Potassium Dihydrogen Phosphate Buffer Research Articles

RP-HPLC-UV Detection Method for the Simultaneous Determinat ion of Econazole Nitrate, Triamcinolone Acetonide and Benzoic Acid in Ternary Mixture

Background:Econazole nitrate and triamcinolone acetonide are one of the topical antifungal corticosteroid fixed combinations. Although this combination has been literately investigated, yet their ternary mixture with benzoic acid as preservative lack of a simple method for its determination.Objective:The objective of this work is to present simple, accurate, precise and low cost reversed-phase liquid chromatography (RP-LC) method for the separation and determination of econazole nitrate, triamcinolone acetonide and benzoic acid in a ternary mixture.Methods:Separation was conducted on Mediterranea C18 (150mm x 4.6mm, 5μm) column using methanol: 50 mM potassium dihydrogen phosphate buffer (pH 2.60±1), (70:30, v/v) as a mobile phase at ambient temperature. Isocratic flow rate at 1 ml/min was used and UV detection was carried out at 230 nm. The Linearity, accuracy and precision of the developed method investigated as per ICH guidelines.Results:Good separation of two drugs, benzoic acid as preservative and excipients of dosage form was achieved with acceptable retention times (< 8 min). Validation parameters tested including linearity, accuracy and precision were found to be satisfactory over the concentration range (10-200 µg/ml) for econazole nitrate, (1-20 µg/ml) for triamcinolone acetonide and (2-40 μg/ml) for benzoic acid.Conclusion:The proposed method showed good separation and demonstrated adequate specificity, robustness and accuracy for the quantification of the tested drugs in laboratory prepared mixtures and in pharmaceutical preparations.

Solubility Enhancement and Pharmacokinetic Assessment of Chemically Modified Lamotrigine in Rat Blood Plasma by HPLC

Aims:
 
 Synthesis/preparation of Lamotrigine (LMN) complexes with β-CD, Caffeine, Nicotinamide, EDTA and
 Development of a new reverse phase liquid chromatographic (HPLC) method for the investigation of Lamotrigine in rat plasma after oral administration and pharmacokinetic assessment of Lamotrigine.
 
 Study Design: The present work describes the formation of LMN drug complexes with β-Cyclodextrin, Caffeine, Nicotinamide and Disodium EDTA. Physical mixture, kneading and solvent evaporation methods were used to prepare LMN complexes (In ratios 1:1, 1:2, 2:1). Further characterization was performed by UV, FTIR, PXRD, and DSC. A reverse phase HPLC method was developed for the investigation of LMN in rat plasma using internal standardization method after oral administration of LMN and its complexes.
 Place and Duration of Study: Department of Pharmaceutical Chemistry, Smt. Kishoritai Bhoyar College of Pharmacy, RTMN University, Nagpur, between July 2018 and June 2019.
 Methodology: LMN complexes with β-CD, Caffeine, Nicotinamide, EDTA was prepared in three ratios i.e. 1:1, 1:2 and 2:1 and characterized by UV, FTIR, PXRD, and DSC. In-vitro Solubility study was performed by saturation solubility study, further % practical yield, drug content, melting point was determined. In-vitro dissolution study of prepared complexes was performed in dissolution apparatus using the paddle method, according to USP Type II. Dissolution studies were carried out using 900 mL 0.1M HCl at 37± 0.5°C at 50 revmin−1 (US FDA guidelines).The interaction of LMN with these hydrophilic complexing agents was characterized by UV, FTIR, PXRD and DSC.
 A reverse phase HPLC bioanalytical method was developed and validated as per ICH guidelines for the quantitative determination of LMN in rat plasma using internal standardization method (HTZ) after oral administration of LMN and its complexes. The method was successfully applied for the pharmacokinetic study in rat. The pharmacokinetic parameter like AUCt, AUCi, MRTi, Cmax, Tmax, t1/2, were calculated using pharmacokinetic software PK solver 2. The efficient separation was carried out for High Performance Liquid Chromatography (HPLC) method on Eclipse XDB-C18 (150×4.6×5 µ) column using a mobile phase consisting of filtered and degassed mixture of potassium dihydrogen orthophosphate buffer (pH 7.0) and Methanol in the ratio 65:35 v/v at a flow rate of 0.8 mL/min and UV detection at 307 nm.
 Results: The LMN complexes were successfully prepared and characterized by UV, FTIR, PXRD, and DSC from which solvent evaporation method was found to be best as per result of in-vitro dissolution study. In-vitro dissolution study reveals that LMN-Caffeine (C1) and LMN-NTM (N1) complexes showed 100.14 and 100.01% drug release at 15 min and 20 min respectively as compared to pure drug (LMN) which shows only 50.56% drug release at 75 min.
 LMN concentration in blood plasma reached (Cmax) was found to be 19.4732 µg/mL at Tmax of 5h, Whereas Cmax of LMN complexes were found to be 48.4876 (B1), 72.2160 (C1), 62.2739 (N1) and 49.3170 (E1) µg/mL at Tmax of 5h out of which complex C1 and N1 in the present study resulted in a sharp increase in Cmax. All complexes showed 4 to 5 time enhancement of Cmax as compared to LMN.. The results demonstrated that complexes of Lamotrigine were successful strategy to improve the solubility and dissolution behavior of Lamotrigine. The complex B1 shows maximum t1/2 and MRTi of 36.224 h and 52.441 h as compared to C1, N1 and E1 having t1/2 of 14.1575, 16.258 and 21.702 h and MRTi of 19.997, 22.994 and 30.883 h respectively. Hence B1 required lesser dosing frequency as compared to other complexes.
 Conclusion: The Lamotrigine complexes were prepared and confirmation of prepared complexes was done by physical characterization (FTIR. DSC, PXRD and UV) and solubility determination by saturation solubility study. The bioanalytical method was developed for estimation of plasma drug concentration of LMN. The method was validated according to ICH guidelines to estimate the mean plasma concentration of LMN after oral administration using internal standardization method (HTZ). Method was reproducible and high recovery of LMN from its complexes was achieved. The method was found to be highly satisfactory sensitive, accurate, linear and specific.

HPLC Method Development and Validation for Estimation of Chlorthalidone in Tablet Dosage Form

The present work relates to development and validation of simple, precise and rapid high-performance liquid chromatographic (HPLC) method for the analysis of Chlorthalidone in tablet dosage form. Method was developed for qualitative and quantitative estimation of Chlorthalidone in tablet dosage form. The chromatographic separation was achieved by using mobile phase 20 mM potassium dihydrogen orthophosphate buffer pH 4.0: methanol (30:70 %v/v) on HiQ Sil C8 (4.6 mm*250 mm* 5μm) column. The mobile phase was pumped at a flow rate of 1.0 ml/min and the eluent was monitored at 230 nm. Retention time was 3.334±0.042 min. Linearity was observed in the concentration range of 5-30 μg/ml with a correlation coefficient (R2) of 0.9915. All the parameters were validated as per ICH guidelines and found to be suitable for routine analysis of drug in pharmaceutical dosage form. Keywords: Chlorthalidone, Quantitative and Qualitative estimation, HPLC.

Validation of HPLC and Liquid-Liquid Extraction Methods for Warfarin Detection in Human Plasma and its Application to a Pharmacokinetics Study

A reversed-phase HPLC method to determine total plasma warfarin was developed and validated. Warfarin was extracted from human plasma using a two-step liquid-liquid extraction method. The residue was reconstituted with a phenylbutazone standard solution, which was used as the internal standard. The analytical column was a Purospher STAR RP-18e (4 x 4mm I.D., 5m particle size). The mobile phase consisted of acetonitrile: potassium dihydrogen orthophosphate buffer solution at pH 6.5 [30:70 (v/v)] with a flow rate of 1mL/min. Both warfarin and phenylbutazone were detected using a photodiode array detector. The lower limit of quantification was 100ng/mL, while the limit of detection was 20ng/mL. The linearity of the assay was good (r2=0.992) in the concentration range from 0.1 - 6.0µg/mL. The extraction recovery of warfarin was 93.53 ± 12.40%. Both the intraday and interday quality control assay for warfarin demonstrated good precision and accuracy, with all of the percentage coefficients of variation being less than 15%. Warfarin was stable in human plasma for up to three months of storage. The validated method was successfully applied to four human samples for a pharmacokinetics study.

STABILITY-INDICATING REVERSE-PHASE HIGH- PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD DEVELOPMENT FOR SIMULTANEOUS ESTIMATION OF SOFOSBUVIR AND DACLATASVIR IN PURE AND PHARMACEUTICAL FORMULATION

Objective: The objective of the study was to develop a novel, simple, sensitive, accurate, precise, and stability-indicating reverse-phase (RP) liquid chromatographic method for simultaneous estimation of sofosbuvir and daclatasvir in pure and pharmaceutical formulation.
 Methods: In the present work, chromatographic separation was done using stationary-phase discovery column (250 mm×4.6 mm; 5 μm particle size) and mobile phase consisting of 0.1N potassium dihydrogen orthophosphate buffer:acetonitrile (55:45, v/v), with the flow rate of 1 ml/min, and the detection of column effluents was achieved with photodiode array at 260 nm.
 Results: The retention time of sofosbuvir and daclatasvir was found to be 2.32 min and 3.06 min, respectively. Stability-indicating studies were conducted according to the guidelines of International Conference on Harmonization (ICH) Q1A R2, and validation of the method was done as per the ICH guidelines Q2R1. The linearity of the method was in the concentration range of 100–600 μg/ml and 15–90 μg/ml for sofosbuvir and daclatasvir, respectively. The correlation coefficients were found to be 0.9996 and 0.9996 for sofosbuvir and daclatasvir, respectively. The limit of detection and limit of quantification were found to be 0.19 μg/ml and 0.59 μg/ml for sofosbuvir and 0.02 μg/ml and 0.05 μg/ml for daclatasvir, respectively.
 Conclusion: The stability-indicating RP high-performance liquid chromatographic (RP-HPLC) method was novel, simple, precise, accurate, and sensitive for simultaneous estimation of sofosbuvir and daclatasvir in pure and pharmaceutical formulations. Hence, the developed method was adopted for qualitative and quantitative analysis of sofosbuvir and daclatasvir in pure and pharmaceutical formulations.

SIMPLE LIQUID CHROMATOGRAPHY METHOD WITH UV DETECTION FOR DETERMINATION OF BROMAZEPAM IN SOLID PHARMACEUTICAL DOSAGE FORMS

Bromazepam is a psychoactive drug belonging to class of benzodiazepines with well-known hypnotic and sedative effects. It acts on the central neural system as an inhibitor of the neurotransmitter gamma aminobutyric acid. It is frequently prescribed for treatment of severe anxiety, to reduce tension, agitation and depression. Dissolution testing (the process by which a solid solute enters in to a solution) is a requirement for all solid oral dosage forms and is used in all phases of research and development for product release and stability testing. Tablet dissolution test is a standardized method for measuring the rate of drug release from a dosage form and it simulates the percentage of active substance that can be absorbed into the blood circulation. The direct determination of Bromazepam in pharmaceutical dosage forms using HPLC with UV detector to carry out dissolution test, have not yet been described. Development of HPLC method with UV detection for direct determination of in-vitro dissolution test of Bromazepam tablets, which can be used in the same time as method for determination of assay of Bromazepam in Bromazepam tablets, can make analytical procedure easier and quicker. A simple, selective, linear, precise and accurate RP-HPLC method has been developed and validated for assay and in-vitro dissolution test of Bromazepam tablets. The method was validated according to the guidelines set by the International Conference of Harmonization for validation of analytical procedures. The chromatographic separation was carried out using reversed phase HPLC LiChrospher RP Select B column (125 x 4.0 mm i.d.; 5μm) at temperature of 50oC. Mobile phase was consisting of the mixture of methanol, acetonitrile and potassium dihydrogen phosphate buffer (pH 7.0, adjusted with 0.5M Potassium hydroxide), with the ratio of 45:5:50 (v/v/v) and flow rate of 1.0 ml/min. The detection was carried out at 239 nm. System suitability tests were performed through evaluation of different parameters (retention time, tailing factor, retention factor and selectivity) on freshly prepared standard solution of bromazepam. The retention time of bromazepam in 0,1M HCl was 3.5 min. High percentage of recovery shows that the method is free from the interferences from excipients in test samples. Linearity of response was calculated as a ratio of peak areas of bromazepam vs. concentration in 0,1M HCl and spiked tablets in the concentration range of 0.0018 – 0.016 mgmL-1. The response was linear over the concentration range of 0.0018 – 0.016 mgmL-1 and coefficient of correlation was greater than 0.99. Good linearity shows that the proposed method may be useful for quickly and routinely determination of the percentage of dissolved bromazepam from bromazepam tablets and it can be a method of choice for assay determination in the same time.

Kinetic Degradation Study of Dapagliflozin Coupled with UHPLC Separation in the Presence of Major Degradation Product and Metformin

A novel ultra-performance liquid chromatography with UV detector technique was established for simultaneous determination of two antidiabetic drugs, dapagliflozin (DAPA) and metformin (MET), followed by a stress degradation study. Main degradation product was chromatographically separated and precisely characterized via LC-MS/MS. Chromatographic separation done on a Symmetry® Acclaim™ RSLC 120 C18 column (100 mm, 2.1 mm, 2.2 µm), column temperature was maintained at 60 °C. Mobile phase was a mixture of potassium dihydrogen phosphate buffer, pH (3.5)—acetonitrile (50:50, v/v) at flow rate of 0.4 mL/min. The method has displayed an adequate detection at concentration ranges of 1–50 µg/mL for dapagliflozin propanediol monohydrate and 0.5–100 µg/mL for metformin hydrochloride. DAPA was then exposed to different stress conditions include alkaline, acidic, oxidative and ultraviolet light. A study of the degradation kinetics in alkaline medium for DAPA has proved that the degradation follows a pseudo-first-order reaction. The proposed method was effectively applied for the analysis of laboratory prepared mixtures as well as a combined pharmaceutical formulation with 1:200 ratio of DAPA: MET. No significant difference was found regarding accuracy and precision upon statistical comparison between the obtained results and those of the reported method. Validation was conducted in compliance with the ICH guidelines proving that method is selective, linear, precise and accurate. The simplicity and sensitivity of this method allows its use in the quality control tests of the two cited drugs.

Method Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Estimation of Escitalopram Oxalate and Clonazepam in Bulk and its Pharmaceutical Formulations

This article refers to simple isocratic reverse-phase high-performance liquid chromatographic method (RP-HPLC) developed for the simultaneous quantification of Escitalopram Oxalate (EST) and Clonazepam (CZP) in active pharmaceutical ingredient and pharmaceuticals. The separation of the two drugs was attained using a C₁₈ column (250mm×4.6mm, 5µ) as a stationary phase. The mobile phase was used as a mixture of methanol; acetonitrile; and 0.05M potassium dihydrogen orthophosphate buffer (pH 4 adjusted by orthophosphoric acid) with an isocratic ratio of 40:20:40 v/v. Detection was made by using PDA detector at 210 nm. Escitalopram Oxalate (RT= 4.428 minutes) and Clonazepam (RT= 6.532 minutes) were separated in a single chromatographic run with resolution of 8.719. The calibration plot indicated good linear relationship with r2 = 0.998 for Escitalopram Oxalate in concentration range of 32 µg/ml - 48 µg/ml and r2 = 0.999 for Clonazepam in concentration range of 16 µg/ml - 24 µg/ml. The retrievals for Escitalopram Oxalate and Clonazepam were found to be 99.75% and 99.00%, respectively. The established analytical method was validated and found acceptable as per ICH guidelines for linearity, precision, accuracy, specificity, limit of detection, limit of quantification, robustness and stability. Escitalopram Oxalate and Clonazepam individually as well as in combination were exposed to different stress conditions like acid, base, thermal, photolytic and oxidation degradation and peaks of a degraded product were well determined from peaks of pure drug. This method is modest, quick and appropriate for routine quality control analysis.
 Keywords: Reverse Phase – HPLC; Escitalopram Oxalate; Clonazepam; Validation; Degradation study.

CLEANING VALIDATION OF A SIMPLE AND RAPID REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF ASPIRIN AND ROSUVASTATIN

Objective: This study describes a new, simple, precise, accurate, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) cleaning validation method for simultaneous estimation of rosuvastatin and aspirin.
 Methods: The proposed RP-HPLC method was carried out on AGILENT-ZORBAX RP-Inertsil column (250 mm × 4.6 mm, 5 μm) in an isocratic mode utilizing potassium dihydrogen phosphate buffer (pH 2.5 with OPA):acetonitrile (50:50,v/v) as mobile phase, at a flow rate of 1.5 ml/min. Detection was carried out at 243 nm using UV detector.
 Results: The method was found specific as there was no swab interference. The Beer–Lambert’s law was obeyed in the concentration range of 0.5–20 μg/ml for both rosuvastatin and aspirin. The mean percentage recoveries at 100% level were 89.4% for rosuvastatin and 82.1% for aspirin. The limit of detection and limit of quantification for rosuvastatin and aspirin were 0.03 μg/ml and 0.1 μg/ml, respectively. The method was found to be robust and precise with percentage RSD <2.0%.
 Conclusion: A simple, novel, and economical RP-HPLC method for cleaning validation has been developed for the simultaneous estimation of rosuvastatin and aspirin. The method was validated as per ICH guidelines for specificity, linearity, accuracy, precision, and robustness. The developed method can be used as a sensitive analytical tool for ensuring the effectiveness of the cleaning procedure adopted.

RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF RITONAVIR, OMBITASVIR AND PARITAPREVIR IN TABLET DOSAGE FORMS AND THEIR STRESS DEGRADATION STUDIES

Objective: The objective of the present study was to develop and validate a novel reverse phase high performance liquid chromatographic (RP-HPLC) method, for simultaneous determination of ritonavir (RIT), ombitasvir (OMB) and paritaprevir (PAR) in bulk mixtures, and in tablets.
 Methods: Determination of the drugs ritonavir (RIT), ombitasvir (OMB), and paritaprevir (PAR), was carried out applying Hypersil BDS C18 column (250 mm X 4.6 mm i.e., 5 µm particle size), with photodiode array detector at λmax of 254 nm. The mobile phase applied for the current study composed of two solvents, i.e. A (0.01N % w/v potassium di-hydrogen orthophosphate buffer, pH 3.0 adjusted with dilute orthophosphoric acid) and B (acetonitrile). The mobile phase was pumped at a flow rate of 1.0 ml/min in the isocratic mode. The validation study with respect to specificity, linearity, precision, accuracy, and robustness, limit of detection (LOD) and limit of quantification (LOQ) was carried out employing the ICH guidelines.
 Results: Ritonavir, ombitasvir, and paritaprevir showed linearity of response between 12.5-75 μg/ml for ritonavir, 3.125-18.75 µg/ml for ombitasvir and 18.75–112.5 µg/ml for paritaprevir, with a correlation coefficient (R2) 0.999, 0.999,0.999 for RIT, OMB, and PAR respectively. The % recovery obtained was 99.82±0.14 % RIT, OMB 100.03±0.96 % and for 99.96±0.26 % PAR. The LOD and LOQ values for RIT, OMB, PAR were obtained to be 0.02, 0.019and0.02, µg/ml and 0.07, 0.06 and 0.07 µg/ml, respectively. The method also exhibits good robustness for different chromatographic conditions like wavelength, flow rate, mobile phase, and injection volume.
 Conclusion: The method was successfully employed, for the quantification of RIT, OMB, and PAR, in the quality control of in-house developed tablets, and can be applied for the industrial use.

Stability Indicating RP-HPLC-PDA Method for Simultameous Quantification of Glucosamine, Methyl Sulphonyl Methane and Diacerein Tablets

A simple, fast, accurate and specific RP-HPLC-PDA method has been developed for the simultaneous quantification of, glucosamine, diacerein and methyl sulphonyl methane and in bulk and tablet dosage form. The chromatographic separation was performed on a reverse phase Altima C18 Column (150×4.6mm, 5μm particle size) consisting mobile phase of potassium dihydrogen ortho phosphate buffer: acetonitrile (65: 35 v/v), with a flow rate 1ml/min, temperature 30°C and UV detection wavelength 254nm. The retention times of glucosamine, diacerein and methyl sulphonyl methane were observed as 2.13min, 3.48min and 4.16 min respectively. The developed method was validated by validation parameters such as linearity, range, accuracy, precision and robustness. The results obtained for validation parameters are within the limits as per ICH guidelines. The linearity of the drugs were obtained in the range of 37.5 ppm-225 ppm for Glucosamine, 12.5 ppm-75 ppm methyl sulphonyl methane and 2.5 ppm-15 ppm for diacerein. %RSD from precision studies were 0.8, 0.6 and 0.8, mean percentage recovery from accuracy studies were found to be 99.53%, 99.83% and 100.09% for Glucosamine, methyl sulphonyl methane and diacerein, respectively. LOD, LOQ values obtained from regression equations of Glucosamine, Methyl sulfonyl methane and diacerein were 0.47ppm, 0.53ppm, 0.14ppm, and 1.42ppm 1.60ppm, 0.41ppm respectively. The method designed and validated can be successfully used for the regular quantification of Glucosamine, Methyl sulfonyl methane and diacerein in tablet and bulk forms.

DoE Approach: A validated Stability Indicating RP-HPLC Method Development for the Separation of Diasteromeric Analogs and Process Impurities of Carfilzomib

Abstract A novel reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for the determination and separation of thirty diastereomers and six process related impurities of Carfilzomib (CFZ) active ingredient using YMC-Pack ODS-A (150 mm x 4.6 mm, 3µm) column and mobile phase comprising of pH 5.5 potassium di-hydrogen phosphate buffer, acetonitrile and methanol. The flow rate and detection wavelength were 0.9mL min−1 and 220 nm respectively. Design of Experiment (DOE) approach was strategically designed to optimize the critical chromatographic parameters like column temperature, flow rate and composition of methanol in the mobile phase to achieve the separation of diastereomers and process impurities. The developed method was validated for linearity, range, precision, accuracy, specificity, selectivity and intermediate precision.

Simultaneous Estimation and Validation of Canagliflozin and Metformin Hydrochloride in Bulk and Pharmaceutical Dosage Form by using RP-HPLC

The first reversed phase high performance liquid chromatographic method for simultaneous determination of, Canagliflozin and Metformin has been developed and validated to be a simple, sensitive, rapid, specific, precise, and accurate method. Chromatographic separation was achieved on C18 column (250×4.6 mm-5μm p.s). Methanol and potassium dihydrogen phosphate buffer in a ratio [90:10 v/v] as a mobile phase at flow rate of 0.9ml/min. UV detection was operated at 222 nm and injection volume was 20 μl. The proposed method showed good linearity, accuracy, precision and was successfully applied for determination of the drugs in laboratory prepared pharmaceutical dosage forms. The current method has been statistically validated according to the ICH guidelines and this method has been subsequently developed and applied successfully to determine the levels of Metformin hydrochloride and Canagliflozin in a combined formulation and in the routine quality control analysis with good accuracy and sensitivity.

Quantitative Estimation and Validation of Dapagliflozin and Metformin Hydrochloride in Pharmaceutical Dosage form by RP-HPLC

A simple and more economic RP-HPLC method was developed and subsequently validated for the simultaneous determination of Metformin and Dapagliflozin in bulk and pharmaceutical dosage form. The chromatographic conditions were standardized using a Cosmosil C18 column with 250mm in length and internal diameter of 4.6mm with size 5μm. The analyte detection was carried out by using a UV detector set at a wavelength of 228 nm. The mobile phase consisted of Methanol: Potassium dihydrogen phosphate buffer with pH 3.0 (80:20%v/v) and retention time of Metformin and Dapagliflozin was found to be 3.6 min and 5.2 min respectively. The calibration curves of two drugs were linear with correlation coefficients of 0.999 and 0.998 over a concentration range of 100–500μg/ml for Metformin and 1–5μg/ml for Dapagliflozin. This method has been validated and shown to be accurate, precise, specific, sensitive, linear, robust and fast. Metformin and Dapagliflozin were subjected to different degradation stress conditions. The degradation products were well resolved from that of pure standard drugs (Metformin and Dapagliflozin) with significant different retention time values. The current method has been statistically validated according to the ICH guidelines and this method has been subsequently developed and applied successfully to determine the levels of Metformin and Dapagliflozin in a combined formulation and in the routine quality control analysis with good accuracy and sensitivity.

Development and Validation of HPLC method for simultaneous estimation of Rifampicin and Ofloxacin using experimental design

ABSTRACTA accurate, precise and robust isocratic HPLC method has been developed and validated for simultaneous determination of Rifampicin and Ofloxacin. The chromatographic separation was carried out on Kinetex C18, 100 A Phenomenex column with a mixture of 0.03M Potassium dihydrogen phosphate buffer pH 3.0: Acetonitrile (55:45) as mobile phase at 230 nm. The retention times were 2.91 and 4.87 min for Ofloxacin and Rifampicin, respectively. Calibration plots were linear over the concentration range 1–5 and 2–10 µg/ml for Rifampicin and Ofloxacin, respectively. The method was validated for linearity, sensitivity accuracy, precision, and robustness. Percent recoveries were found to be close to 100% with low variability. Fractional factorial design with four factors was chosen for robustness testing. The volume of acetonitrile and flow rate showed significant effect on retention factor of both the drugs and asymmetry factor of ofloxacin. The method may be adopted for routine analysis at industry.

A NOVEL, SIMPLE, RAPID RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF FERULIC ACID, QUERCETIN, PIPERINE AND THYMOL IN AYURVEDIC FORMULATION

Objective: A simple, accurate, precise and robust reverse phase high performance liquid chromatography (RP-HPLC) method was developed for simultaneous estimation of ferulic acid, quercetin, piperine and thymol in marketed ayurvedic formulation.Methods: The selected markers were resolved using shim pack GIST C-18 column, with mobile phase acetonitrile: 0.04 M potassium dihydrogen ortho phosphate buffer (pH 3.0 adjusted with ortho phosphoric acid) in a ratio of 60:40 v/v at a flow rate of 1.0 ml/min. The detection was carried out at 264 nm.Results: The retention time of ferulic acid, quercetin, piperine and thymol were found to be 2.98, 3.35, 7.83 and 9.72 min respectively. The developed method was validated according to the guidelines provided in ICH Q2 (R1) in term of linearity, precision, limit of detection, limit of quantification, accuracy and robustness. Linear response for all selected markers was obtained in the concentration range of 12-28 µg/ml with a correlation coefficient (r2) greater than 0.999. The mean % recovery was found to be 99.30 for ferulic acid, 98.77 for quercetin, 100.93 for piperine and 100.25 for thymol.Conclusion: The developed method was applied for quantification of these marker in marketed ayurvedic formulation. This method can be used to evaluate other formulations containing these selected phytoconstituent, thus conforming the quality and safety of ayurvedic or polyherbal formulations.

Stability indicating RP-HPLC method for simultaneous determination of Grazoprevir and Elbasvir in bulk and tablet dosage form

A simple, rapid and sensitive isocratic RP-HPLC method was developed for the simultaneous estimation of Grazoprevir and Elbasvir in bulk and their Pharmaceutical dosage form using Waters C18(250 x 4.6 mm x 5 μ particle size) analytical column in an isocratic mode with mobile phase comprising Acetonitrile: 0.25M Potassium dihydrogen orthophosphate buffer (pH 4.5) (55:45, v/v). The flow rate was 1.5 ml/ min and effluent was monitored at 235nm. The retention times were found to be 2.390 min for Grazoprevir and 4.603 min for Elbasvir. The assay exhibited a linear dynamic range of 100 - 300 μg/ml for Grazoprevir and 50-150 μg/ml for Elbasvir. The calibration curve was linear (r2= 0.9998 for Grazoprevir and r = 0.9999 for Elbasvir) over the entire linear range. Recovery was found to be 99.03% for Grazoprevir and 99.34 % for Elbasvir. The proposed method was statistically evaluated and validated as per ICH guidelines and can be applied for routine quality control analysis of Grazoprevir and Elbasvir in Pharmaceutical dosage form.

METHOD DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE LIQUID CHROMATOGRAPHIC METHOD FOR THE SIMULTANEOUS ESTIMATION OF SELECTED ANTIDIABETIC DRUGS IN THE PRESENCE OF THEIR DEGRADATION PRODUCTS

Objective: This study was designed to conduct forced degradation and validation studies for the simultaneous estimation of metformin, sitagliptin, pioglitazone, and glimepiride. Methods: Analytes were separated on an Agilent XDB-C18, 150 × 4.6 mm, 5 μm column using an isocratic elution mode having mobile phase composition of 20 mM potassium dihydrogen phosphate buffer (pH 4.0):acetonitrile (65:35% v/v). Analytes were detected at a wavelength of 225 nm. The optimized method was validated as per the ICH Q2 guidelines. Results: The retention times of metformin, sitagliptin, pioglitazone, and glimepiride were 3.47, 4.83, 5.83, and 9.44 min, respectively. The linearity was 25–100 μg/ml for metformin, 2.5–10 μg/ml for sitagliptin, 1–4 μg/ml for pioglitazone, and 0.75–3 μg/ml for glimepiride. The correlation coefficient for calibration curves was >0.99, and accuracy was between 98 and 102% for each analyte. Inter- and intra-day precisions were calculated <2% relative standard deviation for each analyte. Conclusion: A significant degradation was observed in the presence of acidic, basic, neutral, oxidative, and photolytic stress conditions. The method is simple, precise, accurate, robust, and reproducible and was able to successfully separate and quantify metformin, sitagliptin, pioglitazone, and glimepiride in the presence of their degradation products.

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE DETERMINATION OF ALVIMOPAN IN RAT PLASMA

Objective: The present investigation demonstrates a simple, sensitive and accurate high pressure liquid chromatographic (HPLC) method for the determination of alvimopan (AMP) in rat plasma.Methods: The chromatographic separation was achieved within 10 min by using acetonitrile: potassium dihydrogen phosphate buffer pH 3.0 adjusted with orthophosphoric acid (50:50) as mobile phase on Altima Grace Smart C-18 column (5μ; 250 × 4.6 mm) at a flow rate of 1.0 ml/min with injection volume 50 µl. The drug was extracted from plasma by liquid-liquid extraction using a mixture of methanol: acetonitrile (50:50) as a solvent. The retention times of drug and internal standard were found to be 5.17 and 6.74 min, respectively. This method was validated as per the United States Food and Drug Administration (US-FDA) guidelines.Results: The results of the validation parameters were found to be within the acceptance limits. The method was linear in the concentration range from 5-1000 ng/ml (r2= 0.9998), and the extraction recovery was found to be 78.71±3.86% for AMP. The lower limit of quantification was found to be 5ng/ml, and the stability of recovered samples at different conditions was found to be more than 95%.Conclusion: The developed method possess good selectivity, specificity, there was no interference found in the plasma blanks at retention times of AMP and Internal Standard (IS). We found a good correlation between the peak area and concentration of the drug under prescribed conditions. Furthermore, the method can also be used to estimate the pharmacokinetic parameters of AMP.Keywords: Alvimopan, Liquid-liquid extraction, Method development, Matrix effect, Plasma, Recovery, Stability, Validation

Six Sigma quality approach for HPLC-UV method optimization

We report a robust HPLC-UV method for ascorbic acid (AS), paracetamol (PA) and guaifenesin (GU) based on response surface/tolerance analysis approach to meet Six Sigma quality of the performance of a chromatographic method. Our goals were the bringing specifications into the optimizations and achieving robustness during the development phase. The critical method attributes (CMAs) were resolution between the peak pairs (Rs), the analysis time(T), tailing factor (Tf), number of theoretical plates (N), and back pressure(P).The critical method parameters (CMPs) included a percentage of organic modifier, a flow rate of the mobile phase, and a temperature of column oven. The separation was performed on a CN column, using an isocratic mode with a mixture of 50 mM potassium dihydrogen phosphate buffer adjusted with ortho-phosphoric acid to pH 2.2 as the aqueous component of the mobile phase and methanol in the ratio 85.9:14.1 (v/v) at 1 ml/min flow rate, with UV detector at 275 nm and a column temperature of 45 °C. The method was validated using the accuracy profiles. Finally, the method was applied to pharmaceutical dosage form.