Isocratic Reversed-phase High Performance Research Articles

Establishment and reliability evaluation of the design space for HPLC analysis of six alkaloids in Coptis chinensis (Huanglian) using Bayesian approach

Coptis chinensis (Huanglian) is a commonly used traditional Chinese medicine (TCM) herb and alkaloids are the most important chemical constituents in it. In the present study, an isocratic reverse phase high performance liquid chromatography (RP-HPLC) method allowing the separation of six alkaloids in Huanglian was for the first time developed under the quality by design (QbD) principles. First, five chromatographic parameters were identified to construct a Plackett-Burman experimental design. The critical resolution, analysis time, and peak width were responses modeled by multivariate linear regression. The results showed that the percentage of acetonitrile, concentration of sodium dodecyl sulfate, and concentration of potassium phosphate monobasic were statistically significant parameters (P < 0.05). Then, the Box-Behnken experimental design was applied to further evaluate the interactions between the three parameters on selected responses. Full quadratic models were built and used to establish the analytical design space. Moreover, the reliability of design space was estimated by the Bayesian posterior predictive distribution. The optimal separation was predicted at 40% acetonitrile, 1.7 g·mL−1 of sodium dodecyl sulfate and 0.03 mol·mL−1 of potassium phosphate monobasic. Finally, the accuracy profile methodology was used to validate the established HPLC method. The results demonstrated that the QbD concept could be efficiently used to develop a robust RP-HPLC analytical method for Huanglian.

Stability-Indicating Method and LC-MS-MS Characterization of Forced Degradation Products of Sofosbuvir.

Sofosbuvir is a novel direct acting antiviral agent against hepatitis C virus. In the present work, a rapid, specific and reproducible isocratic reversed phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the determination of sofosbuvir in the presence of its stressed degradation products. Sobosbuvir was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per international conference on harmonization (ICH) conditions. The drug showed degradation under oxidative, photolysis, acid and base hydrolysis stress conditions. However, it was stable under thermal and neutral hydrolysis stress conditions. Chromatographic separation of the drug from its degradation products was performed on Inertsil ODS-3 C18 (250 mm×4.6 mm i.d., 5 µm) column using a green mobile phase of methanol:water 70:30 (v/v). The degradation products were characterized by LC-MS-MS and the fragmentation pathways were proposed. The developed method was validated as per ICH guidelines. No previous method was reported regarding the degradation behavior of sofosbuvir.

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Bromhexine and Erythromycin in Bulk and Pharmaceutical Dosage Forms

A simple, rapid, accurate and precise isocratic reversed phase high performance liquid chromatographic method has been developed and validated for simultaneous estimation of Bromhexine and Erythromycin in tablet dosage forms. The chromatographic separation was carried out on Hypersil BDS C18 column (150 mm x 4.6 mm I.D., 5 µm particle size) with a mixture of 0.01N phosphate buffer and acetonitrile in the ratio of 35:65 v/v as a mobile phase at a flow rate of 1.0 mL/min. UV detection was performed at 224 nm. The retention times were 4.619 minutes and 2.329 minutes for Bromhexine and Erythromycin respectively. Calibration plots were linear (r2=0.999 for both Bromhexine and Erythromycin respectively) over the concentration range of 2-12 µg/mL for Bromhexine and 62.5-375 µg/mL for Erythromycin. The method was validated for linearity, precision, accuracy and robustness. The proposed method was successfully used for simultaneous estimation of Bromhexine and Erythromycin in tablet dosage forms. Validation studies revealed that the proposed method is specific, rapid, reliable and reproducible. The high % recovery and low % RSD values confirm the suitability of the proposed method for routine quality control analysis of Bromhexine and Erythromycin in bulk and tablet dosage forms. Keywords: Bromhexine, Erythromycin, Estimation, HPLC

Analytical Method Development and Validation of RP-HPLC Method for Simultaneous Estimation of N-acetyl cysteine and Cefexime from its Fixed Dose Combination

A simple novel validated isocratic reverse phase high performance liquid chromatographic method has been developed for the simultaneous estimation of N-acetyl cysteine (NAC) and cefexime from its fixed dose combination (FDC). The fixed dose combination of NAC and cefexime is used in the treatment of chronic obstructive pulmonary disease. Separation was achieved on Acclaim C18 (150 mm x 4mm.) with 25 mM potassium dihydrogen phosphate (pH 2.7) and acetonitrile in the ratio 90:10 v/v. The mobile phase was pumped at 1mL/min and quantification was carried out at 220 nm using a UV detector. The retention time of NAC and cefexime was 2.7min and 11.47 min respectively. The method was validated as per ICH Q2R1 guidelines. The method demonstrated linearity from10-90 µg/ml and accuracy was between 101.16-101.96%. The precision of the method was less than 2% in all the cases. The method was further applied in the assay of marketed formulation.

OPTIMIZATION OF RP-HPLC METHOD BY 32 – CENTRAL COMPOSITE DESIGN FOR THE VALIDATION AND ESTIMATION OF DOXYCYCLINE HYCLATE IN BULK DRUG AND FORMULATIONS

An accurate, sensitive, precise, economic and rapid isocratic Reverse Phase High Performance Liquid Chromatography method was developed complying Quality by Design (QbD) trends and validated for determining doxycycline hyclate in bulk drug, tablet and capsule dosage form. The method was optimized using Minitab software with 3 factors (pH of the buffer, flow rate and percentage of buffer in the mobile phase), 2 level (higher limit and lower limit) Central Composite Design (CCD). The results of randomized 20 runs were analyzed for optimum composite desirability to give optimum conditions such as, pH 6.5, flow rate 0.9 mLmin-1 and 30:70 V/V 0.05M potassium dihydrogen orthophosphate buffer adjusted to pH 6.5 using orthophosphoric acid and methanol using C8 column 250 X 4.6 mm X 5.0 μm, injection volume of 10uL, ambient column temperature and ultraviolet detection using photo diode array detector at 360nm as constants. The method was validated as per ICH guidelines and was found linear over a concentration range of 10-100 μg/mL (r2 = 0.999) with the limits of detection and quantification being 2.45 μg/mL and 7.55 μg/mL respectively.

DETERMINATION AND VALIDATION OF NARINGIN IN RAT PLASMA BY RP-HPLC

A simple, accurate isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of the plasma concentration of naringin, a flavanone glycoside in rat plasma. An assay procedure involved simple liquid–liquid extraction of naringin from plasma directly into methanol. The organic layer was separated, filtered and injected onto a Phenomenex-C18 column (4.6 × 250 mm, 5 μm). The mobile phase acetonitrile and water (30:70, V/V) was used at a flow rate of 1.0 mL/min for the effective separation of naringin. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector at 283 nm. The peak area of analyte was used for quantification of plasma samples. The retention time of naringin was 4.7 min. The standard curve for naringin was linear (r2 = 0.9987) in the concentration range 100-2000 ng/mL. The absolute recoveries of naringin were in the range 85–115% from rat plasma. The lower limit of quantification (LLOQ) of naringin was 30 ng/mL. The intra and inter-assay precisions in the measurement of quality control samples of 100, 400, 900 and 1600 ng/mL, were in the range of 0.19-5.44% and 0.27-2.35% relative standard deviation (RSD), respectively. Accuracy in the measurement of quality control samples was in the range 98.40-107.9% of the spiked nominal values. The plasma concentration of naringin was increased with increase in test dose levels. From the results, maximum plasma concentration of naringin was found to be 289.03 and 757.3 ng/mL with 15 and 30 mg/kg, oral doses, respectively at 2 h. This method is simple and helps in determination of naringin in rat plasma.

Method development and validation of Rupatadine fumarate and Montelukast sodium by RP- HPLC

A simple, sensitive, rapid and selective isocratic reversed phase High Performance Liquid Chromatographic method has been developed for simultaneous estimation of Rupatadine fumarate & Montelucast Sodium from pharmaceutical dosage form using a mobile phase consisting mixture methanol: acetonitrile: buffer 40:30:30, (pH adjusted to 3.2 using ortho phosphoric acid) at the flow rate of 1.0 mL/min. A Hypersil BDS C8, (250 mm X 4.6 mm, 5 particle diameters) column was used as stationary phase. The retention time of Rupatadine Fumarate and Montelucast Sodium was 3.97 min. and 2.79 min. respectively. The eluent were detected at 270 nm. The proposed method is precise, accurate, selective and rapid for the simultaneous determination of Rupatadine Fumarate & Montelucast Sodium.

A novel validated RP-HPLC-DAD method for the estimation of Eluxadoline in bulk and pharmaceutical dosage form

The present study describes about novel approach to develop and validate a rapid, accurate, precise, simple, efficient and reproducible isocratic Reversed Phase-High Performance Liquid Chromatographic (RP-HPLC-DAD) method for the estimation of Eluxadoline in bulk and pharmaceutical dosage form. Eluxadoline were separated using Hypersil C18 column (250mm×4.6 mm, 5μm particle size), Waters Alliance e2695 HPLC system with 2998 PDA detector and the mobile phase contained a mixture of Acetonitrile and Water (70: 30, v/v). The flow rate was set to 1ml/min with the responses measured at 260nm. The retention time of Eluxadoline was found to be 3.481min. Linearity was established for Eluxadoline in the range of 10–125μg/ml with correlation coefficient (r2=0.999). The percentage recoveries were between 99.92% to 100.07%. Validation parameters such as specificity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ) were evaluated for the method according to the International Conference on Harmonization (ICH) Q2 R1 guidelines. The developed method was successfully applied for the quantification and hyphenated instrumental analysis.

Stability indicating studies on NMITLI 118RT+ (standardized extract of withania somnifera dunal).

Background:Withania somnifera Dunal (Ashwagandha) is an Indian medicinal plant of great medicinal value; used in many clinically proven conditions. NMITLI-118RT+ is a candidate drug under a Council of Scientific and Industrial Research (CSIR) networking project. It is a chemotype of W. somnifera's root extract, which has been used for the present study.Objectives:The present investigation aims to develop and validate a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) system for the detection and estimation of Withanolide A (marker compound) and its analytical application for stability indicating studies on NMITLI-118RT+.Material and Methods:A validated RP-HPLC method for Withanolide A was established on a Waters HPLC system and the same was used on NMITLI-118RT+ for quantification and fingerprinting purposes, and for establishing forced degradation, isothermal stress tests, and drug-excipient testing protocols as per International Conference on Harmonization (ICH) guidelines.Results:A validated method was established, which could detect the marker at a retention time of around 6.3 minutes, with a linearity range of 2-100 μg/mL, by varying the amounts of the said marker, which were estimated in four different batches of NMITLI-118RT+. Photostability as per ICH guidelines suggested a slight loss of the active constituent and maximum degradation was afforded with alkali followed by acid, and then peroxide, in the forced degradation studies. In the drug-excipient studies, the maximum amount of active constituent could be detected in the samples with ethyl cellulose and the least with hydroxy propyl cellulose.Conclusion:The method developed here was simple and rapid. The various stability indicating studies carried out in the present investigation would be useful for formulation development and were suggestive of deciding the recommended storage conditions for NMITLI-118RT+.

RP-HPLC Method Development and Validation for Quantitative Analysis of Metformin and Gliclazide in Pharmaceutical Formulations

A rapid and sensitive isocratic reversed phase high performance liquid chromatographic method has been developed for quantitative analysis of metformin hydrochloride and gliclazide in bulk as well as pharmaceutical dosage forms. The method was validated according to FDA, ICH and USP guidelines with respect to accuracy, precision, specificity and linearity. For metformin, the method was developed by using the mobile phase comprising of sodium dihydrogen phosphate solution (NaH2PO4) (pH 3.0) and acetonitrile (90:10, v/v) at a flow rate of 0.5 mL/min over C18 bonded silica column (3.9 x 300 mm, 5 µm) at ambient temperature. On the other hand, the same mobile phase was used in the ratio of 20:80 (v/v) at a flow rate of 0.6 mL/min for gliclazide. The recovery was found to be more than 97% for metformin and 102% for gliclazide that demonstrated the accuracy of the protocol. Intraday and interday precisions of the new method were less than the maximum allowable limit (%RSD 2.0) specified by the USP, ICH and FDA. The method revealed linear response with correlation coefficient value of 0.999 in both cases.

VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF FEBUXOSTAT AND DICLOFENAC POTASSIUM IN BULK DRUG AND IN BI LAYER TABLET FORMULATION

A simple, rapid, accurate and precise isocratic reverse phase high performance liquid chromatography (RP - HPLC) method has been developed for simultaneous estimation of febuxostat and diclofenac potassium in combined dosage form by using phenomenex C18 column (statio nary phase) having dimension of 4.6 × 250 mm and particle size of 5 μm and mobile phase containing a mixtur e of 0.02 M potassium dihydrogen orthophosphate buffer (adjusted to pH 7 with sodium hydroxide): acetonitrile: methanol (35:9:56 v/v/v) at a flow rate of 1 ml/min and detection was carried out at 290 nm. The retention times of febuxostat and diclofenac pot assium were 6.01 ± 0.02 minutes and 7.10 ± 0.02 minutes respectively. The developed method was validated as per ICH guideline for specificity, linearity, accuracy and precision , limit of detection and limit of quantification. Linearity studies for the deve loped method were found in the range of 5 to 30 μg/ml for febuxostat and 12.5 to 75 μg/ml for diclofenac potassium respectively. The accuracy of the method was studied by recovery study and found to be in the range of 98 % - 102 %. The % RSD for intraday a nd inter day precision was found less than 2. The new RP - HPLC method was successfully applied to marketed formulation of Febuxostat and Diclofenac potassium without any interference from excipients.

Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Lamivudine and Tenofovir Disproxil Fumerate in Combined Dosage Form

Objective: A highly sensitive isocratic reverse phase high performance liquid chromatographic method was developed and validated for the estimation of Lamivudine and Tenofovir disproxil fumerate in Bulk drug and Pharmaceutical dosage forms. Method: Separation of Lamivudine and Tenofovir successfully achieved on symmetry COLUMN-ENABLE C18 G 5 μM, 250×4.6mm or equivalent utilizing HPLC Methanol and Water ph adjusted with 3.2 in the ratio of 70:30 v/v as mobile phase at a flow rate of 1mL/min and the eluates was monitored at260 nm. Result: Chromatogram showed peak at a retention time of 3.048min and 5.354min. The method was validated for system suitability, linearity, precision, accuracy, specificity, ruggedness, robustness, LOD and LOQ. Recovery of Tenofovir disproxil fumerate and Lamivudine were found to be in the range of 100.46% and 100.66% and showing linearity in the range of 10- 50 μg / ml. The S/N for LOD and LOQ for estimation of Lamivudine and Tenofovir disproxil fumerate were found to be 1.488μg / ml and 4.510 μg /ml and 3.552μg/ml and 9.763μg/ml respectively. Conclusion: Proposed method can be successfully applied for the quantitative determination of Tenofovir disproxil fumerate and Lamivudine in Bulk drug and Pharmaceutical dosage form.

Optimization and validation of a fast RP–HPLC method for the determination of dobutamine in rat plasma: Pharmacokinetic studies in healthy rat subjects

A novel isocratic reverse phase high performance liquid chromatography (RP–HPLC) with photo diode array (PDA) detection method for the determination of dobutamine (DBT) in rat plasma was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Homoveratrylamine was used as an internal standard. Methanol was used as the extracting solvent for the preparation of plasma samples. Samples were separated on a Symmetry C18 (250mm×4.6mm i.d., 5μm) analytical column. Acetonitrile and 15mM potassium dihydrogen phosphate (pH 5.0 with 0.3% TEA) (20:80, v/v) was used. The column oven temperature was optimized at 35°C and the flow rate was 0.8mL/min. The detection wavelength was fixed at 230nm for entire analysis. The calibration curve was found to be linear over the concentration range of 50–2000ng/mL (r2=0.9992). The limit of quantification (LOQ) of the method was 50ng/mL. The % RSD values of accuracy and precision values for intra and inter days were <15% at quality control (QC) concentrations. Recovery, stability and robustness were studied within the acceptable range according to ICH guidelines. The method was efficiently applied to a pharmacokinetic study in healthy Wistar rats.

RP-HPLC method for the estimation of ceftiofur hydrochloride in bulk form

AimThe aim of the present work was to develop a simple and reliable isocratic Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method and to validate the method for the quantitative estimation of Ceftiofur Hydrochloride in bulk form. ExperimentalAn isocratic reverse phase high performance liquid chromatography was performed on a Hypersil BDS C18 column (250 × 4.6 mm, 5 μ) with a 60:40% (v/v) mixture of disodium hydrogen orthophosphate buffer (pH adjusted to 6.8 with ortho-phosphoric acid) and acetonitrile as mobile phase, at a flow rate of 1.5 mL/min. The detection was made at 292 nm by using a Ultra-violet spectrophotometric detector. Results and discussionThis method was validated for linearity and range, system and method precision, accuracy, specificity, robustness and ruggedness. All the results were satisfactory. The retention time of the ceftiofur hydrochloride peak was found at 7.64 minutes. The method was linear in the range of 50–150 μg/mL and found to be specific and accurate with the mean recovery of 100.02%. Assay content of ceftiofur hydrochloride was determined and the mean assay was found to be 99.36%. ConclusionThe proposed method is highly sensitive, precise and accurate and can be used for the reliable quantitation of ceftiofur hydrochloride in bulk form and could be used for routine analysis.

A reversed phase high performance liquid chromatographic method for determination of rapamycin.

Easily degradating and various isomeric forms of rapamycin (Sirolimus) face the determination of this compound to many challenges. In this study, we developed and validated the isocratic reversed phase high performance liquid chromatographic (RP-HPLC) method for rapamycin. Separation was performed on a C8 column (MZ, 15 × 4.6 mm, 5 μm particle size) using methanol:water (80:20 v/v) as the mobile phase with the flow rate of 1 mL/min. The column temperature was set at 57°C and the detection was carried out at the wavelength of 277 nm. The method was linear over a concentration range of 0.025-2 μg/mL. The coefficient of variation of intra- and inter-day, assessed at three concentration levels of 0.075, 0.3 and 0.900 μg/mL, was less than 2%. Limit of quantification (LOQ) was found 25 ng/mL. The method with high percent recovery and short retention time of rapamycin, was found to be simple, rapid and reproducible.

Simultaneous Estimation of Lamivudine and Stavudine by using Isocratic Reverse Phase High Performance Liquid Chromatography Method

A simple, precise, fast Reversed Phase High Performance Liquid Chromatographic (RP-HPLC) assay method by external standard method was developed for the determination of Lamivudine and Stavudine in a solid dosage form. The chromatographic separation was performed using Phenomenex Bondalone 300 x 3.90 mm, 10 μ column. Analytes were resolved by using mobile phase 5:95 mixture of Acetonitrile: water at UV 265 nm as detection wavelength.

METHOD DEVELOPMENT AND VALIDATION FOR DETERMINATION OF IMPURITIES IN DEFERASIROX BY RP-HPLC TECHNIQUE

A simple and isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was developed and validated for determination of Deferasirox and its impurities. The method was validated as per International Conference on Harmonization (ICHQ2A (R1)) guidelines for system suitability, precision, linearity, specificity, solution stability, robustness and ruggedness, limit of detection and limit of quantitation. Deferasirox was analyzed by using Inertsil ODS-3V (150 mm X 4.6 mm, 5 μm) at 35°C column temperature, with isocratic elution. The analysis was performed at a wavelength of 245 nm using dual λ absorbance detector. Efficient UV detection at 245 nm enabled determination of Deferasirox without any interference of impurities Salicylic acid &amp; Salicylamide. The retention time (RT) for Deferasirox was around 8.7 min. The impurities of Deferasirox are key starting materials. These impurities act as potential impurities. Thus identifying these impurities at low level is crucial. The calibration curves were linear over acceptable concentration range. The method developed was unique in determining the impurities even at low levels than that of specifications. The developed method was successfully applied to estimate the amount of Deferasirox.

Validated high performance liquid chromatography (HPLC) method for the determination of sumatriptan in rabbit plasma: Application to pharmacokinetic study

A new, sensitive and specific isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method with fluorescence detection was developed and validated for the determination of sumatriptan in rabbit plasma using sulpiride as an internal standard (IS). Sumatriptan was extracted from plasma by a liquid-liquid extraction with a mixture of tert-butyl methyl ether, dichloromethane and ethyl acetate (2:2:3, v/v). Chromatographic separation of the analyte and internal standard was achieved on a Phenomenex C4 (250 × 4.6 mm, 5 µm) analytical column maintained at 40°C. The mobile phase was composed of 25 mM ammonium acetate (pH 6.5) and acetonitrile (85:15, v/v), pumped isocratically at a flow rate of 0.9 ml/min. Column eluent was monitored at excitation and emission wavelengths of 225 and 350 nm. The calibration curve was linear over a concentration range of 1 to 300 ng/ml (r2 = 0.9999) with a limit of quantification, 1 ng/ml. The intra-day and inter-day precision and accuracy were between 2.24 and 4.28% and -1.10 and 2.86%, respectively. The mean recoveries of sumatriptan and sulpiride were 89.92 and 91.03%, respectively. Sumatriptan containing plasma samples were stable at -20°C for 14 days. The validated method was successfully applied for pharmacokinetic study after a single oral administration of sumatriptan (50 mg) to rabbits. Key words: Sumatriptan, rabbit plasma, reverse phase-high performance liquid chromatography (RP-HPLC), liquid-liquid extraction, pharmacokinetics.

Isocratic RP-HPLC Method for Simultaneous Separation and Estimation of Zofenopril and Hydrochlorthiazide in Pharmaceutical Dosage Forms

A simple, rapid, sensitive and accurate isocratic reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and subsequently validated for the simultaneous determination of Zofenopril and Hydrochlorthiazide in combined dosage form. Chromatographic separation of the two drugs was performed on a Purospher BDS C18 column (250 mm × 4.6 mm id, 5 μm particle size). The mobile phase comprising of acetonitrile methanol: 0.02M NaH22PO4buffer (40:20:40) was delivered at a flow rate of 1.0mL/min. The pH of the mobile phase is adjusted to 7.2 with Sodium hydroxide solution. Detection was performed at 245 nm.The separation was completed within 10 min and the retention time of hydrochlorthiazide is 4.62 and Zofenopril is 6.86 min respectively. Calibration curves were linear with R2between 0.99-1.0 over a concentration range of 100-600 μg/ml for Zofenopril calcium and 50-300 μg/ml for hydrochlorthiazide..The developed method was successfully applied to determi

RP-HPLC ASSAY OF IBUPROFEN IN PLASMA USING DIFFERENT EXTRACTION PROCEDURES

An isocratic reverse phase high performance liquid chromatographic (RP-HPLC) method with ultra violet detection at 220 nm was used for a simple, selective and precise quantitation of a non-steroidal anti-inflammatory drug i.e. ibuprofen (IBF) from rat plasma. The procedure employed mefenamic acid as an internal standard (IS). A good chromatographic separation between IBF, the internal standard and interfering endogenous peaks was achieved using a Octadecyl Silane column and acetonitrile (ACN) - water - methanol - ortho-phosphoric acid (OPA) (in ratio of 58: 37: 5: 0.05 v/v/v/v) as the mobile phase at a flow rate of 1.0 ml/min. Two different extraction procedures for RP-HPLC quantitation of IBF were analysed. In the first method acetonitrile was used for extraction of the drug from plasma whereas the second method involved extraction of drug from plasma using diethyl ether. The second method proved to be more suitable for quantitation of the drug from plasma. The applicability of procedure includes the requirement of less sample volume, lesser organic solvent consumption and good plasma clean up for pharmacokinetic studies, in case of small animals, neonates and children as well.