Reversed-phase High-performance Liquid Chromatographic Method Research Articles

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).

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.

Analytical Method Development and Validation of Simultaneous Estimation of Pure and Tablet Dosage Form by RP-HPLC

The aim of this analytical research is to establish and validate the RP-HPLC (Reverse Phase High Performance Liquid Chromatography) method for concurrently quantifying Montelukast Sodium, Fexofenadine Hydrochloride, and Acebrophylline in a pharmaceutical formulation. The method utilized Rosagiline mesylate as an internal standard, employing a Shim-Pack Solar C18 Column (4.6 x 150 mm, 5 µm) as the stationary phase and a mobile phase composed of acetonitrile, methanol, and 10 mM Na2HPO4 buffer (50:30:20 % v/v/v, pH 5.5). The flow rate was maintained at 1.0 mL/min, and detection occurred at 210 nm. Validation followed ICH guidelines. The linear concentration ranges were 5-30 µg/ml, 10-60 µg/ml, and 10-100 µg/ml at 210 nm for Montelukast Sodium, Fexofenadine Hydrochloride, and Acebrophylline, respectively. Retention times were 7.643 min, 2.117 min, 3.863 min, and 3.050 min for Montelukast sodium, Fexofenadine Hydrochloride, Acebrophylline, and Rosagiline mesylate respectively. The RP-HPLC analysis of marketed formulations yielded concentrations within the ranges of 98.3–100.9%, 99.46–101.23%, and 99.82–101.74% for Montelukast sodium, Fexofenadine Hydrochloride, and Acebrophylline, respectively. Recovery fell within the range of 95.81-101.35% for both drugs. The proposed method has been validated as per ICH guidelines and successfully applied to the estimation of Montelukast, Fexofenadine and Acebrophylline in their combined tablet dosage form.

A new RP-HPLC method for the simultaneous detection and quantification of minocycline and ascorbic acid in pure forms and combined pharmaceutical dosage forms

We report the first reverse-phase, high performance liquid chromatography (RP-HPLC) method for the rapid and simple simultaneous detection and quantification of minocycline and ascorbic acid in raw and pharmaceutical dosage forms. Detection was performed on a Sunniest C-18 column (5 μm particle size, 150 mm × 4.6 m mi.d(, at UV wavelength of 255 nm. The mobile phase consisted of potassium dihydrogen phosphate pH 2.8: Acetonitrile: Methanol (85:10:5), with 0.6 v/v% TEA and adjusted with orthophosphoric acid to a pH of 2.8 in an isocratic elution mode, with a flow rate of 0.8 mL/min. Performed analysis on the method proved that the newly developed method was sensitive, selective, accurate, precise, and robust. The method demonstrated good linearity with r2 = 0.9999 (n = 7) for both drugs and was validated in the sample concentration ranges of 1–100 and 5–500 µg mL−1 for minocycline and ascorbic acid, respectively. Both intra- and inter-day precisions were less than 2 % for both drugs, with limits of detection of 1.59 and 7.89 µg/mL and limits of quantification of 4.83 and 23.89 µg/mL, for minocycline and ascorbic acid, respectively. The applicability of the method was demonstrated by accurately determining the drug content of two commercial pharmaceutical formulations.

A simple and quick run time RP-HPLC method for simultaneously analysis of pyrazosulfuron ethyl and pretilachlor content on GR formulation

Aim of the study was to develop and validate simple and quick run time reverse phase HPLC method for the simultaneous determination of pyrazosulfuron ethyl and pretilachlor in the pesticide formulation of GR. The method was executed toward using the Agilent Eclipse XDB C-18 column (150 x 4.6 mm; 3.5 μm), alongside the mobile phase comprising of 0.1 % (ortho phosphoric acid) with water: acetonitrile (30:70) at a column rate of flow 1.2 mL per minute and determination has been carried out in 220 nm. The retention time for Pyrazosulfuron Ethyl as well as Pretilachlor have been determined at 1.99 as well as at 4.58 minute, correspondingly with runtime 7 min. The method's effectiveness has been validated using SANCO recommendations for LOD, robustness, accuracy, precision, linearity, selectivity and specificity. There are several analytical methods reported for individual content estimation, but single method is not available to determine simultaneous content. The developed method has been utilized to estimate pyrazosulfuron ethyl and pretilachlor content in industries.

A sensitive, expandable AQC-based LC-MS/MS method to measure amino metabolites and sphingolipids in cell and serum samples

Sphingolipids are a major lipid species found in all eukaryotes. Among structurally complex and diversified lipids, sphingoid bases have been heavily linked to various metabolic diseases. However, most current LC-MS-based methods lack the sensitivity to detect low-abundant sphingoid bases. The 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization reagent, which efficiently forms covalent bonds with amino groups, has been widely used for amino acid detection. Nevertheless, the commonly used reverse-phase HPLC method for amino acid analysis is not suitable for amphipathic sphingolipids. To address this issue, we report a robust reverse-phase HPLC-MS/MS method capable of separating and detecting hydrophilic amino acids and sphingoid bases in a single run with high sensitivity. This method is also inclusive of other amino metabolites with an expandable target list. We tested this method under various conditions and samples, demonstrating its high reproducibility and sensitivity. Using this approach, we systematically analyzed human serum samples from healthy individuals, dyslipidemia, and type II diabetes mellitus (T2DM) patients, respectively. Two sphingolipids and five amino acids were identified with significant differences between the control and T2DM groups, highlighting the potential of this method in clinical studies.

LC–MS/MS-based phospholipid profiling of plant-pathogenic bacteria with tailored separation of methyl-branched species

Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC–MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.Graphical