Robust Reversed-phase High-performance Liquid Chromatography Research Articles

Quality by Design Approach for the Development and Validation of a Robust RP-HPLC Method for the Estimation of Aloe-Emodin

Introduction/Objective: The current study examines the methodical Quality by Design (QbD) that facilitated the creation of an easy-to-use, quick, affordable, and stability-indicating reversed- phase high-performance liquid chromatography (RP-HPLC) technique for the efficient analysis of aloe-emodin. Methods: The chromatographic conditions were optimized with the Design Expert software 11.0 version, i.e., flow rate, buffer concentration, and column temperature. Results: The results of the linearity graph show R2 = 0.9988. The LOQ was 0.07949 μg/mL and the LOD was 0.02623 μg/mL. According to ICH rules, the technique validation parameters were within the allowed range. Utilizing the Design Expert 11.0 version, the Box–Behnken design experimental design explains the relationships between flow rate, buffer concentration, and column temperature at three distinct levels. The responses were monitored: the retention time (Rt), tailing factor (Tf), and number of theoretical plates (NTPs). Conclusion: The suggested approach was appropriate for quantitative determination and may be used in clinical pharmacokinetic investigations, biopharmaceutics, accredited testing laboratories, and quality control departments in enterprises.

High‐Performance Liquid Chromatography–Based Standardization of Stigmasterol in Moringa oleifera: Method Development and Validation Through Design of Experiment Approach

ABSTRACTThe primary objective of this investigation was to develop and validate a robust reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method for quantifying stigmasterol in both Moringa oleifera powder and tablets. The analysis was conducted using a Box–Behnken design. Stigmasterol separation was achieved with a Phenomenex Luna C‐18 column (4.6 × 150 mm2, 5 µm) and a mobile phase consisting of methanol and 0.1% formic acid in an 80:20% v/v ratio. The separation was performed at a flow rate of 1 mL/min, and stigmasterol was detected at a wavelength of 210 nm using a photodiode array detector. Method validation followed the guidelines set forth by the International Council for Harmonisation (ICH) Q2 (R1). This rigorous validation process evaluated critical parameters, including linearity, accuracy, system suitability, precision, and robustness. The results for each parameter were well within the acceptable range, confirming the reliability and efficacy of the established RP‐HPLC method. The findings of this study highlight the potential of the RP‐HPLC method as a dependable tool for the routine analysis of stigmasterol in herbal formulations.

Development of a Robust and Reliable RP-HPLC Method for the Estimation of Finerenone in Tablet Dosage Form

An advanced analytical method was explored, specifically tailored for estimating finerenone, a vital drug found in tablet formulations intended to reduce the risk of chronic kidney disease in patients with type-2 diabetes. This sophisticated technique employs an Inertsil ODS 3V C18 column (5 μm particle size x 250 x 4.6 mm) for precise separation of analyte. The mobile phase consists of an ammonium dihydrogen phosphate buffer (pH 4.5) and acetonitrile (60:40), which is a carefully balanced mixture that flows consistently at 1.0 mL/min. UV detection is achieved at a wavelength of 238 nm. Finerenone’s characteristics are unveiled with a brief retention time of 4.06 minutes, highlighting the efficiency of the method. The method demonstrates linearity within the concentration range of 2.5 to 15 μg/mL, offering a comprehensive scope for accurate analysis. This robust reversed-phase high-performance liquid chromatography (RP-HPLC) method undergoes thorough validation following International Council for Harmonisation (ICH) guidelines, ensuring its reliability. Notably, the accuracy is impressive, with a mean recovery falling comfortably within the acceptable range of 99.76 to 101.09%. In summary, our analytical approach stands out for its simplicity, precision, sensitivity, rapidity, and robustness in estimating finerenone in tablet formulations.

Quality by design‐engineered reversed‐phase high‐performance liquid chromatography method development and validation for simultaneous estimation of neomycin sulfate and beclomethasone dipropionate in bulk and pharmaceutical dosage form

AbstractThe aim of this study was to develop and validate a robust reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method for the simultaneous estimation of neomycin sulfate (NEO) and beclomethasone dipropionate (BECLO) in both bulk drug and pharmaceutical dosage forms. The analysis was conducted using the Box‐Behnken design. The separation of NEO and BECLO was conducted on a Phenomenex Luna C‐18 column (4.6 × 150 mm, 5 µm), employing a mobile phase comprising a mixture of methanol and trifluoroacetic acid in a ratio of 88:12% v/v. The separation was performed at a flow rate of 0.6 mL/min. NEO and BECLO were analyzed at a wavelength of 240 nm employing a photodiode array detector. The validation of the methodology followed the guidelines outlined in the International Council for Harmonization Q2 R (1). The validation process involved assessing critical parameters such as linearity, accuracy, system suitability, precision, and robustness. The results for each parameter were found to be within the acceptable range. The results indicate that the established RP‐HPLC method can effectively be employed for the routine analysis of NEO and BECLO in bulk drug and pharmaceutical dosage forms.

RP-HPLC Method for Quantification of Bilastine and Monteleukast Sodium in Pharmaceutical Dosage form

A simple, accurate, precise and robust reverse phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous estimation of Bilastine and Monteleukast Sodium in bulk and pharmaceutical dosage form. Chromatographic separation was performed on an Zodiac sil RP C18 column (100mm × 4.6mm, 3µm) with mobile phase consist a mixture of 75:25% v/v acetonitrile: phosphate Buffer pH adjusted to 5.2 with ortho-phosphoric acid in an isocratic elution mode, at a flow rate of 1 ml/min. The detection was monitored at 272nm. The retention time of Monteleukast Sodium and Bilastine were found to be 3.668 min and 2.746 min respectively. Linearity range was found between 20-100µg/ml for Bilastine with correlation coefficient of 0.999 and 5-25µg/ml for Monteleukast Sodium with correlation coefficient of 0.999. This method was validated with respect to linearity, precision, accuracy, ruggedness, limit of detection, limit of quantification and robustness. This method was successfully applied for the simultaneous estimation of Bilastine and Monteleukast Sodium in bulk and pharmaceutical formulation.

Analytical Method Development and Validation Protocol for Antiepileptic Agent – Rufinamide

This study developed and validated a new, sensitive, appropriate, clear, accurate, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for determining Rufinamide in bulk medication and tablet formulation. The separation was performed using an HPLC method with a UV detector and Openlab EZ chrome workstation programme, Kromasil C18, 250 mm X 4.6mm ID, 5 μm column, Methanol: 0.025% TFAA (60:40%V/V) was pumped at a flow rate of 1.0 mL/min and detected at 212 nm. The new RP-HPLC method resulted in a 3.22minute retention time for Rufinamide, which was optimized by trial and error. Over a concentration range of 0.5-7.5 μg/mL, the linearity of the established method was confirmed with a correlation coefficient (r2) of 0.99994. The precision of the approach was determined to have a percentage RSD of less than 2.0 percent. The % recoveries were found to be within the acceptable range. The LOD and LOQ were determined to be 0.10 ug/mL and 0.31 ug/mL, respectively. The developed and validated RP-HPLC system takes less time and can be used in the industry for routine quality control/analysis of bulk drug and marketed Rufinamide products.

Development and validation of RP-HPLC method for estimation of brexpiprazole in its bulk and tablet dosage form using Quality by Design approach

BackgroundA new, sensitive, suitable, clear, accurate, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of brexpiprazole in bulk drug and tablet formulation was developed and validated in this research. Surface methodology was used to optimize the data, with a three-level Box-Behnken design. Methanol concentration in the mobile phase, flow rate, and pH were chosen as the three variables. The separation was performed using an HPLC method with a UV detector and Openlab EZchrom program, as well as a Water spherisorb C18 column (100 mm × 4.6; 5m). Acetonitrile was pumped at a flow rate of 1.0 mL/min with a 10 mM phosphate buffer balanced to a pH of 2.50.05 by diluted OPA (65:35% v/v) and detected at 216 nm.ResultThe developed RP-HPLC method yielded a suitable retention time for brexpiprazole of 4.22 min, which was optimized using the Design Expert-12 software. The linearity of the established method was verified with a correlation coefficient (r2) of 0.999 over the concentration range of 5.05–75.75 g/mL. For API and formulation, the percent assay was 99.46% and 100.91%, respectively. The percentage RSD for the method’s precision was found to be less than 2.0%. The percentage recoveries were discovered to be between 99.38 and 101.07%. 0.64 μg/mL and 1.95 μg/mL were found to be the LOD and LOQ, respectively.ConclusionThe developed and validated RP-HPLC system takes less time and can be used in the industry for routine quality control/analysis of bulk drug and marketed brexpiprazole products.Graphical abstract

Development, validation, and application of an RP-HPLC method for concurrent quantification of Minoxidil and Finasteride in a topical solution for hair regrowth

A topical solution comprising of Minoxidil (MXL) and Finasteride (FNS) for alopecia is formulated in the present work, which essentially contains a lipid-Lauroglycol FCC as a penetration enhancer. The objective of the proposed work was to develop a rapid, simple, and robust reverse phase high performance liquid chromatographic (RP-HPLC) method to determine MXL and FNS in the said formulation. Herein, the chromatographic conditions were optimized based on the theoretical principles of separation and physicochemical properties such as pKa and log P of both the Active Pharmaceutical Ingredients (APIs). The separation was accomplished on an Inertsil® ODS-3 C18 column (150mm×4.6mm; 5μm of particle size) at 25°C by using a mobile phase composed of 70:30 v/v ratio of Methanol and Milli-Q water along with 0.5% Triethylamine at pH 6.4 adjusted with Ortho Phosphoric Acid. Drug peaks showed a good resolution at 210nm. The retention times for MXL and FNS were found to be 2.40min and 6.39min, respectively. The developed method was found to be linear (R2≥0.998) in a concentration range of 5-100μg/mL for both the drugs. The method was validated according to the ICH guidelines Q2 (R1). The ability of the method to differentiate between the types formulations was demonstrated by the in vitro diffusion data performed using a highly sophisticated Strat-M® membrane. The cumulative amount of drug released (MXL and FNS) at the end of 24hours was maximum for the topical formulation containing lipids prepared using isopropyl alcohol and propylene glycol as the base.

Development and Validation of Fast, Simple, Cost-effective and Robust RP-HPLC Method for Simultaneous Determination of Lisinopril and Amlodipine in Tablets

A new Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method for simultaneous determination of lisinopril dihydrate and amlodipine besylate in tablets was developed and validated. This method operates under moderate chromatographic conditions, avoiding high column temperatures, ion-pair reagents, gradient elution or mobile phases with very low pH value as it is usually recommended by the majority of the existing RP-HPLC methods used for the same purpose. The developed method uses a C18 column, UV-detection and mobile phase consisting of methanol and phosphate buffer with pH 7.3. The method provides chromatograms with excellent peak symmetry and is very simple, selective, accurate, reproducible, robust and cost-effective. The rather moderate experimental conditions make the method favorable with regards to time, chemicals and consumables consumption and consequently very useful for application in the quality control laboratories in pharmaceutical companies. The method performance has been demonstrated using different reversed-phase columns and with different neutral mobile phases showing that it can be adjusted and used in different laboratories for analysis of different combinations of lisinopril with other antihypertensive active ingredients.

STABILITY INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF DIETHYLCARBAMAZINE CITRATE, GUAIPHENESIN AND CHLORPHENIRAMINE MALEATE

Objective: The present work describes the development and subsequent validation of a simple, precise and stability–indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate in tablet dosage forms.Methods: A simple, accurate, precise and robust RP-HPLC method was developed and validated for the estimation of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate. The chromatographic separation of all the three active components was achieved by using luna phenyl-hexyl column (250 mmx4.6 mm, dp=5 µm) with a mobile phase consisting of isocratic method with 0.1% triethylamine as buffer along with orthophosphoric acid adjusted to PH 2.5: acetonitrile (50:50v/v) at a flow rate 1.0 ml/min and ultraviolet detection at 210 nm.Results: The retention time of chlorpheniramine maleate, guaiphenesin and diethylcarbamazine citrate were 2.86, 4.89 and 7.76 min respectively. Validation of the proposed method was carried out according to an international conference on harmonization (ICH) guidelines. The established method was linear in the range of 1-15, 0.6-9, 0.02-0.3 µg/ml and correlation coefficient was 0.999, 0.9991, and 0.993 for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate respectively.Conclusion: The proposed method can be used for the quantitative analysis of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate.

Single robust RP-HPLC analytical method for quantification of curcuminoids in commercial turmeric products, Ayurvedic medicines, and nanovesicular systems

ABSTRACTA single robust reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated as per International Conference on Harmonization guidelines for the accurate quantification of curcuminoids in commercial turmeric products, Ayurvedic medicines, and nanovesicular systems. The proposed chromatographic method was found to be specific, linear (r2 ≥ 0.999), precise at intra- and inter-day levels (percentage relative standard deviation <2.0%), accurate (percentage recovery 99.14–102.29%), and robust. The limits of detection and quantification were found to be 7.40 and 24.70 ng mL−1 for curcumin, 9.24 and 30.80 ng mL−1 for demethoxycurcumin, and 6.48 and 21.61 ng mL−1 for bisdemethoxycurcumin, respectively. Among different commercial turmeric products and Ayurvedic medicines tested, the contents of curcumin (3.54 ± 0.06–25.8 ± 0.08 mg g−1), demethoxycurcumin (1.28 ± 0.02–9.97 ± 0.03 mg g−1), and bisdemethoxycurcumin (0.50 ± 0.01–5.97 ± 0.01 mg g−1) varied significantly. The developed method was effectively applied to the determination of encapsulation efficiency of curcuminoids (ranged between 84.33 ± 3.50 and 96.59 ± 2.53%) in the nanovesicular systems. In conclusion, the reported method is suitable for the analysis of curcuminoids in a wide variety of turmeric products and used for the quality control of products that contain curcuminoids.

Use of Systematic Approach for Development of RP-HPLC Method for Simultaneous Determination of Lopinavir and Ritonavir

A simple and robust reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the quantitative estimation of Lopinavir and Ritonavir in bulk and formulation. The systematic approach, one of the parts of QbD was use for the analytical method development. Chromatographic separation was conducted on Phenomenex Gemini C18 (250 mm×4.6 mm, 5 µ) column at 25°C using Sodium dihydrogen phosphate buffer: acetonitrile (pH adjusted to 5.3±0.1 with acetic acid) in the ratio of 45:55 v/v. The flow rate was 1 ml/min and UV detection was carried out at 262 nm. The retention times of Lopinavir and Ritonavir were 8.90 min and 5.36 min respectively. The method was found to be linear in the range of 5 30 µg/ml for lopinavir and 2 10 µg/ml for ritonavir. The developed method was validated for specificity, accuracy, precision, LOD, LOQ, robustness as per ICH guidelines.

Using UV-absorbance of intrinsic dithiothreitol (DTT) during RP-HPLC as a measure of experimental redox potential in vitro

Many in-vitro experiments performed to study the response of thiol-containing proteins to changes in environmental redox potentials use dithiothreitol (DTT) to maintain a preset redox environment throughout the experiments. However, the gradual oxidation of DTT during the course of the experiments, and the interaction between DTT and other components in the system, can significantly alter the initial redox potential and complicate data interpretation. Having an internal reporter of the actual redox potential of the assayed sample facilitates direct correlation of biochemical findings with experimental redox status. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used, well-established tool for analysis and purification of biomolecules, including proteins and peptides. Here, we describe a simple, robust, and quantitative RP-HPLC method we developed and tested for determination of the experimental redox potential of an in-vitro sample at the time of the experiment. It exploits the specific UV-absorbance of the oxidized intrinsic DTT in the samples and retains the high resolving power and high sensitivity of RP-HPLC with UV detection.

Separation and determination of resibufogenin and cinobufagin in Chansu using reversed‐phase liquid chromatography with γ‐cyclodextrin as mobile‐phase modifier

A simple, accurate, sensitive, and robust reversed-phase high-performance liquid chromatography (HPLC) method employing cyclodextrins as mobile phase additives has been developed in order to separate and determine resibufogenin and cinobufagin. Various factors affecting the separation for them, such as the nature of cyclodextrins, organic solvent, the concentration of γ-cyclodextrin, and temperature, were systematically studied. γ-cyclodextrin, as an effective mobile phase additive, can markedly improve the separation for resibufogenin and cinobufagin. The role of γ-cyclodextrin in the developed HPLC method is attributed to the formation of the inclusion complex between resibufogenin (or cinobufagin) and γ-cyclodextrin. So, the apparent formation constant (K(f) ) of the resibufogenin (or cinobufagin)/γ-cyclodextrin inclusion complex and the thermodynamic parameters of the inclusion process also were investigated. Resibufogenin (or cinobufagin) forms the 1:1 inclusion complexes with γ-cyclodextrin, and the resibufogenin/γ-cyclodextrin complex is more stable than the cinobufagin/γ-cyclodextrin complex. The K(f) values of resibufogenin and cinobufagin decrease with the increase of the temperature. The thermodynamic parameters of the inclusion reveal that the inclusion process between resibufogenin (or cinobufagin) and γ-cyclodextrin is spontaneous, exothermic, and enthalpically driven. Finally, the optimized method was successfully applied to separate and determine of resibufogenin and cinobufagin in the different Chansu (Bufonis venenum) samples.