Potassium Dihydrogen Phosphate Buffer Research Articles

RP-HPLC Method development and Validation for Rapid estimation of Diazepam in Bulk and Pharmaceutical Dosage Form

The main objective of the study is to establish simple, precise and rapid RP-HPLC method for determination of diazepam and its decomposition products present in bulk and pharmaceutical dosage form. Various parameters that can potentially affect the analytical method were investigated and optimized. The compounds were well resolved in an isocratic method using the mobile phase composition of potassium dihydrogen phosphate buffer(0.05M) along with diluted o-phosphoric acid adjusted to pH 4.5: methanol (40:60v/v) at a flow rate of 1 ml/min using C18 (25cm x 0.46 cm) Hypersil BDS column. The detection was carried out at wavelength of 248nm.The retention time of diazepam was 3.3 min. The developed method was validated by evaluating various parameters such as linearity, precision, accuracy, robustness, specificity, limit of detection, and limit of quantification according to the international council for harmonization guidelines. The standard calibration curve was obtained in the concentration range of 20µg/ml for diazepam. The % of label claim was found out to be 93.66 for diazepam. Observation of all these parameters leads to the point that developed RP-HPLC method is accurate, precise, linear, specific and robust. It can be successfully adopted for routine quality control analysis of Diazepam in bulk and pharmaceutical dosage form without any interference.

Development and validation of reversed phase HPLC method for determination of apremilast in bulk and pharmaceutical dosage form

The present study describes a novel Reversed Phase High-Performance LC method for the determination and estimation of Apremilast (APR) in bulk and pharmaceutical formulation. The chromatographic separation was carried out on Agilent 1120LC Compact system with Binary gradient system connected to UV- Absorbance Detector. Acetonitrile: (30mM) Potassium dihydrogen phosphate buffer pH adjusted to 3.0 with o-phosphoric acid (60:40 v/v), and flow rate 1.0ml/min with an injection volume of 20 μL was selected as it gave sharp symmetric peak for APL with minimal tailing and with desired elution graph.The separation was carried out at a room temperature and the eluents were observed by UV - Vis detector set at 236 nm. The retention time of APR obtained was at 4.46 minutes. The calibration curve for APR was linear (r2= 0.995) over thetested concentration range of 10 - 60 μg/ ml with LOD and LOQ of 0.93 μg/ml and 2.82μg/ml respectively. A recovery of APR in tablet formulation was observed in the range of 98.40 -100.00 %. Thus the proposed method for APR was found to be feasible for the estimation of APR in bulk as well as pharmaceutical dosage form.

EXPLORATION OF STABILITY-INDICATING ASSAY UHPLC METHOD FOR SIMULTANEOUS ANALYSIS OF SITAGLIPTIN AND METFORMIN IN PURE MATERIAL AND PHARMACEUTICAL DOSAGE FORMS

The scientific novelty of designed work was to develop a specific and precise stability-indicating ultra high performance liquid chromatography (UHPLC) assay method for simultaneous quantification of Sitagliptin and Metforminin extendedrelease fixed dose combinations (FDCs). The reversed-phase UHPLC resolution was analyzed with the assistance of UPLC BEH C18 (150 mm 2.1 mm) with 1.7 µm particle size column at ambient temperature using a solvent system in a proportion of (90:10 % v/v) acetonitrile: potassium dihydrogen orthophosphate buffer; pH 3.0±0.2 was adjusted with 0.1 % ortho phosphoric acid (OPA), with flow rate of 0.4 mL/min of the solvent system. The analytes were supervised at 267 nm by employing photodiode array recognition. The retention times of Sitagliptin and Metformin were found to be1.903±0.01 and 1.301±0.022, respectively. The Sitagliptin and Metformin have confirmed the linearity ranges of 5- 30μg/mL, and 100-600 μg/mL respectively, with 0.9996 and 0.9996 determination coefficients. The UHPLC method was effectually validated concerning the accuracy, precision, sensitivity, robustness, ruggedness, selectivity, and specificity. Moreover, the anticipated UHPLC method's capability to analyze the Sitagliptin and Metformin with no obstruction from degradation products.

Simple and Rapid Method Development for Glycopyrrolate Injection Using High-Performance Liquid Chromatography (HPLC)

The main aim of the present work is to develop a simple and rapid method for determining the impurity profile of related substances in glycopyrrolate from glycopyrrolate injection using high-performance liquid chromatography (HPLC). This method makes use of the C8 (Kromasil 100 250 mm x 4.6mm, 5μ) particle size column for rapid separation of related substances of glycopyrrolate from glycopyrrolate injection along with the related substances using ultraviolet and photodiode array detectors (UV/PDA), pH2.9±0.05 potassium dihydrogen phosphate buffer solution with the column temperature was 45°C, the gradient elution mode, and the wavelength of detection was 222 nm. The procedure was tested in order to prove its efficacy for the intended use. In the case of glycopyrrolate, peak responses were shown to be linear throughout impurity values of 0.05 to 0.75 ppm. For glycopyrrolate, the correlation coefficient was found to be 0.999. This method will be beneficial for determining the impurity profile of glycopyrrolate-related compounds in the injection dosage form of glycopyrrolate. The sample and standard solutions are found to be stable at refrigerator temperature for up to 48 h, while the standard solution is found to be stable on the bench-top for up to 48 h. The results for specificity (degradation), precision, accuracy, LOD, LOQ, and robustness were determined to be satisfactory. The method is simple, rapid, and selective; the described method is precise for estimation of related substances in glycopyrrolate injection. Our results demonstrated that the developed method warrants further development for use in the pharmaceutical industry, such as regular quality control analysis.

Determination of Total Radiochemical Purity of [18F]FDG and [18F]FET by High-Performance Liquid Chromatography Avoiding TLC Method

The goal of this work was to present two high-performance liquid chromatography (HPLC) method that could be applied for the determination of the total radioactive purity of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET). The separation of [18F]fluoride ions, [18F]FET and [18F]FET intermediate was accomplished on LiChrosper RP-18, 250 × 4 mm, 5 µm (Merck) analytical column. For mobile phase 10 mM potassium dihydrogen phosphate buffer at pH7 (A) and acetonitrile (B) was used: 0–2 min: 15% B; 2–12 min: 85% B; 12–15 min: 15% B, respectively. Analysis of [18F]FDG was performed using LiChrosper 100 NH2, 250 × 4.5 mm, 5 µm (Merck) analytical column. The initial mobile phase composition was 10 mM KH2PO4 buffer (pH7) and acetonitrile (15:85, v/v) and the acetonitrile ratio was decreased to 15% at 2 min after the sample injection and held for 5 min. Complete elution of [18F]fluoride ions from stationary phases could be achieved by adding 10 mg/mL K[19F]F to radioactive samples in a ratio 1:1 during the sample preparation. Recovery of [18F]fluoride ions ranged from 99.5 to 100.6%. The validation of the developed methods showed good results for linearity (r2 = 0.9981–0.9996), specificity (RS = 3.7–10.2), repeatability (%Area RSD% = 1.2–4.3%) and limit of quantitation (LOQ = 1.6–4.5 kBq). During the cross-validation similar radiochemical purity values were obtained by the novel HPLC methods and thin layer chromatography performed according to the recommendations of the Ph. Eur. monographs.

A Reverse Phase Stability Indicating HPLC Method Development for Estimation Assay of Benzyl Alcohol and Glycopyrrolate in Glycopyrrolate Injection

Background: The present work is focused on developing a simple stability indicating method for the estimation of the assay of benzyl alcohol and glycopyrrolate in glycopyrrolate injection using reverse high performance liquid chromatography. Methods: A simple, selective, rapid, precise, and gradient reversed-phase high-pressure liquid chromatography procedure has been proposed to estimate the assay of benzyl alcohol and glycopyrrolate for glycopyrrolate injection. It was developed on a Kromasil 100-5, C8 250 mm x 4.6 mm, 5 μm column using mobile phase-A, containing pH 2.5 potassium dihydrogen phosphate buffer and methanol in a ratio of 80:20 v/v. and mobile phase-B, containing pH 2.5 potassium dihydrogen phosphate buffer and methanol in a ratio of 10:90 v/v at a flow rate of 1.5 mL/min. The detection of glycopyrrolate and benzyl alcohol was carried out at 222 nm and 254 nm. A sharp peak was obtained for glycopyrrolate and benzyl alcohol at a retention time of about 8.4 and 4.8 min. Results: The method was validated for specificity (blank, system suitability solution, standard, and sample and stress samples for any interference), precision (six replicate sample preparations), robustness (by slightly varying critical method parameters), and solution stability, and the results were found satisfactory. Conclusion: The method is specific, precise, accurate, and robust for estimation of the assay of benzyl alcohol of glycopyrrolate in glycopyrrolate injection.

DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF METFORMIN, SAXAGLIPTIN, AND DAPAGLIFLOZIN

Objective: A new combination of Metformin, Saxagliptin, and Dapagliflozinfixed-dose combination of antidiabetic medication is being used as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes. Methods: Chromatography was carried out using Kromasil C18 250×4.6, 5.0 mm column with a flow rate of 1.0 ml/min. The mobile phase comprised of pH 4.8 0.01 N Potassium dihydrogen phosphate buffer and Acetonitrile in the ratio of 65:35 is used. The developed method has been validated for various parameters such as precision, accuracy, linearity, robustness, limit of detection (LOD), limit of quantification (LOQ), and solution stability. The stability-indicating capability of the method was established by forced degradation studies under stress conditions such as acid, base, peroxide, UV, thermal, and humidity. Results: The retention times of Metformin, Saxagliptin, and Dapagliflozin were found to be 2.294, 2.869, and 3.887 min, respectively. The method was specific and linear (R2>0.999) for MET, SXG, and DGF. The LOD and LOQ were found to be 13.85 μg/ml and 41.95 μg/ml for MET, 0.06 μg/ml and 0.19 μg/ml for SXG and 0.19 μg/ml and 0.57 μg/ml for DGF, respectively. The mean % recovery obtained was found to be 100.21% for MET, 99.83% for SXG, and 99.97% for DGF, respectively. Conclusion: Hence, the chromatographic method developed was said to be rapid, simple, specific, sensitive, precise, accurate, robust, and reliable that can be effectively applied for routine analysis in research institutions, quality control departments in industries.

High Performance Liquid Chromatographic Method for the Determination of Mesalamine in Bulk Drug and Pharmaceutical Dosage Forms

A simple, rapid and sensitive reverse phase high performance liquid chromatographic method has been developed for the determination of Mesalamine in bulk drug and pharmaceutical dosage forms. It was carried out using a mobile phase of methanol and 0.025 M potassium dihydrogen phosphate buffer (65:35 v/v) on a kromosil C18 column (250 X 4.6 mm, 5µm) in an isocratic mode at a flow rate of 1.0 ml/min. with detection at 250 nm using a UV detector. The linearity was obtained in the concentration range of 20 –100 µg/ml. The developed method was found to be accurate for the determination of Mesalamine in tablet dosage forms.

Validated Stability-Indicating Assay UHPLC Method for Simultaneous Estimation of Doravirine, Lamivudine and Tenofovir Disoproxil Fumarate in Pure Material and Pharmaceutical Matrix

An efficient, robust, and specific stability-indicating UHPLC assay method was acquainted for simultaneous determination of antiretroviral agents doravirine (DOV), lamivudine (LVD), and tenofovir disoproxil fumarate (TDF). The determination of cited agents was performed on UPLC system using UPLC BEH C18 (150 mm × 2.1 mm) with 1.7 µm particle size column at ambient temperature using a solvent system in a proportion of (60:40 % v/v) acetonitrile: potassium dihydrogen orthophosphate buffer; pH 4.5 ± 0.2 was adjusted with 0.1 % OPA. The PDA detector was subjected to a wavelength of 235 nm. DOV, LVD, and TDF were experienced in various stress procedures of forced degradation experiments, as recommended by the ICH, to assess the established method's stability-indicating practicability. Using the optimized procedure, selected agents were successfully distinguished from the peaks of their respective degradation products. Furthermore, the calibration curves for DOV, LVD, and TDF in over wide concentration ranges of 2–12μg/mL, 5–30μg/mL, and 5–30μg/mL, respectively, with determination coefficients (r2) of 0.997, 0.9994, and 0.9994. The method validation assays had less than 2 % relative standard deviations for accuracy, precision, repeatability, and robustness.

BIOANALYTICAL RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF CLOPIDOGREL BISULFATE IN WISTAR RAT PLASMA AND ITS APPLICATION TO PHARMACOKINETIC STUDY

Objective: A novel, simple, precise, accurate, sensitive, and reproducible HPLC method for determining clopidogrel bisulfate in Wistar rat plasma was developed and validated. Methods: The chromatographic separation was performed using Xterra C18 (250 x 4.6 mm, 5μ) column. Mobile phase composed of Acetonitrile ACN: 0.05M potassium dihydrogen orthophosphate buffer pH 4.2 and in the ratio of 75:25% v/v at a flow rate of 1.2 ml/min. Detection was carried out using a PDA detector at 220 nm. The bioanalytical clopidogrel method was validated as per ICH guidelines. Results: The selected chromatographic condition was found to efficiently separate clopidogrel bisulfate (RT-2.838 min). The calibration curve was linear over the concentration range 40-200 ng/ml in Wistar rat plasma with a correlation coefficient of 0.999, respectively. The precision study revealed that the cumulative percentage variation was within the acceptable limit, and accuracy research showed the value of mean percent recovery between 99.72-99.83 %. Conclusion: A simple, rapid, specific, accurate, and precise analytical method was developed and validated using Wistar rat plasma. The technique was strictly validated according to the ICH guidelines. Acquired results demonstrate that the proposed strategy can be effortlessly and advantageously applied for routine analysis of clopidogrel in the Wistar rat plasma.

Stability indicating Rp-UPLC method development and validation for the simultaneous estimation of fosnetupitant and palonosetron in bulk and injection dosage form

A stability indicating UPLC method has been developed and validated for the simultaneous determination of fosnetupitant and palonosetron in bulk and in injection dosage form. This combination is used for the prevention of acute and delayed nausea and vomiting associated with initial and repeated courses of highly emetogenic chemotherapy for cancer. The chromatographic analysis was performed on an HSS, RP C18 column (2.1 x 100 mm, 1.8 µm) with an isocratic mobile phase composed of 0.25 M potassium dihydrogen orthophosphate buffer (pH 6.5), pH adjusted with dilute sodium hydroxide:acetonitrile (55:45 v/v), at a flow rate of 0.5 mL/min, and the eluents were monitored at an isosbestic point of 286 nm. The developed method was validated according to the ICH guidelines pertaining to specificity, precision, accuracy, linearity and robustness, and the stability indicating nature of the method was established by forced degradation studies. The retention times of fosnetupitant and palonosetron were observed at 1.390 and 2.404 min, respectively. The developed method proved to be accurate and precise. Linearity was established between 4.70 and 14.10 µg/mL for fosnetupitant and between 0.05 and 0.15 µg/mL for palonosetron. The LOD and LOQ were 0.115 and 0.385 µg/mL, respectively, for fosnetupitant, and 0.005 and 0.016 µg/mL, respectively, for palonosetron. Therefore, the proposed UPLC method was reliable, reproducible, precise and sensitive for the quantification of fosnetupitant and palonosetron.

Stability indicating RP-HPLC method for the determination of Tenofovir in pharmaceutical formulation

Stability indicating high performance liquid chromatography (HPLC) method was developed for the assay of Tenofovir in bulk and solid dose formulation. The HPLC separation was achieved on kromasil C18 (100mm × 4.6mm, 5 μm) column using a mobile phase of Methanol: Potassium dihydrogen orthophosphate buffer (30:70,v/v) at a flow rate of 1 ml min-1 and UV detection at 260 nm. Peak elutes at 7.33 appropriate. The method was validated for linearity, repeatability, accuracy, precision, robustness, limit of detection and limit of quantification. The accuracy was between 99.14 - 99.97%. The highest R.S.D. amongst interday and Intraday precision was found 0.808 and 0.473 respectively.The assay was linear over the concentration range of 10-50 μg/ml (R≈0. 999). The method was robust as no significant change in chromatographic parameters. LOD and LOQ was found to be 0.90 and 2.71 respectively. The stress studies were performed per ICH guidelines to confirm its Stress testing was carried out in presence of acid, base, hydrogen peroxide, heat and light to demonstrate specificity of the method as per ICH guidelines. The developed method could separate the potential degradation products from the Tenofovir peak. It was concluded that highest degradation occurs in basic condition. This proposed method was suitable and practical for analysis the content of Tenofovir in pharmaceutical products and could be of benefit for the prediction shelf life of Tenofovir in marketed formulations.

DoE Screening and Optimization of Liquid Chromatographic Determination of Nicotinic Acid and Six Statins: Application to Pharmaceutical Preparations and Counterfeit Detection.

An isocratic reversed-phase high performance liquid chromatographic method has been developed and validated to simultaneously determine nicotinic acid, pravastatin sodium, rosuvastatin calcium, atorvastatin calcium, pitavastatin calcium, lovastatin sodium and simvastatin sodium in focus on counterfeit drug detection. Thin-layer chromatography, nuclear magnetic resonance and mass spectrometry have been additionally performed to verify the identification of adulterants of counterfeit herbal medicines. Chromatographic separation was carried out on Inertsil® ODS-3 C18 (4.6 × 150mm, 5μm) with isocratic mobile phase elution containing a mixture of acetonitrile: methanol: 25mM potassium dihydrogen phosphate buffer, pH2.86 adjusted with 0.1M o-phosphoric acid (48: 30: 22, v/v/v), at a flow rate of 1mL/min and with UV detection at 238nm. The design of experiment methodology, Plackett-Burman and Box-Behnken designs, was used to screen and optimize the mobile phase composition. The validation of the method was also carried out under the International Conference on Harmonization guidelines. The developed method was sensitive, accurate, simple, economical and highly robust, in addition to the comprehensiveness and novelty of this method for separating the seven drugs. The results were statistically compared with the reference methods used Student's t-test and variance ratio F-test at P < 0.05.

Environmental benign AQbD based estimation of ketoconazole and beclomethasone by RP-HPLC and multi-analytical UV spectrophotometric method

Hazardous chemicalsare one of the most significant adverse effects of drug analysis. Chemicalwaste generatedat every stage of the analysisfound to bein significant concentrations during quality testing. Green analytical chemistry provides a safe environment for drug analysis. Three simple and environmentally sustainable UV spectrophotometric andliquid chromatographic techniques were established to simultaneously measure Ketoconazole and Beclomethasone dipropionate in their bulk and cream formulation. Firstly, the spectrophotometric estimation deals with three green methods: absorption ratio, the first-order absorption ratio, and the area under curve methods. The second approach was green rotatable centralcomposite analytical quality by design-based RP-HPLC method with an optimized mobile phase of ethanol: 0.1 M potassium dihydrogen phosphate buffer (pH 2.5) 33: 67 v/v. The separation was accomplished using an ODS reversed-phase column (250 × 4.6 mm, 5 µm) operating at a 1.0 mL/min flow rate. The determination was performed using the concentrations ranging for Ketoconazole 140–260 µg/mL and Beclomethasone 1.75–3.75 µg/mL (R2 = 0.9991–0.9999). The results showed that the Forced degradation experiments indicate that the drugs degrade under their intended circumstances. Green solvents were utilized in the developed techniques since they significantly impact environmentally benign analytical methods. The proposed techniques have been validated in linearity, accuracy, and precision based on International Council on Harmonization guidelines and green assessment tools, with acceptable results. These techniques shall be employed in place of methods that utilized toxic chemicals for determination and are easily adaptable for routine analysis.

A new stability indicating RP-UPLC method for simultaneous estimation of Doravirine, Lamivudine and Tenofovir disoproxil fumarate in bulk and their combined pharmaceutical formulation

BackgroundTo establish a simple, sensitive, accurate, precise, efficient, economical RP-UPLC method for simultaneous estimation of Doravirine, Lamivudine and Tenofovir disoproxil fumarate in bulk and their combined pharmaceutical formulations. Optimization of Chromatographic separation was achieved on analytical column HSS C18 (100 × 2.1 mm, 1.8 μ) maintained at temperature 30 °C and mobile phase consisting of 0.01 N Potassium dihydrogen orthophosphate buffer (pH-4.8) and acetonitrile in the ratio 60:40 v/v and at a flow rate 0.3 mL/min in isocratic mode. The injection volume was set as 1 µl detection wavelength is 260 nm. The proposed method validation was done as per International Council on Harmonization Q2 (R1) guidelines.ResultsDoravirine, Lamivudine and Tenofovir disoproxil fumarate were eluted at retention times of 1.2, 1.5, and 1.8 min respectively. The proposed method was identified an excellent linearity over concentration range of 12.5–75.0 µg/mL for Doravirine and 37.5–225.0 µg/mL for Lamivudine and 37.5–225.0 µg/mL for Tenofovir disoproxil fumarate. The percentage relative standard deviation for intra-day and inter-day precision of the present method was less than 2% for Doravirine, Lamivudine and Tenofovir disoproxil fumarate. Accuracy of the present method was evaluated by recovery studies which were in the range of 99.62–99.88% for Doravirine and 98.78–99.44% for Lamivudine and 99.67–100.52% for Tenofovir disoproxil fumarate. The limit of detection and limit of quantification were found to be 0.249 µg/mL and 0.756 µg/mL for Doravirine and 0.24 µg/mL and 0.727 µg/mL for Lamivudine and 0.797 µg/mL and 2.966 µg/mL for Tenofovir disoproxil fumarate. Forced degradation studies were carried out under various stress conditions like acid, base, peroxide, thermal, photo and neutral conditions.ConclusionsThe present method makes sure about no degraded impurity peak interference at the retention time of analyte peak hence can be applied for quality control investigation of Doravirine, Lamivudine and Tenofovir disoproxil fumarate in bulk and pharmaceutical formulations.

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF AZELNIDIPINE AND TELMISARTAN IN TABLET DOSAGE FORM BY RP- HPLC

A simple, accurate and precise method for the simultaneous determination of azelnidipine and telmisartan in bulk drug and pharmaceutical dosage has been developed by RPHPLC method. Separation was performed on a Hyperchrom ODS C18 HPLC Column (250*4.6mm) column and Buffer 0.05M Potassium dihydrogen orthophosphate (KH₂PO₄) Buffer (pH-4.0): Methanol (60:40) as a mobile phase, at a flow rate 1ml/min and UV detection wavelength 215 nm. The calibration of the method was performed by concentration range of 20-60μg/ml for telmisartan and 40-120 μg/ml for azelndipine. The validation of proposed method was carried out for accuracy, precision, ruggedness, specificity for both alzenidipine and telmisartan the method can be used for routine quality analysis of titled drug in tablet formulation.

STABILITY INDICATING HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF EMTRICITABINE, DOLUTEGRAVIR AND TENOFOVIR ALAFENAMIDE IN THEIR COMBINED PHARMACEUTICAL DOSAGE FORM

A simple, rapid, precise and accurate reversed phase HPLC stability indicating method was developed for simultaneous determination of emtricitabine (ECT), dolutegravir (DOL) and tenofovir alafenamide (TEN) in their combined dosage marketed formulation. The chromatographic separation was achieved on Hypersil BDS C18 (250  4.6 mm, 5.0 µ) column using 0.5 M potassium dihydrogen phosphate buffer: acetonitrile (80: 20, v/v), pH 3.5 ± 0.1 maintained by 0.1 % o-phosphoric acid as mobile phase at a flow rate of 1.0 mL/min and monitored at 260 nm. All three analytes were resolved at retention times of 5.777, 6.783, 8.393 min for ECT, DOL, and TEN respectively. The assay performance of the present developed method was validated for various parameters like system suitability, linearity, accuracy, precision, robustness, limit of detection and quantification. The calibration curves were linear over concentration ranges of 12.5-75.0, 50.0-300, and 6.25-37.5 μg/ml for DOL, ECT, and TEN, respectively with correlation coefficients  0.9995. All three analytes were subjected to various stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found in alkaline condition. The present method demonstrated to be effective stability-indicating by resolution of the analytes from their forced-degradation products. The validated method was successfully applied for the simultaneous estimation three drugs in their combined pharmaceutical dosage form Spegra.

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF RELATED SUBSTANCES PRESENT IN THE PHARMACEUTICAL DOSAGE FORM OF EMTRICITABINE, TENOFOVIR ALAFENAMIDE AND DOLUTEGRAVIR

A novel, rapid and an accurate reversed phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantification of emtricitabine and tenofovir related impurities namely emtricitabine s-oxide (emtricitabine impurity-A) and tenofovir (S)-propanol (tenofovir impurity-A), respectively in the combined pharmaceutical dosage form of emtricitabine, dolutegravir and tenofovir alafenamide. The separation was accomplished on Inertsil ODS C18 (250  4.6 mm, 5μ) column using 0.5 M potassium dihydrogen phosphate buffer: acetonitrile (80: 20, v/v), pH 4.5 ± 0.1 maintained by 0.1 % o-phosphoric acid as mobile phase delivered at a flow rate of 1.0 mL/min. Detection was carried out at 260 nm. Emtricitabine impurity-A and tenofovir impurity-A analytes were eluted at retention times of 10.5 and 16.6 min, respectively. The developed method was successfully validated for various parameters like system suitability, linearity, accuracy, precision, robustness, limit of detection and quantification in accordance with ICH guidelines. The calibration curves were linear over the concentration range of 0.125-7.500 μg/ml for emtricitabine impurity-A and tenofovir impurity-A with correlation coefficients  0.998. The validated method was successfully applied for the simultaneous estimation of emtricitabine s-oxide (emtricitabine impurity-A) and tenofovir (S)-propanol (tenofovir impurity-A) in fixed-dose combination comprising of emtricitabine, dolutegravir and tenofovir alafenamide.

Analytical method development and validation for simultaneous estimation of Fimasartan Potassium Trihydrate and Cilnidipine in synthetic mixture by HPLC for the treatment of hypertension stage-II

BackgroundA specific, accurate, precise, robust, and cost-effective HPLC method was developed and validated for quantitative analysis of Fimasartan Potassium Trihydrate and Cilnidipine in fixed-dose combination. The isocratic elution was accomplished by Symmetry C18 column (150 mm × 4.6 mm, 5 µm) at 25 °C. Mobile phase composition is Methanol: Acetonitrile: Potassium Dihydrogen Phosphate buffer (pH 3) (60:05:35%v/v/v) at a flow rate of 1.0 mL/min, injection volume 20 µL with DAD detection at 240 nm.ResultFimasartan Potassium Trihydrate and Cilnidipine were eluted with retention time 2.65 min and 5.51 min respectively. This method was validated as per ICH guideline (Q2 R1). The calibration plots were over the concentration range of 15–90 μg/mL and 2.5–15 μg/mL for Fimasartan Potassium Trihydrate and Cilnidipine with correlation coefficient 0.9992 and 0.9989 respectively. Accuracy was obtained between 99.51–101.65% and 100.06–101.20% for Fimasartan Potassium Trihydrate and Cilnidipine respectively. LOD were found to be 0.97 μg/mL and 0.57 μg/mL and LOQ were found to be 2.95 μg/mL and 1.75 μg/mL for Fimasartan Potassium Trihydrate and Cilnidipine respectively.ConclusionThe results showed that the developed method is reliable for the routine analysis for simultaneous determination of Fimasartan Potassium Trihydrate and Cilnidipine.

Stability Indicating Assay Method Development and Validation of Simultaneous Estimation of Chlorzoxazone, Diclofenac Sodium and Paracetamol in Bulk Drug and Tablet by RP-HPLC

A simple, accurate, specific and stability-indicating RP-HPLC method was developed for simultaneous determination of chlorzoxazone, diclofenac sodium and paracetamol, using C18 Vydac Monomeric 120A (250 × 4.6mm, 5μ) at 40ºC. The mobile phase contains a mixture of 20mM potassium dihydrogen phosphate buffer (pH 6.2 adjusted with potassium hydroxide) and acetonitrile (30:70 v/v). The flow rate was 1ml/min and detection was carried out at 275nm using PDA detector. The retention time of paracetamol, chlorzoxazone and diclofenac sodium were 3.28mins, 13.27mins and 15.61mins respectively. The analytical curve was linear over a concentration range of 0.65- 6.5μg/ml for paracetamol, 1-10μg/ml for chlorzoxazone and 0.1-1μg/ml for diclofenac sodium. The drugs in bulk and tablet were subjected to acid and alkali hydrolysis, oxidation, thermal and photolytic degradation. This method can be successfully employed for simultaneous quantitative analysis of Chlorzoxazone, Diclofenac sodium and Paracetamol in bulk drug and tablet formulation.