Validation Of High-performance Liquid Chromatography Method Research Articles

CUTTING-EDGE DEVELOPMENTS IN HPLC FOR IDENTIFYING PROCESS IMPURITIES: A REVIEW ON METHOD OPTIMIZATION AND VALIDATION

High-Performance Liquid Chromatography (HPLC) is a widely utilized column chromatography technique in biochemistry and analytical sciences for the separation, identification, and quantification of active compounds. Renowned as a leading separation technology, HPLC plays a pivotal role in detecting, separating, and quantifying pharmaceutical drugs. The development and validation of HPLC methods are essential in novel drug discovery, development, manufacturing, and various human and animal studies. This review highlights the comprehensive processes involved in the development and validation of HPLC methods. Method development is influenced by factors such as the chemical structure of molecules, synthetic pathways, solubility, polarity, pH, pKa values, and the activity of functional groups. Validation, as per ICH Guidelines, encompasses key parameters including accuracy, precision, specificity, linearity, range, limits of detection and quantification, robustness, and system suitability testing.

Validated HPLC-UV Method for the Quantification of a Novel BCr-Abl 1 Inhibitor, Vodobatinib, in Rat Plasma: Application to a Pharmacokinetic Study.

Vodobatinib is a Bcr-Abl 1 inhibitor, currently entering into Phase 2 clinical trials as a potential drug to treat glioblastoma patients. In the present work, a validated high-performance liquid chromatography (HPLC) detection method for the quantification of vodobatinib in rat plasma was established. Sample preparation involved liquid-liquid extraction method. The chromatographic separation of vodobatinib and the IS was accomplished on an XBridge C18 column using 10 mM ammonium acetate and acetonitrile in gradient condition. The flow-rate and injection volume were 0.8 mL/min and 20 μL, respectively. The eluent was detected at 310 nm. Vodobatinib and the IS eluted at 7.5 and 5.9 min, respectively, with a total run time of 10 min. Validation tests were performed according to FDA requirements. The established method showed good linearity in the range of 50.5-6534 ng/mL. Interday and intraday coefficients of variations were < 5.11%. Stability studies, dilution integrity, and incurred sample reanalysis met the acceptance criteria. Subsequently, the validated HPLC method was used to study the disposition kinetics of vodobatinib in rats.

Simultaneous determination of naphthalimide-labelled monosaccharides in P. cyrtonema Hua. polysaccharides utilizing the HPLC-UV technique.

4-Amidogen-1,8-naphthalimide (ANA), a novel pre-column derivatization reagent, has been successfully synthesized and utilized for the highly sensitive analysis of monosaccharides. ANA reacts with the reducing carbonyl groups of saccharides, facilitating monosaccharide detection. The resulting monosaccharide derivatives were meticulously investigated using High-Performance Liquid Chromatography (HPLC) coupled with ultraviolet (UV) spectroscopy. The derivatization process was conducted in an environmentally friendly solvent mixture of EtOH and AcOH at a constant temperature of 100 °C for a duration of 4 hours across all monosaccharides. After derivatization, the separated adducts were eluted through a ZORBAX SB-C8 column within a mere 22 minutes at a flow rate of 0.8 mL min-1, with detection occurring at a wavelength of 416 nm. By employing this derivatization technique, the comprehensive analysis of the monosaccharide composition in P. cyrtonema Hua. polysaccharides (PCPs) following strong acid hydrolysis was achieved. The monosaccharide profiles of PCPs predominantly consist of glucose (Glc), galactose (Gal), mannose (Man), xylose (Xyl), and galacturonic acid (GalA), with a molar ratio of Glc : Gal : Man : Xyl : GalA of 20.26 : 12.76 : 24.24 : 41.91 : 0.83. The LOD values for the six sugars ranged from 27.37 to 84.37 μg mL-1. The validated HPLC method, characterized by its exceptional precision and accuracy, has successfully facilitated both rapid qualitative and quantitative determination of PCPs, thereby establishing a robust quality control protocol for P. cyrtonema Hua.

Validation of the High-Performance Liquid Chromatography Method for Determining Quercitrin in Capsicum annuum L. Cultivar Dangjo.

Capsicum annuum L. cultivar Dangjo (DJ), developed as a novel crop, possesses potential health benefits, such as reducing blood glucose levels. DJ contains flavonoid glycosides, bioactive compounds that have been found in various plants and have promising pharmacological effects. A representative flavonol glycoside is quercitrin, a standard compound. Notwithstanding, its adoption by the food industry, it is necessary to standardize the quantification of quercitrin in DJ. Thus, an analytical method needs to be developed and validated to quantify quercitrin accurately. In this study, we established a high-performance liquid chromatography (HPLC) method for quantifying quercitrin in DJ extracts (DJEs); then, we validated the method, to ensure its accuracy and reliability. Our results demonstrated that the HPLC method effectively detecteds quercitrin in DJE samples, consistently reporting retention times and peak spectra similar to those in the standard solutions. The linearity assessment revealed a linear response within the concentration range of 2.5 to 15.0 μg/mL, which was supported by strong correlation coefficients (R2>0.9997). Accuracy assessment via recovery studies produced satisfactory results (89.02%－99.30%), with a relative standard deviation (RSD) within acceptable limits (0.50%－5.95%). Precision analysis confirmed the repeatability and reproducibility of the method, with RSD values within the Association of Official Agricultural Chemists (AOAC) standard criteria (≤8%). Overall, our study provides a validated HPLC method for quercitrin quantification in DJEs, facilitating its standardization and ensuring the accuracy of the analysis. This method is potentially valuable for quality control and further research on the health-promoting properties of DJ.

High-performance liquid chromatography method for measuring Captopril: an empirical study on hydrogel film permeability test.

High-performance liquid chromatography (HPLC) has emerged as a highly sensitive and versatile analytical technique for quantifying antihypertensive drugs, such as Captopril (CAP). This study focused on the optimization and validation of an HPLC method for quantifying CAP in an in vitro hydrogel permeability test. The main objective of this study was to develop and validate an HPLC method for quantifying CAP in an in vitro hydrogel permeability test. The HPLC method employed a C18 column (Waters, Sunfire, 5μm; 250 × 4.6mm) and a mobile phase consisting of methanol-water (85% v/v orthophosphoric acid) in a 55:45 (v/v) ratio at a flow rate of 0.5 mL/min. The UV-Vis detector was configured to detect CAP at a wavelength of 220nm. The hydrogel film used in the permeability test was prepared using poly (vinyl alcohol)/poly (vinyl caprolactam) (PVA/PNVCL) with citric acid as a crosslinking agent. All results met the validation parameters according to ICH Guideline. The HPLC method showed consistent retention time (4.7-4.9min), linearity (1-50µg/mL; r = 0.9995), accuracy (98.11-101.78%), precision (RSD ≤ 2%), and LoD/LoQ (0.19/0.62µg/mL). The developed HPLC method was successfully applied to an in vitro permeability test using horizontal diffusion cells. The results demonstrated that CAP permeated through the swollen hydrogel film, with a cumulative drug permeation exceeding 30%. This highlighted the method's utility in assessing drug transport properties through hydrogels. The validated HPLC method demonstrates robustness and reliability for quantifying CAP in the hydrogel permeability test.

A simple HPLC method for the determination of plasma progesterone levels in the third trimester of human pregnancy.

Progesterone is a steroid hormone primarily associated with pregnancy. A simple, rapid, and reliable high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of progesterone in human plasma. The method consists of a simple liquid-liquid extraction of progesterone and internal standard (estriol) from human plasma using a mixture of hexane and diethyl ether. The chromatographic determination of progesterone was performed using an acetonitrile-water (70:30, v/v) mobile phase with a C18 reversed-phase column. The method achieved an extraction recovery of greater than 96.4% from spiked plasma samples. Intra- and inter-day precision were generally acceptable, with relative SD% less than ≤6.60% and accuracy (relative error %) better than 3.64%. The developed and validated method was used to successfully quantify progesterone levels in plasma samples collected from women during the third trimester of pregnancy. Furthermore, a statistical comparison was conducted between progesterone concentrations in plasma samples obtained from 2 groups of pregnant women: group 1 (n = 9) at 30-35 weeks and group 2 (n = 9) at 36-41 weeks. The developed and validated HPLC method described in this study enables the successful determination of progesterone in human plasma, offering advantages such as shorter analysis time, simplicity, cost-effectiveness, and potential routine use during pregnancy.

Stability-Indicating and RP-HPLC Method for Analyzing Amlodipine Besylate in Commercially Available Drug Products in Kurdistan, Iraq

This study aims to develop and validate a stability-indicating HPLC method for the simultaneous determination of Amlodipine (Amlo) in bulk and tablet dosage forms. The method’s goal is to evaluate the stability and degradation of Amlo under various stress conditions. The method was optimized using high-performance liquid chromatography (HPLC) with a UV detector. The mobile phase consisted of water and methanol in a 50:50 (v/v) ratio. An Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used for separation. The analysis was carried out at room temperature (25°C) with a 10 μL injection volume, a flow rate of 1.2 mL/min, and detection at 360 nm. Method validation was conducted in terms of linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). The stability of Amlodipine in tablet forms, under various stress conditions (acidic, alkaline, oxidative, thermal, and photolytic), was also evaluated. The finding showed excellent linearity (R² = 0.994) in the concentration range of 5–25 μg/mL. Precision was demonstrated by a relative standard deviation (RSD) of less than 0.8%. The LOD and LOQ were found to be 0.65 μg/mL and 2.16 μg/mL, respectively. Accuracy was assessed at three different levels, yielding recoveries above 98%. The method successfully detected the degradation products of Amlo under the applied stress conditions. The developed and validated HPLC method is simple, precise, and accurate for the simultaneous determination of Amlodipine in bulk and tablet forms. It is reliable for evaluating the stability and degradation of Amlo under different stress conditions, making it suitable for routine quality control and stability testing of Amlo-based pharmaceutical products.

Validation of chromatographic methods, TLC and HPLC, to quantify known radiochemical impurities and the radiochemical purity the radiopharmaceutical [177Lu]Lu-PSMA I&T

The Energy and Nuclear Research Institute (IPEN) is studying the production of the radiopharmaceutical [177Lu]Lu-PSMA I&T, in accordance with the good manufacturing practices recommended by the National Health Surveillance Agency (ANVISA), to be used in the therapy of prostate cancer. This work aims to validate chromatographic methods, Thin-Layer Chromatographic (TLC) and high-performance liquid chromatography (HPLC), to determine the radiochemical purity (RCP) of the product. The entire validation process of this work was based on ANVISA's RDC 166, 2017 and the Guide 10, version 1, 2017, guided the statistical treatments adopted. With the selectivity study we can observe that the presence of impurities or excess excipients in the sample does not interfere with the quantification of the product. The proposed methods were linear with linear correlation coefficients (r) above 0.99. The precision and repeatability presented relative standard deviation values lower than specified (RSD <5 %). The small controlled variations in the method suggested for the robustness test also did not affect the radiochemical purity of the product. In view of the results and in accordance with the criteria established by the National Health Surveillance Agency (ANVISA), the two chromatographic methods were validated in accordance with RDC 166, 2017, proving to be selective, precise, linear and robust. The validation of TLC and HPLC methods enables their application in the batch release routine of the new radiopharmaceutical at Radiopharmacy Center of IPEN.

Development and Validation of an HPLC Method for Niraparib Analysis: A Comprehensive Approach.

Background: Niraparib is a significant PARP inhibitor utilized in cancer therapy. This study aims to develop and validate a high-performance liquid chromatography (HPLC) method for the quantification of Niraparib in pharmaceutical formulations and biological samples. Results: A Phenomenex Kinetex XB-C8 column (150 x 4.6 mm, 5 µ) was employed with a mobile phase of 0.1% formic acid in acetonitrile (60:40, % v/v). The analysis was performed at an oven temperature of 30℃ with a flow rate of 0.5 mL/min, and detection was achieved at 240 nm. The method demonstrated excellent linearity (R² = 0.9998) across concentrations from 40 to 60 μg/mL. Validation studies confirmed high accuracy, with a mean recovery of 99.96%. Conclusion: The validated HPLC method is robust and reliable for quantifying Niraparib in various formulations, aiding in the stability assessment and ensuring the efficacy and safety of cancer treatment protocols. This method facilitates informed decisions in pharmaceutical development.

High-Performance Liquid Chromatographic Method Development and Validation for Analysis of Expired and Marketed Doxofylline Tablets.

The objectives of the study were to develop a simple, reliable, sensitive and accurate high-performance liquid chromatography (HPLC) method for doxofylline pure and pharmaceutical dosage forms. HPLC method was developed for the estimation of doxofylline in pure and marketed, recently expired, and 1-year expired tablet formulations. Chromatographic separation of the drug was achieved on a stainless steel column 25 cm × 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm), Cosmosil® C18, column using a mobile phase of Potassium dihydrogen orthophosphate buffer: Acetonitrile pH 5.1 (60:40 v/v) at a flow rate of 1-mL/min. The drug eluted was monitored at 274 nm and the retention time of doxofylline was found to be 3.575 minutes, respectively. The validation of developed methods was done according to ICH Q2 (R1) guidelines. The calibration curve was linear over the range of 5 to 50 µg/mL. The correlation coefficient was found to be 0.9943 for doxofylline. The average retention time was found to be 3.538 minutes. The results were reproducible, showing the effectiveness of the system. The plate number was found to be 4909, which is also within the limit, i.e. ≥ 2000 prescribed in I.P. The dissolution studies of all three marketed, recently expired and 1-year expired formulations were carried out by using 0.01 M HCl and the results showed almost a similar rate of release profile and all three formulations accomplished the drug release within the limit. The developed and validated HPLC method offers a precise, accurate, and efficient analytical tool for the determination of doxofylline in pharmaceutical formulations.

Development and validation of HPLC and GC methods for quantification of cannabinoids and terpenes extracted by ultrasound assisted extraction technique

Cannabis exhibits a wide array of therapeutic effects due to its diverse and potent compounds. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are the primary techniques used to quantify and qualify cannabinoids and terpenes in cannabis. This paper rigorously validates HPLC and GC-MS methods for quantifying cannabinoids and terpenes in cannabis plants. Cannabinoids and terpenes were extracted using an optimised ultrasound assisted extraction (UAE) method. The HPLC and GC-MS methods were validated by assessing linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, precision (inter-day and intra-day), accuracy (inter-day and intra-day), and matrix effect for 12 cannabinoids and 30 terpenes using appropriate standards. The HPLC and GC results fell within acceptable ranges (for HPLC, recovery between 73-121%, precision, accuracy and matrix effect all were below 10%; and for GC, recovery between 79-91%, precision, accuracy and matrix effect all were below 10%) demonstrating the accuracy and reliability of the validated method. The findings of this study can be used effectively to measure these important compounds in cannabis samples, facilitating quality control and standardisation of cannabis-based products for therapeutic applications.

Unveiling Impurities: A Comprehensive RP-HPLC Method for Accurate Atorvastatin Impurity Analysis in Pharmaceuticals Using Accuracy Profile Approach.

Accurate determination of active pharmaceutical ingredients and impurities is essential for ensuring the safety and effectiveness of medications. This study focuses on the validation of a high-performance liquid chromatography (HPLC) method for quantifying Atorvastatin and its impurities, addressing a critical aspect of pharmaceutical analysis. The primary objective is to conduct a comprehensive validation study for the HPLC method, covering specificity assessment, response function establishment, and a detailed analysis of precision, trueness, and tolerance intervals. The emphasis is on demonstrating the method's precision, accuracy, and stability-indicating capabilities across various concentrations and compounds. The HPLC method is validated through rigorous assessments, including specificity, response function establishment, and analyses of precision, trueness, and tolerance intervals. Induced degradation experiments are conducted to explore Atorvastatin's behavior under extreme conditions. Insights into the compound's synthesis and degradation pathways are provided through a proposed mechanism for intramolecular esterification. The results affirm the precision, accuracy, and stability-indicating capabilities of the validated HPLC method. The method effectively differentiates between Atorvastatin and its impurities, showcasing its suitability for pharmaceutical quality control. The validated HPLC method emerges as a robust and reliable tool for Atorvastatin analysis, contributing significantly to pharmaceutical research and quality control. Its application ensures the safety and efficacy of medications, reinforcing its role in pharmaceutical analysis. This study not only validates a crucial HPLC method for Atorvastatin analysis but also provides insights into the compound's behavior under extreme conditions and its synthesis and degradation pathways. The validated method serves as a cornerstone in pharmaceutical research and quality control, ensuring medication safety and efficacy.

Determination of Water-Soluble Vitamins in Palmyrah Sweet Sap and Its Derived Products using RP-HPLC Method

Palmyrah sweet sap, obtained from the inflorescence of the Borassus flabellifer L. palm tree, is a popular natural drink in Sri Lanka, available seasonally from January to August. It contains a blend of water, sugars, and essential vitamins and minerals. The sweet sap is processed thermally to produce sugary products like crude sugar, jaggery, and treacle. It is crucial to analyze them to ensure their nutritional value, due to the possibility of vitamin decomposition during the production stage. This study aimed to quantify the existing vitamins in sweet sap and its derived products using validated high-performance liquid chromatography (HPLC). The HPLC method validation parameters, such as linearity, accuracy, precision, range, limit of detection (LOD), and limit of quantification (LOQ), were determined. The results showed that the derived products from palmyrah sap had high concentrations of vitamins, with thiamine ranging from 2.81 to 35.16 mg/100g, niacin from 4.35 to 45.03 mg/100g, pyridoxine from 4.44 to 87.16 mg/100g, and ascorbic acid from 5.42 to 52.52 mg/100g. The accuracy of the HPLC method for vitamin analysis was between 98.43 and 100.64%, with a limit of detection ranging from 0.357 to 1.152 ppm and a limit of quantification from 1.081 to 13.573 ppm. The study successfully quantified the existing vitamins in the sap and its derived products, with overall results indicating that the vitamins were retained even after thermal processing.

Time-efficient HPLC Validation Methodology for the Qualitative Analysis of 68Ga PSMA-11 in Routine Clinical Usage under Isocratic Method.

Prostate-specific membrane antigen (PSMA) has shown to be a promising agent for prostate cancer imaging under PET-CT. With the automation in radiolabeling with 68Ga, using iTG 68Ge/68Ga generator, it has helped introduce various new diagnostic agents and achieve good manufacturing practices (GMP) simultaneously. However, before any radiopharmaceutical is put into clinical usage, it should always be checked for its radiochemical purity and other quality parameters before injecting in the patient. Chromatography techniques such as Gas Chromatography (GC), High-Performance Liquid Chromatography (HPLC), and Thin-Layer Chromatography (TLC) are the most frequently utilized separation technique for purity analysis. A rapid quality control HPLC based methodology was required for radiopharmaceuticals. In our current setting, we conducted quality control analysis and standardized and validated HPLC method for the routine quality check of 68Ga-PSMA-11. The QC of 68Ga PSMA-11 was performed under ITLC and HPLC. Linearity, accuracy, precision and specificity were assessed and quantified in accordance with International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (Q2 (R1) ICH) guidelines, which can be implemented in resource-limited settings to check the quality. The current HPLC based methodology is rapid, with a retention time of 2.24 min, rendering it a favorable analytical standard operating procedure for QC analysis of 68Ga-PSMA-11.

Quantitative Analysis of 5-hydroxyindoleacetic Acid in Rat Urine by High-performance Liquid Chromatography Method

ABSTRACT Background: 5-hydroxyindoleacetic acid (5-HIAA) is a metabolite of serotonin, a neurotransmitter primarily associated with regulating mood, appetite, and sleep. When serotonin is broken down in the body, it forms 5-HIAA, which is then excreted in urine. Quantification of 5-HIAA levels in urine is often used as a diagnostic tool for certain conditions, particularly carcinoid tumors, which can produce excess serotonin. Elevated levels of 5-HIAA in urine can indicate increased serotonin production, which may be indicative of carcinoid tumors or other conditions affecting serotonin metabolism. However, there is limited work on employing high-performance liquid chromatography (HPLC)–ultraviolet to estimate monoamines in biological samples. Aim and Objectives: The present study explores the detection of 5-HIAA in rat urine samples, developing an easy, precise validated HPLC method. Materials and Methods: Mobile phase combination acetonitrile and o-phosphoric acid in the ratio of 30:70% v/v and flow rate 0.5 mL/min was able to separate 5-HIAA distinctly. Results: The detection was observed at λmax 277 nm with better sensitivity. According to the parameters indicated in the international council for harmonisation of technical requirements for pharmaceuticals for human use guidelines (Q2A; Q2B), the procedure was verified. The linearity range was selected from 20 to 45 μg/mL, r 2 = 0.992, limit of detection (4.65 μg/mL), limit of quantification (15.52 μg/mL), retention time (6.3 min). Conclusion: The technique has demonstrated that it is repeatable and recoverable within the given range. Thus can be used for routine analysis of 5-HIAA in urine samples.

Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer

Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer

Advances in High-Performance Liquid Chromatography (HPLC) Method Development and Validation: A Comprehensive Review

High-Performance Liquid Chromatography (HPLC) stands as the cornerstone technique for the detection, separation, and quantification of drugs in various matrices. Method optimization necessitates a thorough investigation of multiple chromatographic parameters, encompassing sample pretreatment, mobile phase composition, column selection, and detector configuration. This article aims to comprehensively address the processes of method development, optimization, and validation within the realm of HPLC. The inherent advantages of HPLC, including its rapidity, specificity, accuracy, precision, and automation feasibility, render it an indispensable tool for analyzing a wide spectrum of drugs in complex multi-component dosage forms. Furthermore, HPLC method development and validation hold paramount importance across the spectrum of drug discovery, development, and manufacturing, as well as in diverse human and animal studies. The validation of analytical methods during drug development and manufacturing is imperative to ensure their fitness for the intended purpose. Adhering to Good Manufacturing Practice (GMP) requirements, pharmaceutical industries must establish comprehensive validation policies outlining the validation procedures. This article primarily delves into the optimization of HPLC conditions, emphasizing the critical role of method refinement in ensuring robust and reliable analytical methods.

GREEN SYNTHESIS AND ANALYTICAL TECHNIQUE FOR THE SEPARATION OF SUBSTITUTED CHLOROPHENYL HYDRAZINE ISOMERS BY REVERSE PHASE HPLC METHOD

4-Chlorophenylhydrazine hydrochloride (4-CPH) is a crucial intermediate in organic synthesis, utilized in various applications including the production of Carprofen and wastewater treatment. This study focuses on the synthesis and impurity analysis of 4-CPH on green approach, with particular emphasis on the detection and quantification of positional isomers 2-Chlorophenylhydrazine hydrochloride (2-CPH), 3-Chlorophenylhydrazine hydrochloride (3- CPH), and 4-Chloroaniline. A high-performance liquid chromatography (HPLC) method was developed using a Waters X-Bridge C18 column and optimized for resolution and sensitivity. The method demonstrated effective separation, with detection limits for 2-CPH, 3-CPH and 4-Chloroaniline at 0.02%, 0.04%, and 0.02% respectively. The validated HPLC method provides reliable analysis for the quality control of 4-CPH, ensuring low impurity levels in the final product.

A high-performance liquid chromatography method validation and a Box-Behnken experimental design for the extraction optimization of quercitrin from Nectandra reticulata.

The ethanolic extract of Nectandra reticulata contains a high amount of quercetin-3-O-rhamnoside (quercitrin) that has exhibited a significant activity toward Alzheimer's disease, specifically with LXR receptors. In this work, a methodology was validated following the specifications of the International Conference of Harmonization in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), repeatability (intra-assay), intermediate precision (intra-laboratory), reproducibility (inter-laboratory), robustness, and specificity. The effect of location (Oiba, Granada, and Chiquinquira) and the extraction method (percolation, maceration, and ultrasound-assisted extraction) towards the chromatographic profile and quercitrin recovery was studied. Furthermore, a Box-Behnken design was conducted to optimize quercitrin extraction and extraction yield by ultrasonic-assisted extraction. The chromatographic method was validated, with a linear range from 5 to 180 mg quercitrin per L, LOD 0.26 mg L-1, and LOQ 0.86 mg L-1. Accuracy [recovery of 93.8% (w/w)], repeatability (relative standard deviation, RSD, 3.3%), intermediate precision (RSD 5.4%), and reproducibility (RSD 1.4%) were within the acceptable values. The method was robust and specific, except for the variation in the formic acid concentration. The location had a greater influence than the extraction method towards both the chromatographic profile and quercitrin recovery. Quercitrin extraction was maximized at 60% (v/v) ethanol and 50 °C, independent of the solvent : material ratio used. The highest yield values were achieved at 60% (v/v) ethanol and 50 °C, with a solvent : material ratio of 40 mL g-1.

A Sensitive and Specific Analytical Method for Determination of Diclofenac Sodium Using High-Performance Liquid Chromatography

Diclofenac Sodium (DCL-Na) is a widely prescribed nonsteroidal anti-inflammatory drug (NSAID) used to treat conditions such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, and gout. This study aims to develop and validate a simple, precise, and accurate high-performance liquid chromatography (HPLC) method for quantifying DCL-Na in pharmaceutical formulations. The method utilizes a Supelcosil C-18 column with a mobile phase consisting of potassium dihydrogen phosphate solution and acetonitrile. The method validation follows International Conference on Harmonization (ICH) guidelines, evaluating precision, accuracy, specificity, linearity, and range parameters. The developed method demonstrated excellent linearity with an R² value of 0.9996 over a concentration range of 5-50 μg/ml, and the limits of detection (LOD) and quantification (LOQ) were found to be 6.634 μg/ml and 22.114 μg/ml, respectively. Intraday and interday precision tests showed relative standard deviations (% RSD) well within acceptable ranges. The method's practical applicability was confirmed through the analysis of controlled-release tablets, which showed a release of approximately 80% of DCL-Na after 20 hours. This validated HPLC method is suitable for routine quality control of DCL-Na in pharmaceutical products.