Reversed-phase High-performance Liquid Chromatographic Method Research Articles

Green RP-HPLC method for simultaneous quantification of epigallocatechin-3-gallate and rosmarinic acid in lipid-based nanocarriers and biological fluids: Quality by Design-driven optimization and lean six sigma approach

This study presents the development and validation of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for concurrent quantification of EGCG and RA in prepared lipid-based nanocarriers and biological fluids using a QbD approach, greenness assessment, and Six-Sigma methodology. Initially, variations in critical analytical attributes were identified using the Ishikawa fishbone diagram and Risk Priority Number scores. A Taguchi Orthogonal Array design was employed to screen the critical method parameters influencing method development. Systematic optimization of the RP-HPLC method was subsequently achieved using a Box-Behnken design, with the % organic phase, flow rate, and column temperature as the variables. The responses were retention time, tailing factor, and theoretical plates for both EGCG and RA. Chromatographic separation was accomplished with a methanol and 0.1% formic acid (60:40) isocratic flow system on a Phenomenex Luna C18 column (4.6 × 250 mm, 5 μm) at a 1 mL/min flow rate. The method was rigorously validated according to ICH guidelines. Linearity was observed over a concentration range of 2 to 10 µg/mL for both EGCG and RA, yielding correlation coefficients of 0.998 and 0.999, respectively. The LOD and LOQ were determined to be 0.51 µg/mL and 1.54 µg/mL for EGCG, and 0.35 µg/mL and 1.07 µg/mL for RA. Moreover, the %RSD for all validation parameters were consistently below 2%. Forced degradation studies were conducted under acidic, basic, oxidative, and photolytic conditions to elucidate potential degradation pathways and identify degradation products. The developed method was applied to estimate EGCG and RA in prepared lipid-based nanocarriers and biological fluids like blood plasma and urine. Recovery assays in lipid-based nanocarriers, plasma, and urine samples demonstrated excellent recoveries (96.2–102.1%). The method's greenness was assessed using various tools, and its accuracy was evaluated using Six Sigma methodology. Overall, the developed HPLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents, alone and in combination therapies.

Novel analytical approach for baclofen quantification in rodent plasma.

A simple, accurate, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of Baclofen in rat plasma. The method demonstrated high degree of linearity (r2 = 0.9936) across a concentration range of 10-50 μg/mL. Precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness were evaluated according to ICH guidelines. The LOD and LOQ were found to be 0.076197 and 0.23090 μg/mL, respectively. This method provides an efficient approach for Baclofen quantification in plasma, making it suitable for pharmacokinetic and bioavailability studies. The novelty of this study lies in its optimization for routine use in laboratories, ensuring reproducibility with minimal variations across different conditions and analysts.

Eco-friendly RP-HPLC approach for simultaneously estimating the promising combination of pentoxifylline and simvastatin in therapeutic potential for breast cancer: Appraisal of greenness, whiteness, and Box–Behnken design

Abstract Breast cancer affects millions of women worldwide. This study explores the potential of combining pentoxifylline (PTX) and simvastatin (SIM) as a treatment for breast cancer. We aimed to develop six sustainability tools using green and white metrics to evaluate the environmental impact of reversed-phase high-performance liquid chromatography (RP-HPLC) methods for analyzing and separating PTX and SIM in their pure forms. The tools include analytical GREEnness, green analytical procedure index, Complexgreen analytical procedure index, analytical greenness metric for sample preparation, blue applicability grade index, and the RGB 12 algorithm. For the separation, we used a Novapack C8 column (15 × 0.46 cm, 5 µm) at 25°C. The injection volume was 5.0 µL, the wavelength was set to 210 nm, and the total runtime was 5 min. We identified optimal chromatographic conditions efficiently using the Box–Behnken design with minimal trials. We investigated the effects of three factors on retention time and resolution: acetonitrile ratio, pH, and flow rate. We used overlay plots with a 60:40 ratio (v/v) of acetonitrile and bi-distilled water to forecast the most effective mobile phase. The calibration curves for PTX and SIM showed a correlation value of over 0.999 within the range of 5–60 µg·mL−1. The recovery rates ranged from 99.9% to 100.2%, indicating high accuracy. Our RP-HPLC technique proves to be reliable and efficient for the simultaneous estimation of multiple anticancer drugs. We evaluated the environmental sustainability of this approach using green and white metrics, and the recommended method has been thoroughly validated according to International Council for Harmonisation guidelines, making it highly reliable for analyzing new formulations.

Application of Reversed-Phase HPLC Method for the Simultaneous Determination of Lenacapavir and Bictegravir in Tablets Dosage Form

Application of Reversed-Phase HPLC Method for the Simultaneous Determination of Lenacapavir and Bictegravir in Tablets Dosage Form

TLC vs. HPLC: A green comparison for veterinary drug analysis

This work describes two analytical methods for determining lidocaine (LD) and oxytetracycline (OTC) in veterinary dosage form: a traditional Thin-Layer Chromatography (TLC) method and a more environmentally friendly Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method.Initially, the TLC method was used due to its speed, simplicity, and accuracy. It successfully separated LD and OTC in veterinary dosage forms with a linearity range of 0.5–11.0 µg/band for LD and 4.0–34.0 µg/band for OTC. However, TLC proved to be inadequate for resolving the binary mixture in the presence of toxic lidocaine impurity (2,6-dimethylaniline) (DMA), and was less environmentally friendly.Consequently, an RP-HPLC method was developed using a C18 column and a mobile phase consisting of ethanol and phosphate buffer (pH 7) in a 70:30 ratio, with a flow rate of 1.0 mL/min. This method effectively separated OTC, LD and DMA, using a PDA detector at wavelengths of 272, 232, and 210 nm and with retention times of 1.529, 2.77, and 3.581 min for OTC, DMA, and LD respectively. The RP-HPLC method showed linearity ranges of 270.0–972.0 µg/mL for OTC, 0.5–25.0 µg/mL for DMA, and 5.0–18.0 µg/mL for LD.Both methods were validated for linearity, precision, accuracy, robustness, detection limit, and quantitation limit. The greenness of each method was evaluated using NEMI, Eco-scale, and Agree tools, with results demonstrating the RP-HPLC method as the more environmentally sustainable method.

Stability indicating RP-HPLC method development and validation for quantification of impurities in gonadotropin-releasing hormone (Elagolix): Robustness study by quality by design.

The purpose of this research was to establish and validate a reverse phase HPLC method for the determination of Elagolix impurities in pharmaceutical dosage form. Mobile phase A, consisting of 10mM sodium dihydrogen phosphate (pH6.0) and acetonitrile in a 95:5v/v ratio, and mobile phase B, containing 85:10:5v/v/v of acetonitrile, Milli-Q water, and methanol, were used to achieve the method's specificity in the analytical column Kromasil 100-C18 (250 mm × 4.6mm, 5μm). The gradient program includes (%B/Time [min]: 36/0, 36/10, 38/15, 85/55, 85/65, 36/67, and 36/75). The flow rate is 0.8mL/min. The overall run duration is 75.0min, the injection volume is 10.0μL, and the detection is at 210 nm in UV. The samples were subjected to hydrolysis, oxidation, and heat conditions in order to facilitate their forced degradation. The procedure was validated and determined with the standards of ICH guidelines. From the LOQ to a concentration level of 200%, the linearity of the technique was ascertained. An accuracy range of LOQ to 150% was established for the method, and the average recovery was acceptable. Design of experiments, part of the quality by design idea, was used to prove the method's reliability.

Fabrication of a molecularly imprinted poly(methyl methacrylate) decorated graphite electrode for detection of aloe emodin in Aloe vera

In this work, the authors represent a comprehensive approach for the development and validation of a novel graphite-supported molecularly imprinted poly(methyl methacrylate) electrode for the selective and sensitive detection of aloe emodin (AE) in aloe-based cosmetic products. The electrocatalytic activity of the electrode was evaluated using cyclic voltammetry and differential pulse voltammetry techniques in phosphate buffer saline. Surface morphology, structural characteristics, and imprinting endorsement were investigated using field emission scanning electron microscopy, Fourier-transform infrared spectroscopy, and UV-Vis spectroscopy. Under the optimal conditions, the electrode showed an oxidative differential pulse voltammetry peak at -0.2931±0.011 V (vs. Ag/AgCl, saturated KCl). The current was increased linearly with increasing concentrations of AE from 0.0005 to 350 µM, with a low detection limit of 0.0003 µM. The voltammetric analysis has been validated by reverse-phase high-performance liquid chromatography (RP-HPLC) methods. A partial least square regression model was developed to correlate the results obtained from the proposed system with the reference values obtained by RP-HPLC. The model showed a high prediction accuracy of 95.95 % for the detection of AE in cosmetic samples. The developed electrode provides a low-cost, rapid, easy, and convenient method for the detection of AE in cosmetic products, offering potential benefits for quality control and safety assessment in the cosmetic industry.

An Alternative Method of Reversed-Phase HPLC for Identification and Quantitative Determination 0f Ergot Alkaloids

An Alternative Method of Reversed-Phase HPLC for Identification and Quantitative Determination 0f Ergot Alkaloids

Establishment and Validation of a Robust Reversed-Phase HPLC Method for the Determination of Calotropis gigantea in Bulk Material and Marketed Product

Establishment and Validation of a Robust Reversed-Phase HPLC Method for the Determination of Calotropis gigantea in Bulk Material and Marketed Product

Development and validation of a stability-indicating HPLC method for assay of tonabersat in pharmaceutical formulations

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to assay tonabersat and assess its stability in pharmaceutical formulations. Chromatographic separation was achieved using a Kinetex® C18 column (2.6 µm, 150 x 3 mm, 100 Å) at 50 °C, with a 20 µL injection volume. A linear gradient of acetonitrile in water (5 – 33.5 %) was applied for 1 min, followed by a gradual increase to 100 % over 26 min at a flow rate of 0.5 mL/min. Tonabersat and its degradation products were detected at 275 nm and 210 nm, respectively. The optimized method was used to evaluate the stability of tonabersat in lipid-based pharmaceutical formulations at 5 ± 3 °C, 25 ± 2°C/60 ± 5 % RH, and 40 ± 2 °C/75 ± 5 % RH over 3 months. The method was validated as per ICH guidelines and demonstrated linearity in the range of 5 – 200 µg/mL (R2 = 0.99994) with good accuracy (98.25 – 101.58 % recovery) and precision (% RSD < 2.5 %). The limits of detection and quantitation were 0.8 µg/mL and 5 µg/mL, respectively. Forced degradation studies showed significant degradation on exposure to alkaline (90.33 ± 0.80 %), acidic (70.60 ± 1.57 %), and oxidative stress (33.95 ± 0.69 %) at 70 °C, but no degradation was observed on exposure to thermal or photolytic stress. No chemical degradation was observed in either formulation on storage. Thus, the method was sensitive, specific, and suitable for stability testing of tonabersat in pharmaceutical formulations.

A stability-indicating method development and validation for the determination of related substances in novel synthetic decapeptide by HPLC.

In the present scenario, peptide is an emerging field of research having vast therapeutic applications. Diverse impurities may rise from various stages of the synthesis process and storage of the peptides. Because these contaminants may have an impact on the therapeutic safety and effectiveness of peptides in their approaching applications, they must be identified and carefully monitored. Considering the pharmaceutical importance of the extent of peptides, we were motivated to synthesize a decapeptide and establish a novel gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method for its analysis along with efficient separation of its six related impurities. Different buffers, organic modifiers, and columns were used in the tests for good separation of these impurities. To establish a stability-indicating method, a stress study was also conducted. The International Conference on Harmonization (ICH) guidelines have been followed for validation of the developed analytical method. The validated method revealed sufficient accuracy, specificity, linearity, robustness, precision, and high sensitivity for its intended use. The proposed method could be appropriate for routine analysis and stability assessment of the decapeptide, which might be useful for further scientific investigation.

A sustainable RP-HPLC method for concurrent estimation of capecitabine and celecoxib in liposomal formulation: Greenness and whiteness appraisal.

Liposomes have been reported for combination therapy due to their ability to carry both hydrophilic and lipophilic drugs together. The current investigation aims to develop a novel, eco-friendly, and sustainable reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of capecitabine and celecoxib co-encapsulated in liposomes. The method reported herein uses a C18 column (4.6 × 250 mm2, 5 μm) and a mobile phase consisting of water, and acetonitrile/methanol in a ratio of 60:40, containing 0.1% formic acid in both the phases. The flow rate is maintained at 1 mL/min, with an injection volume of 10 μL in the gradient mode. Detection is set at λmax = 240 nm for capecitabine and 252 nm for celecoxib. The developed liposomes are mono-disperse with a surface potential of -6.93 mV. The average size of the liposomes is 142 nm. The percentage entrapment efficiency for capecitabine is 52.39 ± 0.94%, and for celecoxib, it is 77.13 ± 0.74%. The Analytical Greenness Metric of 0.61 and Analytical Eco-Scale Score of 75 signify the greenness of the developed method. Also, the Red-Green-Blue model shows excellent whiteness, with a score of 83.2. Thus, the developed method offers a reliable, accurate, precise, buffer-free, and environment-friendly RP-HPLC approach for the simultaneous analysis of capecitabine and celecoxib co-encapsulated in liposomes.

Analytical Method Validation and Simultaneous Determination of Ketotifen Fumarate and Cyproheptadine Hydrochloride in Divided Powder by RP-HPLC Method

Ketotifen fumarate and cyproheptadine HCl are the most popular prescription for children at “X” hospital in Yogyakarta. The interest for studying this divided powder was related to the stability test towards human health. Hence, it was important to developed a validated analytical method to quantify content of active drug from sample. In this study, a reversed phase HPLC (RP-HPLC) method was validated and applied for determining content of ketotifen fumarate and cyproheptadine hydrochloride in divided powder. The developed RP-HPLC method was met the requirement of selectivity and sensitivity. Calibration curve formulas of ketotifen fumarate and cyproheptadine hydrochloride were y = 27714x-79111 (r=0.9945) and y = 26324x+72581 (r=0.9935) in the range of 10.9-54.5 and 10.6-53.0 µg/mL, respectively. Accuracy and intermediate precision were assessed on both standard solution and samples spiked with standard solution of ketotifen fumarate and cyproheptadine hydrochloride. It was found that both standard solution and the spiked samples solution were met the requirement of recovery of 80-110% and RSD of less than 7.3% for intraday and interday analysis. Content of ketotifen fumarate and cyproheptadine hydrochloride in sample (n=7) were 16.574±0.098 and 30.640±0.035 µg/mL, respectively. It can be concluded that the RP-HPLC method was successfully validated and applied in the determination of ketotifen fumarate and cyproheptadine hydrochloride in divided powder.

Method Development and Validation for the Simultaneous Estimation of Lobeglitazone Sulphate and Glimepiride by HPLC Method.

A simple, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of lobeglitazone sulfate and glimepiride in bulk and tablet dosage forms. The method involved selecting various chromatographic parameters. Specifically, a new method was developed using a 250 mm × 4.6 mm (HSS) reverse-phase C18 column with 5 µm particle size (Shim-pack C18, 250 × 4.6 mm; 5 µm). The mobile phase consisted of 40 volumes of phosphate buffer (pH 3) and 60 volumes of acetonitrile, with methanol as the diluent, running as an isocratic elution. The flow rate was set at 1.0 mL/min, and UV detection was performed at 254 nm. The injection volume was 2 µL, and the total runtime was 10 minutes. The recoveries for lobeglitazone sulfate were found to be in the range of 101% to 100.6%, while those for glimepiride were in the range of 101.5% to 100%. The method’s accuracy is evident from these results. The inter-day and intra-day precision of the new method were both below the maximum allowable limit (RSD% ≤ 2.0), as per International Council on Harmonisation (Q2 R1) guidelines. The method exhibited a linear response, with correlation coefficient (r2) values of 0.9972% for pioglitazone and 0.998 for glimepiride. The validation parameters, such as accuracy, precision, linearity, specificity, stability in the analytical solution and robustness, met the acceptance criteria. Hence, this method can be effectively used for the simultaneous estimation of lobeglitazone sulfate and glimepiride in both bulk and pharmaceutical dosage forms.

A comprehensive review of analytical strategies for validating RP-HPLC methods of aceclofenac and thiocolchicoside in bulk drug and formulation

This comprehensive review explores the analytical method development and validation of Aceclofenac and Thiocolchicoside bulk drugs and formulations using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). Aceclofenac, a nonsteroidal anti-inflammatory drug (NSAID), and Thiocolchicoside, a muscle relaxant, are commonly co-formulated for the treatment of pain and inflammation. The review discusses the principles and strategies involved in RP-HPLC method development, emphasizing factors such as stationary phase selection, mobile phase composition, and detection wavelength optimization. Furthermore, it highlights the importance of method validation in pharmaceutical analysis, covering parameters such as specificity, linearity, accuracy, precision, robustness, and system suitability. The literature review section examines previous studies on RP-HPLC methods developed for Aceclofenac and Thiocolchicoside, summarizing key findings and comparing different methodologies. Challenges and limitations encountered during method development and validation are discussed, along with recent advances in RP-HPLC methodology. Finally, the review outlines future research directions and potential applications of RP-HPLC in studying the pharmacokinetics and bioavailability of Aceclofenac and Thiocolchicoside formulations.

Development and Validation of an UV-Spectrophotometric and Reverse-Phase High-Performance Liquid Chromatography Method for the Estimation of Umifenovir in Bulk and Tablet Formulations

Development and Validation of an UV-Spectrophotometric and Reverse-Phase High-Performance Liquid Chromatography Method for the Estimation of Umifenovir in Bulk and Tablet Formulations

Development and Validation of Methodologies for the Identification of Specialized Pro-Resolving Lipid Mediators and Classic Eicosanoids in Biological Matrices.

Lipid mediators, which include specialized pro-resolving mediators and classic eicosanoids, are pivotal in both initiating and resolving inflammation. The regulation of these molecules determines whether inflammation resolves naturally or persists. However, our understanding of how these mediators are regulated over time in various inflammatory contexts is limited. This gap hinders our grasp of the mechanisms underlying the disease onset and progression. Due to their localized action and low endogenous levels in many tissues, developing robust and highly sensitive methodologies is imperative for assessing their endogenous regulation in diverse inflammatory settings. These methodologies will help us gain insight into their physiological roles. Here, we establish methodologies for extracting, identifying, and quantifying these mediators. Using our methods, we identified a total of 37 lipid mediators. Additionally, by employing a reverse-phase HPLC method, we successfully separated both double-bond and chiral isomers of select lipid mediators, including Lipoxin (LX) A4, 15-epi-LXA4, Protectin (PD) D1, PDX, and 17R-PD1. Validation of the method was performed in both solvent and surrogate matrix for linearity of the standard curves, lower limits of quantitation (LLOQ), accuracy, and precision. Results from these studies demonstrated that linearity was good with r2 values > 0.98, and LLOQ for the mediators ranged from 0.01 to 0.9 pg in phase and from 0.1 to 8.5 pg in surrogate matrix. The relative standard deviation (RSD) for inter- and intraday precision in solvent ranged from 5% to 12% at low, intermediate, and high concentrations, whereas the RSD for the inter- and intraday variability in the accuracy ranged from 95% to 87% at low to high concentrations. The recovery in biological matrices (plasma and serum) for the internal standards used ranged from 60% to 118%. We observed a marked ion suppression for molecules evaluated in negative ionization mode, while there was an ion enhancement effect by the matrix for molecules evaluated in positive ionization mode. Comparison of the integration algorithms, namely, AutoPeak and MQ4, and approaches for calculating signal-to-noise ratios (i.e., US Pharmacopeia, relative noise, peak to peak, and standard deviation) demonstrated that different integration algorithms tested had little influence on signal-to-noise ratio calculations. In contrast, the method used to calculate the signal-to-noise ratio had a more significant effect on the results, with the relative noise approach proving to be the most robust. The methods described herein provide a platform to study the SPM and classic eicosanoids in biological tissues that will help further our understanding of disease mechanisms.

Development of RP-HPLC methods for the analysis of melatonin alone and in combination with sleep-enhancing dietary supplements

Melatonin, a hormone produced by the pineal gland, is widely recognized for its role in regulating sleep-wake cycles. With the increasing prevalence of sleep disorders, melatonin supplements have gained popularity as a natural remedy. Additionally, various sleep-enhancing dietary supplements combine melatonin with other active ingredients to enhance efficacy. Reliable and accurate analytical methods are essential for ensuring the quality, safety, and efficacy of these products. Reverse-phase high-performance liquid chromatography (RP-HPLC) is a powerful analytical technique widely used for the separation, identification, and quantification of compounds in complex mixtures. This article explores the development and validation of RP-HPLC methods for the analysis of melatonin, both as a single ingredient and in combination with other components in sleep-enhancing dietary supplements.

Development and Validation of Reversed-phase High-performance Liquid Chromatography Method for Simultaneous Estimation of Desonide and Curcumin in Topical Dosage Form

Development and Validation of Reversed-phase High-performance Liquid Chromatography Method for Simultaneous Estimation of Desonide and Curcumin in Topical Dosage Form

A novel QbD HPLC approach for the concurrent analysis of amoxicillin, amprolium, and ethopabate in food samples and dosage forms

A simple, rapid, and sensitive reversed-phase HPLC method coupled with photodiode array detection (PDA) was developed for the simultaneous estimation of three co-administered antibacterial drugs, amprolium, ethopabate, and amoxicillin for the first time. An analytical quality-by-design (QbD) approach was adopted to optimize the developed approach. A two-level full factorial design (25 FFD) was implemented to optimize how variable factors affected chromatographic responses. On a Hypersil BDS C18 column, the chromatographic separation was carried out using isocratic elution at 40.1 °C. The mobile phase consisted of methanol: potassium phosphate buffer containing 5.76 mM heptane sulphonate (9.05 mM; pH 3.32, 44.8:55.2 % v/v) pumped at a flow rate of 0.8 mL/min and an injection volume of 20.0 μL. In less than 7 min, the suggested method was able to separate the ternary mixture with a challenging ratio of 1: 15.6: 1 for amoxicillin, amprolium, and ethopabate, respectively. The calibration curves showed excellent linearity over the concentration ranges of 0.50–30.0, 0.30–30.0, and 0.05–20.0 μg/mL, respectively. Recovery percentages ranged from 90.30 % to 111.13 % with %RSD less than 2 % were obtained upon spiking to chicken tissues, liver, and egg samples, indicating the suitability of the proposed method for determining of the studied drugs in complicated food matrices. Furthermore, the proposed method was successfully applied to determine the studied drugs in their commercial formulations. Full validation was carried out according to ICHQ2 (R1) guidelines. Finally, the method's greenness and ecofriendliness were evaluated using various metrics tools, including the Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE).