Development Of RP-HPLC Method Research Articles

Development and Validation of RP HPLC Method for Estimation of Deferiprone and Its Related Impurity in Pharmaceutical Dosage Form

Development and Validation of RP HPLC Method for Estimation of Deferiprone and Its Related Impurity in Pharmaceutical Dosage Form

Stability Indicating RP-HPLC Method Development and Validation for the determination of Pretomanid an anti-bacterial drug

A new simple, rapid, accurate and stability indicating RP-HPLC method have been developed and validated for estimation of Pretomanid is an anti-bacterial agent in pharmaceutical dosage form. The RP-HPLC analysis was performed by using Xtimate C18 column (250mm × 4.6mm; 5µm) with Methanol: Acetonitrile: Ammonium phosphate buffer in the ratio of 55:40:05 (v/v/v) as mobile phase at a flow rate of 1.0mL/min and detected at 262nm wavelength. The calibration curve was linear over the concentration range from 10 to 40µg/mL and correlation coefficient was found to be 0.99. LOD and LOQ were estimated as 0.30µg/mL and 5.0µg/mL respectively. The % recovery was achieved from 98.67% to 99.96%. The method was applied for estimation in its pharmaceutical dosage form and assay result was 98.9% label claim. Proposed method showed excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ, and system suitability results within the acceptance criteria as per ICH guidelines. The Pretomanid drug was subjected to stress condition of Hydrolysis, Oxidation, Photolysis and Thermal degradation, Forced degradation study was carried out and the degradation products were well resolved from the pure Pretomanid drug. The proposed method was successfully applied for the estimation of Pretomanid drugs in commercial dosage form.

A New RP-HPLC Method Development and Validation of Levosulpiride and Rabeprazole

Abstract: The Present study was conducted to obtain a new, affordable, cost-effective and convenient method for RP- HPLC determination of Rabeprazole and Levosulpiride in tablet dosage form. The experiment was carried out according to the official specifications of USP–30, ICH- 1996 and Global Quality Guidelines-2002.The method was validated for the parameters like system suitability , selectivity, linearity, accuracy, precision ,LOD, LOQ, and robustness. 20mg Rabeprazole and 75mg Levosulpiride was dissolve in 100 ml of Diluent (1:1,Methanol:Na2HPO4) and was further diluted to get stock solution of Rabeprazole and Levosulpiride (47.5µg/ml). This is taken as a 100% concentration. Solution containing mixture of Rabeprazole and Levosulpiride of five different concentrations (50%,75%, 100% 125%, and 150% of target concentration) were prepared in the same way. System suitability study of the method was carried out by six replicate analysis of solution containing 100% target concentration of Rabeprazole and Levosulpiride. Various chromatographic parameters such as retention time, peak area tailing factor, theoretical plates (Tangent) of the column and resolution between the peaks were determined and the method was evaluated by analyzing these parameters. . % Recovery was 98.6% for Levosulpiride and 98.7% for Rabeprazole. All the results indicate that the method is highly accurate, retention time for standard sample and commercial product of Rabeprazole and Levosulpiride are same. This proves that, excipients have no effect on the analytical method. On the other hand, blank peak did not overlap drug peak. So the method is highly selective. The method is highly precise as % RSD of peak area was less than 2% in all tests

Derivative Spectroscopic Method and RP-HPLC Method Development and Validation of Levofloxacin hemihydrate

The present work describes the development of a simple, precise and accurate derivative spectroscopic and RP-HPLC method for the estimation of levofloxacin hemihydrate in the tablet dosage form. Levofloxacin hemihydrate showed absorption maxima at 287.36, 287.2 and 296.2nm in zero, first and second-order spectroscopic methods. The RP-HPLC method was optimized by using Orthophosphoric acid: acetonitrile: methanol (50: 20: 30) as solvent at 287nm. The methods showed linearity in the concentration range 1-5 and 2-10 µg/ ml and the correlation coefficient was 0.999 respectively. The method has been validated according to the ICH guidelines. The percentage relative standard deviation was found to be less than 2% in respect of both methods.

RP-HPLC Method Development and Validation for Simultaneous Estimation of Rutin and Quercetin in Morus alba L. leaf extract

Mulberry, which pertains to the Moraceae family and the genus Morus,has been used in medicine for ages because of its chemical composition and pharmacological action. The plant is recognized in traditional Chinese medicine for its antiphlogistic, diuretic, expectorant, and antidiabetic characteristics. The mulberry leaf is high in flavonoids, which have a variety of biological functions, including antioxidant potential. Quercetin, isoquercetin, rutin, isoquercitrin, quercitrin, luteolin, chlorogenic acid, and other flavonoids can be found in their leaves. The major constituents of total flavones in mulberry leaf extract are rutin and quercetin. The goal of the RP-HPLC approach was to design and validate a method for identifying these elements. HPLC (Shimadzu Technologies LC series) with UV-visible detector at 259nm and C18 column (250mm 4.6mm, 5µm particle size) was used to achieve chromatographic separation (Phenomenex Luna). Solvents (acetonitrile and 0.1 percent v/v solution of glacial acetic acid) were used in the isocratic elution mode. The run time was 10 minutes. The column temperature was 37 degrees Celsius with sample insertion volume 20µl and the flow velocity was 1ml/min. For quercetin and rutin, the devised method produced strong linearity, high accuracy, high precision, high repeatability, high robustness, and high ruggedness. For quercetin and rutin, the LOD and LOQs values were determined to be within acceptable levels.

Development and validation of a stability-indicating RP-HPLC method for estimation of Lefamulin in pure and pharmaceutical drug products

BackgroundCommonly occurring serious lung parenchymal infection is community-acquired bacterial pneumonia (CABP). Lefamulin acetate drug products are useful for the treatment of community-acquired bacterial pneumonia (CABP). The main objective of the study was the development and validation of a quick reverse-phase high performance liquid chromatographic analytical testing method for Lefamulin in pure and pharmaceutical dosage forms. The C18 column was the stationary phase for reverse-phase high performance liquid chromatographic separation, and the mobile phase consisted of 0.05 M phosphate buffer with a pH adjustment of 2.5 mixed with an equal volume of acetonitrile. A flow rate of 1.0 mL/min, a column temperature of 30 °C, an injection volume of 10 µL, and a detection wavelength of 210 nm were used as optimum conditions.ResultsThe developed method reported that Lefamulin peak’s retention time was 3.1 min, the average theoretical plate count 5000, and the average peak asymmetry 1.12. In both pure and pharmaceutical dosage forms, the method was found to be accurate, precise, specific, linear calibration curve, and robust. Quantitative assay results from laboratory-made formulations showed similarity to existing commercial products of Lefamulin. Lefamulin is stable in acidic and photolytic environmental circumstances, while it degraded in oxidative, basic, and thermal humidity environment, according to the results of stress studies.ConclusionsThe developed analytical method is fast, economic and stability indicating. It is useful for routine pharmaceutical analysis where the Lefamulin is synthesized in pure form, pharmaceutical tablet formulation, and parenteral dosage form, for their quality and safety.

Development and validation of RP-HPLC method for the simultaneous estimation of rabeprazole sodium and lafutidine in combined dosage form

Development and validation of RP-HPLC method for the simultaneous estimation of rabeprazole sodium and lafutidine in combined dosage form

Development and validation of RP-HPLC method for the simultaneous estimation of rabeprazole sodium and lafutidine in combined dosage form

A simple, rapid, specific, accurate and precise RP-HPLC method was developed for the simultaneous estimation of Lafutidine and Rabeprazole Sodium in combined dosage form. A Hi Q sil C-8 Column (250 mm × 4.6 mm, 5 μm) in Isocratic mode with mobile phase containing Acetonitrile : Phosphate Buffer (70:30 v/v) pH 7.3 was used. The flow rate was 1.0ml/min and effluents were monitored at 230 nm. The retention time of Rabeprazole Sodium and Lafutidine was found to be 3.658 min and 5.408 min respectively. Validation parameters such as linearity, precision, accuracy, limit of detection(LOD), limit of quantification (LOQ), robustness were determined according to the International Conference on Harmonization (ICH) Q2R1 guidelines. The detector response was linear in the range for Lafutidine 10-60 μg/ml and for Rabeprazole Sodium 20-120 μg/ml. The proposed method was successfully applied for the simultaneous estimation of LAF and RAB in pharmaceutical dosage form.

RP-HPLC Method Development for Simultaneous Determination of Phenolic Compounds in Fruit Extracts of Momordica charantia from Different Locations in Malaysia

A sensitive, reproducible, and reliable reversed-phase high performance liquid chromatography (RP-HPLC) method with diode array detection (DAD) was developed and validated. Simultaneous determinations of five compounds; gallic acid, catechin, chlorogenic acid, epigallocatechin gallate and caffeic acid in four types of Momordica charantia extracts; water, ethanol, water:ethanol (1:1) and acetone were conducted. The compounds were successfully separated by C18 column (250 mm × 4.6 mm, 5 µm) with a gradient solvent system of 3% acetic acid in water:methanol:acetonitrile at flow rate of 1.0 mL/min. UV detection was carried out at 280 nm. The standard curves of the five compounds were linear in the range of 0.0396 µg/mL–100 µg/mL. The intra-assay relative standard deviation (RSD) was less than 4.97%, while the inter-assay RSD was less than 4.92%, whereas the accuracy was between 90.96% and 108.92%, respectively. Our optimised RP-HPLC-DAD method was capable to detect flavonoids and phenolic acid contents in four types of M. charantia fruits extracts simultaneously from five locations in Malaysia. The present method is recommended to be used for chemical analyses of phenolic compounds in other Momordica species.

Forced degradation studies and development of RP-HPLC method for related substances of Tenofovir alafenamide in tablet dosage form

The development and validation of an easy and rapid stability-indicating RP-HPLC technique for Tenofovir alafenamide and its impurities. In this newly developed method, chromatographic separation of Tenofovir alafenamide and its impurities was achieved on an Inertsil ODS-3V C18 (250 mm x 4.6 mm, 5 μ) column. The impurities were extracted by a mixture of Mobile Phase A: buffer solution: Acetonitrile: Purified water (20:02:78) and Mobile Phase B: Solvent Mixture and Purified water (75:25), The injection volume was 20 μL, the column temperature was 40°C, the flow rate was 1 mL/min, and the detection was carried out at 262 nm. The retention time of Tenofovir alafenamide, PMPA, PMPA anhydrate, Phenyl PMPA and PMPA isopropyl alaninate were 56.24, 5.69, 8.71, 25.89 and 37.13 respectively. The Correlation Coefficient within the acceptance criteria is not less than 0.999. The evaluated concentrations for Tenofovir alafenamide and its impurities were in the range of 0.5–7.5 ppm. The average recovery value was in the range of 90.2–113.9%. Tenofovir alafenamide’s LOD and LOQ were determined to be 0.1 μg/mL and 0.5 μg/mL respectively. Tenofovir alafenamide solution degradation behaviour was assessed using solution stability studies. A new, accurate, and precise method has been developed, evaluated, and elevated for the detection of impurities in Tenofovir alafenamide tablet dosage form. The results obtained from the validation study established that this method is specific, reliable, precise and effective. As a result, the proposed strategy can serve as an alternate method to determine related substances in routine analysis of tablet dosage form.

Development and Bioanalytical Validation of RP-HPLC Method for the Simultaneous Determination of Perampanel and Carbamazepine in Human Plasma for Therapeutic Drug Monitoring

Development and Bioanalytical Validation of RP-HPLC Method for the Simultaneous Determination of Perampanel and Carbamazepine in Human Plasma for Therapeutic Drug Monitoring

Development and Validation of RP-HPLC Method of Lafutidine (API)

The goal of this study is to develop and validate an RP HPLC technique for estimating Lafutidine in bulk form. A thorough review of the literature found that there are ways available, but none of them are as straightforward, specific, exact, or accurate as the established approach for estimating Lafutidine. The chromatographic method was found to be suitable for effective separation of Lafutidine with good resolution, peak shape. The mobile phase composed of 0.1M ammonium acetate buffer (pH 7.5): Methanol (80:20 v/v), at a flow rate of 1.4 ml/min was selected as it gave well resolved peaks of standard Lafutidine. The optimum wavelength 290nm selected for detection and quantitation. According to ICH criteria, the devised approach was validated.
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RP-HPLC Method Development for Simultaneous Estimation of Brimonidine Tartrate and Timolol Maleate in Ophthalmic Preparation

Introduction: A simple, precise, rapid, and reproducible RP-HPLC method is developed for the simultaneous estimation of brimonidine tartrate and timolol maleate present in ophthalmic dosage forms.
 Method: Gradient RP-HPLC is carried out at 35℃ on column C18 (25 cm X 4.6 mm, 5 µm) with mobile phase a ACN and buffer (25 ml ACN & 475 ml buffer) and mobile phase B water and ACN (37.5 ml water & 212.5 ml ACN) at a flow rate of 1.2ml/min. detection is carried out using DAD detector at 248 nm. Parameter such as linearity, accuracy, precision, specificity, LOD, LOQ and robustness are studied as per ICH guidelines Q2 (R1). 
 Results: The linearity range of Brimonidine tartrate and Timolol maleate are 10-50 µg/ml and 4-20 µg/ml. The accuracy for both Brimonidine Tartrate and Timolol Maleate are in range of 100-101%. The RSD for Brimonidine Tartrate and Timolol Maleate are less than 2 for precision, specificity, robustness respectively. 
 Conclusion: This RP-HPLC method can be highly suitable for analysis of brimonidine tartrate and timolol maleate without interference in combined dosage form.

RP-HPLC Method Development for Simultaneous Estimation of Oral Iron Chelator Deferiprone and its related Impurity

Purpose: This study aims to develop and validate a RP-HPLC method for simultaneous estimation of deferiprone and its synthesized impurity. Method: Structure of impurity was confirmed by spectral analysis while their purity was confirmed by HPLC analysis. The chromatographic separation was achieved on Agilent Zorbax Bonus-RP (250 x 4.6mm, 5µ) using mobile phase of 20 ACN-80 0.1% TFA. Detection was carried out at 280 nm using PDA detector with flow rate of 1 ml/min and temperature of 30℃. Result: The method was validated as per ICH guidelines. The percent recovery was found to be within limit and %RSD for deferiprone and the impurity was found to be 0.09 and 0.48. The standard curve was linear over the concentration range of 60-140µg/ml and shows correlation coefficient (r2) of 1. The LOD and LOQ values for deferiprone were found to be 0.21µg/ml and 0.62µg/ml respectively. The method was found to be robust even by change in flow rate of ±0.2ml/min. Conclusion: Validation studies demonstrated that the proposed HPLC method is specific, reliable, rapid and reproducible. The developed method can be successfully employed for routine analysis for deferiprone in pharmaceutical dosage form.

Development And Validation of RP-HPLC Method For Simultaneous Estimation of Amlodipine and Valsartan In Its Bulk And Tablet Dosage Form by Using The Quality By Design Approach

Development And Validation of RP-HPLC Method For Simultaneous Estimation of Amlodipine and Valsartan In Its Bulk And Tablet Dosage Form by Using The Quality By Design Approach

Development and Validation of Robust, Highly Sensitive and Stability-Indicating RP-HPLC Method for Estimation of Deferasirox and its Degradation Products.

A rapid, simple and highly sensitive stability-indicating reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of Deferasirox (DFS). The chromatographic separation was achieved using a C-18 (250 × 4.6 mm, 5μm) stationary phase and a mobile phase composed of 0.1% orthophosphoric acid and acetonitrile at a flow rate of 1mL/min. The detection was carried out at a wavelength of 245nm with a constant injection volume of 10μL throughout the analysis. With an R2 value of 0.9996, the calibration curve was determined to be linear over an appropriate concentration range of 50-500ng/mL. According to the International Conference on Harmonization (ICH) Q1 (R2) guideline, DFS was evaluated under stress conditions that included hydrolytic (acid, alkali and neutral), oxidative and thermal degradation. The findings demonstrated that significant degradation was observed in acidic degradation conditions, whereas drug substance was found to be stable when exposed to neutral, basic, oxidative and thermal degradation. The developed method was validated as per ICH guidelines. The developed method was employed successfully to estimate the amount of DFS in bulk and pharmaceutical formulation.

RP-HPLC Method Development and Validation for Simultaneous Estimation of Escitalopram Oxalate and Atazanavir Sulphate in Bulk and Dosage Form

Background: This study was designed for the estimation of escitalopram oxalte and atazanavir sulphate in the experimental tablet formulation. The separation was performed at 210 nm by using Inertsil ODS 3V (250 * 4.6 mm, 5.0 m) column and acetonitrile/phosphate buffer (pH =3.55) as the eluent under isocratic elution conditions at a flow rate of 1.0 mL/min. Results: Escitalopram oxalate and atazanavir sulphate peaks were eluted at 2.636 and 3.319 minutes and no interference was observed in the diluent blank at the respective retention times, indicating that the developed method was specific and selective. A linear relationship was found for escitalopram oxalte (2.5 - 15 µg mL-1) and atazanavir sulphate (30 - 150.0 µg mL-1), with coefficient of regression greater than 0.999. Based on the RSD values (< 2.0%) and recovery results (> 98%), the developed method is accurate. At room temperature and in the refrigerated conditions, standard and sample solutions of escitalopram oxalate and atazanavir sulphate have been found to be stable for 48 hours. Conclusion: The established method is linear, precise, accurate, specific, and robust in the studied concentration range. Accordingly, the proposed method can be applied to the analysis of experimental tablet formulations as well as bulk drug samples.

Stability indicating RP-HPLC method development and validation for simultaneous estimation of cilnidipine and bisprolol fumarate in synthetic mixture

A new simple, precise, accurate and selective stability indicating RP-HPLC method has been developed and validated for estimation of Cilnidipine and Bisoprolol fumarate in synthetic mixture. The method was carried out on Hypersil ODS C 5µ column (250 x 4.6 mm) with a mobile phase consisting of Acetonitrile: 0.025 M Dibasic phosphate buffer pH 3.0 with phosphoric acid (70: 30 v/v) and flow rate of 1 mL/min. Detection was carried out at 245 nm. The retention time for Cilnidipine and Bisoprolol fumarate was found to be 3.04 min and 15.114 min, respectively. The Cilnidipine and Bisoprolol fumarate followed linearity in the concentration range of 5 - 25 µg/mL (r= 0.998) and 2.5 - 12.5 µg/mL (r= 0.9993). The developed method was validated for linearity and range, accuracy, precision, and assay. Cilnidipine and Bisoprolol fumarate was subjected to acid and alkali hydrolysis, oxidation and thermal degradation. This indicates that the drug is susceptible to acid, base, oxidation and thermal conditions. The degraded product was well resolved from the pure drug with significantly different Retention time. The proposed method can be used for routine analysis of Cilnidipine and Bisoprolol fumarate in synthetic mixture.

Utility of Quality by Design approach in RP-HPLC method development for quantification of lamivudine and effavirenz in combination formulation

Objective: For the measurement of lamivudine(LAM) and effavirenz (EVZ) in combination formulation, a uncomplicated and reliable liquid chromatographic approach has been proposed.
 Material and Method: Multivariate optimization of the RP-HPLC method, experimental conditions were accomplished using the design of experiments (DoE). The crucial method parameters were determined by a risk assessment. The mathematical models were created using three independent variables: percentage of acetonitrile, percentage of methanol, and buffer pH. The impacts of these independent elements were thoroughly investigated using the central composite design (CCD), which was utilized to analyze the response surface methodology. 
 Result and Discussion: The LAM and EVZ retention time and resolution were both concurrently optimized using the desirability function. Acetonitrile, methanol, and phosphate buffer (pH 7.0) in the proportions of 40:20:40 v/v each were used in the optimized and anticipated data from the contour diagram, with detection occurring at a wavelength of 215 nm. Baseline separation of both pharmaceuticals with high resolution and a run time of under 6 minutes was accomplished under these ideal conditions. The validated test parameters followed ICH recommendations. As a consequence, the findings demonstrated that the Quality by Design methodology could be successfully used to optimize the RP-HPLC technique for the concurrent quantification of LAM and EVZ.

DoE-enhanced development and validation of eco-friendly RP-HPLC method for analysis of safinamide and its precursor impurity: QbD approach

Design of Experiment (DoE) is an effective methodology for studying the relationship between variables and their dependent responses. In this study, DoE was employed for method development and optimization. A binary combination of safinamide mesylate (SAF) and its synthetic precursor p-hydroxybenzaldehyde (PHB) were analyzed by RP-HPLC. Method development focused on evaluating the effect of three independent factors; % EtOH, pH, and flow rate; on performance and efficiency of separation. A rotatable central composite design (CCD) was applied to determine crucial variables and optimize key performance parameters.Significant interest has been shown in green analytical chemistry as a means to reduce the use of hazardous organic solvents without degrading chromatographic performance. A green separation and quantification were performed using a Waters VDSpher PUR 100 C18-E column (5 µm, 250 × 4.6 mm) at a flow rate of 1 mL/min, utilizing abinary combination of mobile phase A: B (60:40, v/v). Phase A of the mobile phase consisted of 0.02 M ammonium formate and 0.02 M ammonium acetate, with the pH adjusted to 5.5 using formic acid. EtOH was utilized as mobile phase B, and 20 µL was employed as the injection volume. The technique was validated in accordance with ICH criteria. SAF had a linear range of 4–100 µg/mL, while PHB had a linear range of 0.2–10.0 µg/mL. Both analytes had R2 values of 0.9999, with detection limits of 0.95 and 0.06 µg/mL, respectively. The designed method could be applicable for regular QC analysis of SAF and its pharmaceutical product. GAPI and AGREE algorithms, which use 12 principles of green analytical chemistry, were used to evaluate the method greenness.