by using 10μg/mL LPV, the amplitudes at 279.2nm were found proportional to SBV concentrations. Method III depends on ratio-difference measurement where the peak to trough amplitudes between 229.2 and 268.4nm were measured and correlated to LPV concentration. Similarly, the amplitudes between 268.6 and 229.2nm in the SBV ratio spectra were recorded. For method IV, the two compounds were separated using HPTLC sheets of silica gel and a mobile phase composed of chloroform-methanol (94:6) followed by densitometric measurement of LPV and SBV spots at 331 and 267nm respectively. Method V depends on HPLC-DAD. Effective chromatographic separation was achieved using Thermohypersil C8 column (4.6×250mm, 5μm) with a mobile phase consisting of 0.01M sodium dihydrogen phosphate (pH 2.5) and methanol (20:80) at a flow rate 1.2mL/min and detection at 332 and 262nm for LPV and SBV respectively. Analytical performance of the developed methods was validated according to the ICH guidelines with respect to linearity, ranges, precision, accuracy, detection and quantification limits. The validated methods were successfully applied to the simultaneous analysis of LPV and SBV in mixtures of different proportions and their combined tablet dosage form.

Speed of sound and density of water + 1,5-pentanediol mixtures in a wide temperature range. Comparison with mixtures of water with diethylene glycol, propoxyethanol and diethylene glycol dimethyl ether

Densities (ρ) and speed of sound (u) were measured in the binary liquid mixture of water and 1,5-pentanediol (PD) over the entire mixture composition range. Densitometric measurement were conducted in a temperature range of 278.15–333.15K under atmospheric pressure, using a precise sealed vibrating-tube densimeter. Acoustic study was carried out in a range of 288.15–318.15K. The values of molar volume (Vm), adiabatic compressibility coefficient (βs), temperature coefficients of these parameters, excess of compressibility coefficient (βsE), excess molar volume (VmE) and derivatives (∂H/∂p), (∂S/∂p) and (∂Cp/∂p) were determined from the experimental data. The values of molar volume were used to determine the values of partial molar volumes of the mixture components. Changes in the measured and calculated values of the physicochemical parameters as a function of temperature and composition of the mixture were analysed in terms of interactions in two-component systems. The obtained results were compared with those received in our earlier investigations of the mixtures of water with diethylene glycol and propoxyethanol as well as with those found in the literature survey concerning water–diethylene glycol dimethyl ether mixtures.

Examination of printability parameters of �Pa free offset printing

Fountain solution system in offset printing system has significant impacts on the printing quality and fountain is the indispensable part of this system. The most important chemical added into the fountain solution is isopropyl alcohol. Fundamental features of isopropyl alcohol are reducing surface tension and adjusting and stabilizing pH. However, in the course of time, the disadvantages of using alcohol have emerged. In the present study, the quality levels of IPA-based and IPA-free offset prints were compared. Specially designed test scale printing was carried out after IPA-based and IPA-free fountain solutions were prepared with the same ink on matt-coated paper under optimal printing conditions. Densitometric and spectrophotometric measurements were carried out on the prints and it was ensured that they were printed in accordance with ISO-12647-2:2013 offset printing standards. Special test scales that were printed were read through scale readers and colour profiles were obtained. Comparisons were carried out on the obtained IPA-free and IPA- based printing colour gamut. Microscopic images of the smallest dots were taken and edge sharpening was examined. Print brightness measurements were carried out for the selected areas on the test scale. The study practically demonstrated that printing performed by using IPA-free fountain solution produced a better and wider colour gamut, edge sharpening was better in the IPA-free systems as the number of prints increased and the prints made by using IPA-free fountain solution were brighter.

Development of a Validated Comparative Stability-Indicating Assay Method for Some H2-Receptor Antagonists.

A comparative force degradation high performance thin layer chromatography (HPTLC) method was developed and validated for some H2-receptor antagonists. The studied H2-receptor antagonists were ranitidine (RAN), nizatidine (NIZ) and famotidine (FAM). The degradation behaviors of the studied H2-receptor antagonists were studied under different stress conditions (hydrolytic, thermal and oxidative) conditions as well as storage conditions according to International Conference on Harmonization (ICH) recommendations. A stability-indicating HPTLC method was optimized in order to separate the analyte from the degradation products formed under various stress conditions. Full separation of the drugs from their degradation products was successfully achieved on an HPTLC precoated silica gel plates. Densitometric measurements were carried out using a Camag TLC Scanner III in the absorbance mode at 320 nm for RAN and NIZ, and 280 nm for FAM. The limits of detection and limits of quantitation range were 5.47-9.37 and 16.30-31.26 ng/band, respectively, for all investigated drugs. The validation studies were performed according to ICH requirements. The developed method was simple, rapid and reliable hence it could be applied for routine quality control analysis of the investigated H2-receptor antagonists in dosage forms. The kinetic behavior, degradation rate constants and half-lives of the degradation of the investigated drugs were studied and compared at different stress conditions. The present study provides, for the first time, a new vision to compare the degradation kinetics of H2-receptor antagonists at the same degradation procedures.

Long-bone properties and development are affected by caponisation and breed in Polish fowls

ABSTRACT1. The aim of the study was to investigate the effects of caponisation on bone development of males of two native breeds in Poland.2. The weight, length and cross-sectional area of tibiae and femora were measured, densitometric measurements and tests of strength were determined and dimensions were calculated.3. Breed and caponisation did not influence bone weight and length. Higher mechanical strength of the femur was found in entire males, mainly in the Polbar breed. Tibial strength was reduced in capons of the Green Partridge breed. Maximum elastic strength was greater in the Polbar, irrespective of caponisation. Bone cross-sectional area was influenced by breed, while caponisation reduced femoral bone mineral density in both breeds.4. Caponisation thus increased growth rate but had adverse effects on bone development.5. Caponisation had fewer negative effects in the Polbar than in the Greenleg Partridge.

Densitometric and local histogram based analysis of computed tomography images in patients with idiopathic pulmonary fibrosis

BackgroundPrior studies of clinical prognostication in idiopathic pulmonary fibrosis (IPF) using computed tomography (CT) have often used subjective analyses or have evaluated quantitative measures in isolation. This study examined associations between both densitometric and local histogram based quantitative CT measurements with pulmonary function test (PFT) parameters and mortality. In addition, this study sought to compare risk prediction scores that incorporate quantitative CT measures with previously described systems.MethodsForty six patients with biopsy proven IPF were identified from a registry of patients with interstitial lung disease at Brigham and Women’s Hospital in Boston, MA. CT scans for each subject were visually scored using a previously published method. After a semi-automated method was used to segment the lungs from the surrounding tissue, densitometric measurements including the percent high attenuating area, mean lung density, skewness and kurtosis were made for the entirety of each patient’s lungs. A separate, automated tool was used to detect and quantify the percent of lung occupied by interstitial lung features. These analyses were used to create clinical and quantitative CT based risk prediction scores, and the performance of these was compared to the performance of clinical and visual analysis based methods.ResultsAll of the densitometric measures were correlated with forced vital capacity and diffusing capacity, as were the total amount of interstitial change and the percentage of interstitial change that was honeycombing measured using the local histogram method. Higher percent high attenuating area, higher mean lung density, lower skewness, lower kurtosis and a higher percentage of honeycombing were associated with worse transplant free survival. The quantitative CT based risk prediction scores performed similarly to the clinical and visual analysis based methods.ConclusionsBoth densitometric and feature based quantitative CT measures correlate with pulmonary function test measures and are associated with transplant free survival. These objective measures may be useful for identifying high risk patients and monitoring disease progression. Further work will be needed to validate these measures and the quantitative imaging based risk prediction scores in other cohorts.

Simultaneous Determination of Guaifenesin, Salbutamol Sulfate or Dextromethorphan HBr and Guaifenesin Impurity (Guaiacol) by HPTLC Method

Simple, selective and sensitive high-performance thin layer chromatographic (HPTLC) method has been developed and validated for the simultaneous determination of Guaifenesin (GUI) and its impurity Guaiacol (GUA), in two ternary mixtures with Salbutamol Sulfate (SAL) or Dextromethorphan HBr (DEX). The separation was carried out on HPTLC silica gel 60 F254 using ethylacetate: hexane: methanol: ammonia (65:35:15:2, by volume) as a developing system followed by densitometric measurement of bands at 227 nm for the first mixture containing GUI, SAL, GUA and by using ethylacetate: hexane: methanol: ammonia (65:35:5:2, by volume) followed by densitometric measurement at 275 nm for the second mixture containing GUI, DEX, GUA. Calibration curves were constructed in the range of (0.2–4 μg/band), (0.2–2 μg/band) and (0.4–1.2 μg/band) with good correlation for GUI, SAL and GUA, respectively, for the first mixture and in the range of (0.4–2 μg/band), (0.2–2 μg/band) and (0.4–1.2 μg/band) with good correlation for GUI, DEX and GUA, respectively, for the second mixture. The developed method was validated according to ICH guidelines and demonstrated good accuracy and precision. The results were statistically compared to the reported methods with no significant difference, indicating the ability of our method to be used for routine analysis of drug product.

Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and HPLC chromatographic methods

Two chromatographic methods have been established and validated for simultaneous determination of mixture of Dimenhydrinate (DMH) and Cinnarizine (CIN) in their pharmaceutical formulation and in presence of Cinnarizine impurity (1-(Diphenylmethyl) piperazine); CIN impurity. The first method was TLC-densitometric one, depends on separation and quantitation of DMH, CIN and CIN impurity on TLC silica gel 60 F254 plates, using chloroform:methanol:glacial acetic acid:ammonia solution (9.5:0.5:0.1:0.1, by volume) as a developing system followed by densitometric measurement at 235nm. Linear relationships were obtained in the range of 0.2–2, 0.4–1.6 and 0.1–1μg/band for DMH, CIN and CIN impurity, respectively. The studied components were well resolved from each other with significantly different Rf values of 0.35, 0.52 and 0.04 for DMH, CIN and CIN impurity, respectively. The second method was RP-HPLC, separation on C8 column using 0.05MKH2PO4 (pH=3):methanol (35:65,v/v) as the mobile phase at a flow rate of 1mL/min and DAD detection at 240nm. Linear relationships were obtained in the ranges of 3–30, 2–20 and 1–10μg/mL, with significantly different Rt values of 3.27, 6.95 and 2.87min for DMH, CIN and CIN impurity, respectively. The developed methods were validated according to ICH guidelines demonstrating good accuracy and precision. The results were statistically compared with those obtained by reported HPLC method and no significant difference was obtained.

HPTLC analysis of ternary mixture containing ketorolac tromethamine, phenylephrine hydrochloride, and chlorpheniramine maleate

ABSTRACTValidated and selective high-performance thin-layer chromatography (HPTLC) method was developed for the determination of ketorolac tromethamine (KTC), phenylephrine hydrochloride (PHE), and chlorpheniramine maleate (CPM) in bulk drug and in combined dosage form. The proposed method depends on using HPTLC for separation of the drugs followed by densitometric measurements of their spots at 261 nm. The separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using chloroform–methanol–ammonia (7.75:2.25:0.1, v/v) as mobile phase. Linear regression lines were obtained over the concentration ranges 0.12–0.50, 0.075–0.27, and 0.09–0.27 µg band−1 for KTC, PHE, and CPM, respectively, with correlation coefficients higher than 0.999. The method was successfully applied to the analysis of the three drugs in their synthetic mixtures and in their dosage form. The mean percentage recoveries were in the range of 98–102% with percentage relative standard deviation values less than 2%. The method was validated according to ICH guidelines and showed good performances in terms of linearity, precision, accuracy, sensitivity, and stability.

Selection of crude Solidago virgaurea L. ssp. extracts based on two fast and efficient chromatographic methods (high-performance thin-layer chromatography or high-performance liquid chromatography) for the quantification of total saponins

A previous study on Solidago virgaurea extracts showed an inhibiting activity of Candida albicans yeast—hyphal transition due to a mixture of triterpene saponins, leading to applications in the field of oral care products. Such applications require the development of an efficient, fast, and simple quantification method of S. virgaurea total saponins. Two methods were developed: the first was based on high-performance liquid chromatography (HPLC) separation with gradient elution and evaporative light scattering detection (ELSD); the second was based on high-performance thin-layer chromatography (HPTLC) separation with in-situ hydrolysis followed by densitometric measurements at 366 nm. Both calibration curves showed good linear regressions (R2 > 0.99) within the range of the concentrations tested. The limit of detection (LOD) and the limit of quantitation (LOQ) were 2–4 μg and 10–16 μg, respectively. The intra- and inter-day variations were studied and found to remain below 5.6% in terms of relative standa...

Purity Indicating TLC Method for Quantitative Determination of Phenylephrine and Dimethindine Maleate in Presence of Dimethindine Maleate Impurity: 2-ethyl pyridine in Nasal Gel

The aim of this work was to establish, optimize and validate a sensitive, selective and accurate thin layer chromatographic (TLC) method for determination of Phenylephrine (PHE), Dimetindene maleate (DM) and its major toxic impurity; 2-ethyl pyridine (EP), in raw material and nasal gel. The proposed method was applied for analysis of PHE, DM and EP over the concentration range of 1.00–10.0, 0.50–8.00 and 2.00–20.0 µg/band, respectively. The method is based on the separation of three components followed by densitometric measurement of the separated peaks at 254 nm. The separation was carried out on silica gel TLC F254 plates, using toluene: acetone: isopropyl alc.: ammonia (5.3: 2.7: 1.8: 0.4, by volume) as a developing system. The suggested method was validated according to International Conference on Harmonization guidelines and can be applied for routine analysis in quality control laboratories. The results obtained by the proposed method were statistically com-pared with the official method revealing high accuracy and good precision.

Different chromatographic methods for the simultaneous determination of vitamin E and vinpocetine in their combined dosage form and in the presence of the alkaline-induced degradation product of vinpocetine

Accurate, selective, and sensitive thin-layer chromatography (TLC)—densitometry and reversed-phase high-performance liquid chromatography (RP-HPLC) methods have been developed and validated for the simultaneous determination of vitamin E (VIT E) and vinpocetine (VINP) in the presence of the alkaline-induced degradation product of vinpocetine (DEG). The proposed TLC—densitometric method depends on the separation and quantitation of VIT E, VINP, and VINP alkaline-induced degradation product on TLC silica gel 60 F254 plates, using methanol—chloroform—ethyl acetate—glacial acetic acid—ammonia solution (6:2:2:0.5:0.1, by volume) as the developing system followed by densitometric measurement at 235 nm. The studied components were well resolved from each other with significantly different RF values of 0.81, 0.62, and 0.41 for VIT E, VINP, and DEG, respectively. On the other hand, the developed RP-HPLC method was based on the separation of the studied components using 0.05 M KH2PO4 (adjusted to pH = 3) and methanol in gradient elution mode on C8 column at a flow rate of 1.5 mL min−1 and ultraviolet (UV) detection at 235 nm. The studied components were well resolved from each other with significantly different Rt values of 10.90, 2.89, and 1.90 min for VIT E, VINP, and DEG, respectively. The developed methods were validated according to the International Conference on Harmonization (ICH) guidelines demonstrating good accuracy and precision. The results were statistically compared with those obtained by the reported method, and no significant difference was found. The developed methods are the first developed stability-indicating assay methods (SIAMs) for the analysis of the studied binary mixture.

DEVELOPMENT OF RP-HPLC, HPTLC AND UV SPECTROPHOTOMETRY METHODS FOR QUANTIFICATION OF DOLUTEGRAVIR SODIUM IN BULK AND FORMULATION.

In the present study, three quantitative methods for dolutegravir sodium (DTG) by RP-HPLC, HPTLC and UV spectrophotometry were developed. RP-HPLC comprises separation on a C4 column by gradient elution using mobile phase consisting of sodium dihydrogen ortho phosphate (pH 3; 10 mM) and acetonitrile at a flow rate of 1 mL/min. The method was successfully applied to study stability under different stress conditions, as recommended by ICH guidelines. In HPTLC, separation and quantitation depends on aluminium plates pre-coated with silica gel 60 F254 as a stationary phase using chloroform: toluene: methanol: acetic acid (8:1:1:0.1, V/V/V/V) as mobile phase followed by densitometric measurement of the bands at 258 nm. In UV spectrophotometry, DTG was quantified by measuring the absorbance at 258 nm using methanol as solvent. The developed methods were validated as per ICH guidelines. The validated methods were successfully applied for determination of the DTG in pharmaceutical formulatios.

Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts.

Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. This study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA) in the concentration range of 5×10-7 to 5×10-3 M. The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA in the concentration range of 5×10-7 to 5×10-4M as compared to that in the GA-free control cultures; furthermore, the DNA-synthetic activity also rose in a concentration-dependent manner in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA at concentrations between 5×10-7 and 5×10-3 M. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.

A validated direct thin-layer chromatographic separation and enantioselective determination of racemic centrally acting drugs using ion-pair and ligand-exchange chiral selectors: A thermodynamic study of complexation

A rapid, inexpensive, and stereoselective densitometric—thin-layer chromatographic (TLC) method using l-(+)-tartaric acid and l-histidine—Cu complex as chiral mobile phase additive for the enantioseparation of atracurium besylate and atropine sulfate, respectively, and quantitative determination of their chiral switching (eutomer) isomers, cisatracurium besylate and hyoscyamine sulfate, were used in this study. The effect on resolution of different chiral selector concentrations, temperatures, and pH values was investigated. The spots were detected with ultraviolet (UV) lamp followed by densitometric measurements at 280 nm and 215 nm for cisatracurium besylate and hyoscyamine sulfate, respectively. The mobile phases enabling successful resolution were acetonitrile—methanol—dichloromethane—glacial acetic acid—H2O containing 70 mg l-(+)-tartaric acid (7:1:0.5:0.7:1, by volume), pH 5 for atracurium besylate, and methanol—H2O containing 40 mmol l-histidine and 20 mmol copper(II) acetate (8.8:1.2, by volume), ...

A Foveon Sensor/Green-Pass Filter Technique for Direct Exposure of Traditional False Color Images

A direct exposure technique to simulate the vegetation red-magenta hues associated with the traditional Kodak Ektachrome Professional Infrared EIR film is proposed. The method uses the Foveon sensor in full spectrum mode as found in Sigma cameras in combination with green-pass filters to reproduce the red-magenta hues of healthy vegetation without resorting to multi-exposures or channel swaps as is the current norm in single sensor cameras. An array of commercially available green-pass filters are analyzed via spectrophotmetric, densitometric and colorimetric measurements. A calibration technique using color-compensating filters is presented such that any green-pass filter will transmit green wavelengths to a set norm and ensure repeatability. A color model for the Foveon/green-pass filter is developed using quantum efficiencies to explain the color effects observed. Color similarities in near-infrared, blue and black and differences in red and green compared with Kodak Ektachrome Infrared film are discussed. Aerial photography examples are discussed, and the technique is proposed as a more direct means of obtaining traditional false color infrared imagery for pictorial and scientific applications when using Foveon type commercially available digital cameras.

Validated chromatographic methods for the simultaneous determination of Mometasone furoate and Formoterol fumarate dihydrate in a combined dosage form

Two chromatographic methods were developed and validated for the simultaneous determination of Mometasone furoate (MO) and Formoterol fumarate dihydrate (FOR). Combination of MO and FOR is used for the treatment of asthma in patients suffering from reversible obstructive airway disease. The first chromatographic method was based on using aluminum TLC plates pre-coated with silica gel GF254 as the stationary phase and chloroform:ethyl acetate:methanol:toluene:formic acid (5:2:2:2:0.1, by volume) as the mobile phase followed by densitometric measurement of the separated bands at 233nm. The second method is a high performance liquid chromatographic method for the separation and determination of MO and FOR using reversed phase C18 column with isocratic elution. The mobile phase composed of methanol: 0.5% ammonium acetate pH adjusted with acetic acid (80:20, v/v) at a flow rate of 1.0mL/min. Quantitation was achieved with UV detection at 220nm. The specificity of the developed methods was investigated by analyzing the pharmaceutical dosage form. The validity of the proposed methods was assessed using the standard addition technique. The obtained results were statistically compared with those obtained by the reported methods, showing no significant difference with respect to accuracy and precision at p=0.05.

Validated spectrophotometric and chromatographic methods for simultaneous determination of ketorolac tromethamine and phenylephrine hydrochloride.

This work describes five simple and reliable spectrophotometric and chromatographic methods for analysis of the binary mixture of ketorolac tromethamine (KTR) and phenylephrine hydrochloride (PHE). Method I is based on the use of conventional Amax and derivative spectrophotometry with the zero-crossing technique where KTR was determined using its Amax and (1)D amplitudes at 323 and 341nm respectively, while PHE was determined by measuring the (1)D amplitudes at 248.5nm. Method II involves the application of the ratio spectra derivative spectrophotometry. For KTR, 12μg/mL PHE was used as a divisor and the (1)DD amplitudes at 265nm were plotted against KTR concentrations; while -by using 4μg/mL KTR as divisor- the (1)DD amplitudes at 243.5nm were found proportional to PHE concentrations. Method III depends on ratio-difference measurement where the peak to trough amplitudes between 260 and 284nm were measured and correlated to KTR concentration. Similarly, the peak to trough amplitudes between 235 and 260nm in the PHE ratio spectra were recorded. For method IV, the two compounds were separated using Merck HPTLC sheets of silica gel 60 F254 and a mobile phase composed of chloroform/methanol/ammonia (70:30:2, by volume) followed by densitometric measurement of KTR and PHE spots at 320 and 278nm respectively. Method V depends on HPLC-DAD. Effective chromatographic separation was achieved using Zorbax eclipse plus C8 column (4.6×250mm, 5μm) with a mobile phase consisting of 0.05M o-phosphoric acid and acetonitrile (50:50, by volume) at a flow rate 1mL/min and detection at 313 and 274nm for KTR and PHE respectively. Analytical performance of the developed methods was statistically validated according to the ICH guidelines with respect to linearity, ranges, precision, accuracy, detection and quantification limits. The validated spectrophotometric and chromatographic methods were successfully applied to the simultaneous analysis of KTR and PHE in synthetic mixtures of different proportions and laboratory-made ophthalmic solution.