RP-HPLC Method Research Articles

The optimization and validation of a stability indicating rP-HPLC method to analyze the long term and in use stability of two short peptides

In this paper an HPLC stability-indicating assay was developed for the stability study of two short universal cancer peptides derived from telomerase (UCP2 and UCP4) used as vaccine. The optimum HPLC gradient conditions were determined for the identification on the chromatogram of the active pharmaceutical ingredients from the impurity products including isoAspUCP4. This HPLC method was validated as per the International Committee on Harmonization (ICH) Q2 (R1) guidelines. It was showed that this vaccine was physicochemical stable for 2 years when stored in borosilicate glass vials at −20 °C and no formation of isoAspUCP4, an inhibitor of the UCP4 immunogenicity was detected. In addition, it was stable for up to 10 h at 4 °C or room temperature after thawing the samples. As well, this present work was the first report including the lightness and chromaticity measurements in a peptide vaccine and therefore may be a guidance in stability studies.

Development, Optimization, and Stability Study of a Yataprasen Film-Forming Spray for Musculoskeletal Pain Management

Yataprasen (YTPS) remedy ethanolic spray, one of the National Thai Traditional Medicine Formulary, is extensively employed in Thai traditional healthcare to manage musculoskeletal pain and inflammation. Despite its widespread use, the quality and stability of the YTPS formulation, critical to its efficacy, safety, and patient adherence, have not been comprehensively studied. This research developed and optimized a film-forming spray (FFS) formulation of YTPS ethanolic extract and conducted a 6-month stability evaluation. The FFS shares similarities with gel formulations, particularly in its ability to form a cohesive, semi-solid film upon application, enhancing localized drug delivery and prolonged contact time. Key physicochemical properties, including density (0.8450–0.9086 g/cm3), pH (4.72–4.95), spray angle (55.58–60.10°), evaporation time (1.04–1.27 min), and theoretical film thickness (7.72–13.97 µm), were analyzed across varying storage conditions. Active components β-amyrin and stigmasterol demonstrated retention rates of 96.78% and 68.22%, respectively, under refrigerated conditions, with degradation rates accelerating at higher temperatures. Significant variations in density, spray angle, film thickness, and stigmasterol concentration were observed. Additionally, the RP-HPLC method was validated for the accurate and precise quantification of the bioactive compounds such as β-amyrin and stigmasterol, demonstrating excellent linearity within a 10–100 µg/mL range for both compounds with excellent linearity R2 > 0.999. The results confirmed that YTPS-FFS exhibits good stability and that the validated HPLC method is reliable for routine quality control. These findings supported the potential of YTPS-FFS formulation as a standardized and effective dosage form for managing musculoskeletal conditions, advancing its role in modernized traditional medicine.

Stability indicating eco-friendly HPLC method development and validation for the estimation of bisoprolol fumarate and telmisartan

BackgroundTelmisartan and bisoprolol fumarate together are two medications that diminish arterial pressure. The current study comprises an evaluation of the proposed methodology's greenness regarding the HPLC method used to govern the medication mixture regardless of dose form A novel stability suggesting HPLC method's environmental effect was evaluated using the greenness metrics. Stress conditions comprising acidic, alkaline, oxidative, thermal, and photolytic degradation were applied for both of the medications.ResultsThe RP—HPLC method employing a reversed-phase C18 column with a gradient approach, the HPLC chromatography was carried out. The mobile phase consisted of acetonitrile, methanol, and phosphate buffer (60:35:5, %v/v/v), with the stationary phase being the Unisphere C18 column Agela Tech. The RP-HPLC method uses UV detection at 224 nm with chromatographic purification spanning linearities of 2.5–12.5 μg/mL for bisoprolol fumarate and 40.0–200.0 μg/mL for telmisartan, correspondingly. The procedure is accurate and precise, as demonstrated by an outcome that % RSD inside the permissible range. Additionally, various stressors were introduced to the medications. The approach's green credentials with respect to solvent utilization, chemical substances, expenditure of energy, and waste formation have been verified by the greenness data collected during the evaluation. No chromatographic or spectrum impediments caused by formulation additives have been observed.ConclusionBisoprolol fumarate and telmisartan could be measured simultaneously using the devised RP-HPLC method, which was simple, quick, sensitive, accurate, precise, linear, and stability indicating. The proposed approach showed ecological friendliness, robustness, sensitivity, and ease of use. As a result, the devised method could be applied to the regular quality checking of tablets and bulk medications.

Development and Validation of the RP-HPLC Method for Dexamethasone Sodium Phosphate Determination in Nasal Chitosan Microsphere Preparations

The purpose of this work was to provide a robust, sensitive, accurate, and straightforward analytical method for measuring dexamethasone sodium phosphate (DSP) in chitosan microspheres preparedn using the spray drying method. DSP was quantitatively analyzed using RP-HPLC with an ultraviolet detector at 254 nm, a mobile phase that contained a mixture of acetonitrile and 0.1% sodium dihydrogen phosphate monohydrate (50:50) operating isocratically at a flow rate of 1.0 mL/min, and a stationary phase that was a C18 PrincetonSPHER-100 C18-QB 100A HPLC Column (250 × 4.6 mm, 5 um). The ICH recommendations were followed in the validation of the analytical method. DSP had a retention duration of 2.899 minutes and a tailing factor of 0.827. The RP-HPLC method was linear (R = 0.9992) in the 15-60 ug/mL concentration range. The limits of quantitation (LOQ) and detection (LOD) were 4.425 ug/mL and 1.327 ug/mL, respectively. The relative standard deviations for the intra-day and inter-day precisions were 0.057-0.876% and 0.780-0.949%, respectively. The recovery percentages at 50, 100, and 200% concentration levels were within the 99.269-100.980% range. The validated method has been successfully applied to determine DSP entrapment efficiency in chitosan microspheres. A linear, sensitive, accurate, precise, and robust technique of determining DSP in chitosan microsphere preparations is offered by the established RP-HPLC method.

Quality by design assisted a stability indicating RP-HPLC method for the estimation of hesperidin in transfersome and marketed product

Quality by design assisted a stability indicating RP-HPLC method for the estimation of hesperidin in transfersome and marketed product

Green analytical comparison and central composite design optimization for simultaneous estimation of pain management drugs using RP-liquid chromatography

The Central Composite Design method was utilized to validate a precise RP-HPLC method for concurrently determining the quantities of Paracetamol (PC), Diclofenac Sodium (DS), and Eperisone Hydrochloride (EH) in tablet compositions. By employing Design of Experiment (DOE), the experimental parameters were fine-tuned, resulting in an optimized eluent consisting of methanol: water (90:10) with 0.1 % orthophosphoic acid at a eluent velocity of 1 mL/min. The method exhibited exceptional purities: PC (100.83 % ± 0.85), DS (102.01 % ± 0.90), and EH (100.49 % ± 1.29). Regression equations were formulated for PC, DS, and EH as follows: y = 479762x + 151907, y = 2182788x + 2409442, and y = 777144x − 1146334, respectively. The analytical method underwent comprehensive validation, including tests for: Accuracy, Precision, Linearity and Robustness. To assess the method’s environmental impact, several Green Analytical Chemistry (GAC) tools were employed. These tools provided a multifaceted evaluation of the method’s sustainability and eco-friendliness.

A simple, cost-efficient stability-indicating RP-HPLC method for simultaneous estimation of embelin and piperine for routine analysis of marketed polyherbal capsules and tablets

Embelin (EMB) and Piperine (PIP) alkaloids are reported for ­antidiabetic, hypolipidemic, antioxidant, and anti-cancer properties. However, simultaneous analytical methods are scarce. A stability-indicating RP-HPLC method was developed with mobile phase MeOH: 0.1%TEA (pH 4.2 adjusted by OPA) (92:08 v/v ratio), CHEMSIL-ODS, C18 (4.6 × 250mm, 5 µm) column, and 1.2 mL/min ­flowrate. Retention times were 3.2, and 6.2 min for PIP and EMB respectively, at 289 nm, and validated as per Q2 (R1) ICH guidelines. Linearity ranging (2-10 µg/mL), with regression coefficients (r 2), LOD, and LOQ for PIP were r 2 = 0.9979, 0.793 µg/mL, 2.403 µg/mL, and r 2 = 0.9974, 0.851 µg/mL, 2.578 µg/mL for EMB. Developed method precision, robustness, and ruggedness were confirmed by <2%RSD. In tablet and capsule formulations, PIP %purity was 90.36%, 94.33%, and EMB 91.65, 92.85%. Additionally, forced-degradation studies ­indicated method stability through various stressed conditions. Hence, this ­validated method is specific, precise, linear, accurate, and robust, ­suitable for routine analysis of EMB and PIP in polyherbal ­formulations containing Embelia ribes Brum. and Piper nigrum Linn. extracts.

Novel RP-HPLC Method Development and Validation of Budesonide Suppository

In the current study, a simple and cost-effective stability-indicating RP-HPLC method was developed and validated to estimate Budesonide from bulk and pharmaceutical dosage forms.0.1% formic acid and methanol (15:85 v/v) were employed as the mobile phase with an injection volume of 20μl, and detection was carried out at 244 nm. The developed method was validated as per ICH Q2 guidelines. The RT of Budesonide was determined to be 4.3 ± 0.328 minutes, providing a reliable marker for its identification. The method was found to be linear between 2-12 μg/ml concentration with (R²) of 0.997. This demonstrates the method's ability to measure varying concentrations of Budesonide accurately. Additionally, the percentage recovery of Budesonide was approximately 100%, confirming the accuracy of the developed method. The parameters for the system's suitability have also been found to be within acceptable limits. Force degradation studies reinforced the method's selectivity and sensitivity in detecting Budesonide under various degradation scenarios. In conclusion, our proposed RP-HPLC method provides a sensitive, accurate, and precise way to analyze Budesonide in bulk and pharmaceutical dosage forms.

Method development and validation of ornidazole by using RP-HPLC

Analytical way is more suitable, highly precise, safe and selective. Developing analytical method for newly introduced pharmaceutical formulation is a matter of most importance. The technique which is widely used to check the quality of drug is known as ‘CHROMATOGRAPHY’. Our present plan is to develop a new, simple, precise or accurate method for analysis of ORNIDAZOLE formulation after a detailed study a new RP-HPLC method was decided to be developed and validated. Ornidazole is a popular anti-protozoal agent. Different concentrations of drug solutions are prepared for method validation. Injected each level into the chromatographic system and measure the peak area. The accuracy limit is the percentage recovery should be in the range of 98.0% - 102.0%. The total recovery was found to be 100.34% for Ornidazole. The validation of developed method shows that the accuracy is well within the limit, which shows that the method is capable of showing good accuracy and reproducibility.

Development of RP-HPLC method for estimation of indomethacin in sustained-release capsule samples

Development of RP-HPLC method for estimation of indomethacin in sustained-release capsule samples

Novel and Stability Indicating RP-HPLC Method Development and Validation for the Determination of Metformin HCL: A Potential Drug Against Diabetic Disorder, in Pure and Pharmaceutical Dosage Forms

Novel and Stability Indicating RP-HPLC Method Development and Validation for the Determination of Metformin HCL: A Potential Drug Against Diabetic Disorder, in Pure and Pharmaceutical Dosage Forms

A Novel, Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Assay and Organic Impurities of Pyridostigmine Bromide and Assay of Sodium Benzoate in Liquid Oral Formulation

A Novel, Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Assay and Organic Impurities of Pyridostigmine Bromide and Assay of Sodium Benzoate in Liquid Oral Formulation

A Robust RP-HPLC Method for Simultaneous Quantification, Validation and Stability Studies on Vildagliptin (VILD), Dapagliflozin (DAPA) and Metformin Hydrochloride (METF) in its Combined Dosage Formulation

Background: Vildagliptin, Dapagliflozin and Metformin Hydrochloride are anti- hyperglycemic agents. Anti-hyperglycemic agents are drugs that stimulate insulin secretion and lower the glucose level in the blood. They were investigated as a medication for Diabetes Mellitus (DM). Various studies have reported the HPLC, HPTLC, LC/MS methods for the estimation of individuals of VILD, DAPA and METF. However, until date stability indicating HPLC analysis method has not been reported for the estimation of VILD, DAPA and METF in combined glucagon secretion in a glucose-dependent manner. Introduction: The objective of present work was to develop and validate the stability of RPHPLC method for the estimation of Vildagliptin (VILD), Dapagliflozin (DAPA) and Metformin Hydrochloride (METF) in combined dosage form. Method: In this research work, High Performance Liquid Chromatography (HPLC) was used for validation in the chromatography. In the HPLC, Methanol and Potassium Dihydrogen phosphate buffer (85:15 v/v) were used as mobile phases. HPLC analysis was performed at 230 nm. The method was validated with different parameters such as linearity, precision, accuracy, specificity, and robustness, limit of detection (LOD) and limit of quantitation (LOQ). The RF values of VILD, DAPA and METF were 6.24 ± 0.05 min, 8.34 ± 0.05 min. and 3.68 ± 0.05 min, respectively. The accuracies of drug recovery were 0.6398 %,0.3680 %, 0.6398 % and over the range of 1–4 μg/ml, 20-80 μg/ml and 15-85 μg/ml of VILD, DAPA and METF, respectively. Results: Degradation products found in stress conditions did not interfere with the detection of VILD, DAPA and METF; therefore, the proposed technique can be considered stable. Significant degradation products of VILD, DAPA and METF were obtained in an acid at METF at 2.05 ± 0.05 min. (MDP-1) and degradation product for oxidation of METF at 2.11 ± 0.05 min. (MDP-2). When alkaline hydrolysis was conducted, photolytic and thermal conditions were applied alone or in combination with METF, no degradation product of VILD, DAPA and METF was found. Conclusion: The developed and validation method was satisfactorily applied for the analysis of pharmaceutical preparations and proved specific and accurate for quality control of the cited drugs in their combined dosage form.

Risk assessment of antibiotic residues in raw cow’s milk in Bangladesh

ABSTRACT This study evaluated the presence of residual levels of commonly utilised antibiotics, including oxytetracycline (tetracyclines), ciprofloxacin, enrofloxacin, and levofloxacin (fluoroquinolones), in milk samples. Fifty raw milk samples were collected from five farms in Keraniganj, Dhaka. A validated RP-HPLC method was applied to detect and quantify antibiotic residues, demonstrating good linearity with coefficients ranging from 0.999 to 1.0 in the concentration range of 1.25–15.00 µg/mL. The study revealed that oxytetracycline was detected in 90% of the samples, followed by levofloxacin (66%), enrofloxacin (64%), and ciprofloxacin (62%). One farm showed the highest antibiotic prevalence, with oxytetracycline in all samples and levofloxacin, enrofloxacin, and ciprofloxacin in 80% of the samples. About 30% of the oxytetracycline-positive samples exceeded the MRL, while none surpassed the MRL for enrofloxacin and ciprofloxacin. Calculated human health risks appeared to be low, but children might face potential risks due to prolonged exposure.

Analytical Quality by Design aided RP-HPLC method for the estimation of Sunset Yellow in commercial food samples employing green ultrasound assisted extraction: Greenness, Blueness and Whiteness evaluation

Analytical Quality by Design aided RP-HPLC method for the estimation of Sunset Yellow in commercial food samples employing green ultrasound assisted extraction: Greenness, Blueness and Whiteness evaluation

Investigating the stability of a cerebral vasodilator drug using chromatographic methods: Evaluation of methods’ practicality and environmental aspects

A vinca alkaloid; vinburnine (VNB) is utilized as an effective vasodilator. As a cyclic amide-containing drug, it is likely susceptible to hydrolytic degradation. This study examined the degradation profile of VNB, findings indicated that VNB undergoes degradation solely in the presence of alkali, generating a carboxylic acid derivative (DEG). The present study aimed to design and apply green TLC-densitometric and RP-HPLC assays for concurrently measuring VNB and its degradation product for the first time. TLC-densitometric assay was carried out on silica gel 60 F254 TLC plates and a developing system of ethyl acetate: methanol: triethylamine (6:4:0.05, by volume) and detection at 230 nm. RP-HPLC method depended on a C8 column and a mixture of methanol: water (95:5, v/v). The rate of flow was 1 mL/min and UV detection at 230 nm. The proposed assays were used for prediction of the degradation behavior of VNB under the mentioned conditions and then applied for quantitation of VNB in its commercially available capsules. Four distinct metric approaches; National Environmental Method Index (NEMI), Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Blue Applicability Grade Index (BAGI) were utilized to assess the chromatographic method’s ecological effect. Findings obtained from the provided methodologies were contrasted statistically with the stated HPLC method using Student’s t and F-tests. The analysis revealed that there were no significant differences between them. The established methods were verified in accordance with the recommendations of the International Council for Harmonization (ICH), and all the outcomes were deemed to fall within the permissible limit.

Design of Experiment and green analytical chemistry principles for developing a robust and sustainable Stability-Indicating HPLC method for Bempedoic acid impurity profiling

Bempedoic acid (BEM), a drug used to lower cholesterol levels in patients with atherosclerosis. Controlling its process-related impurities is crucial due to their potential toxicity. This study details the development of a RP-HPLC method for the analysis of four process-related substances in BEM. To ensure robust chromatographic performance meeting six sigma quality standards, the method was optimized using a combined approach of response surface methodology (specifically, an I-optimal design) and tolerance analysis, integrating predefined specifications for critical method attributes such as resolution, tailing factor, and number of theoretical plates into the developmental process. Chromatographic separation was achieved using a C18 column. The mobile phase consisted of 0.1 % phosphoric acid in water and acetonitrile, utilizing a stepped gradient elution profile. The flow rate was maintained at 0.9 mL/min, with UV detection performed at 210 nm. The column temperature was set to 20 °C. The method demonstrated linearity for BEM process related impurities in the range of LOQ to 30 μg mL−1. The method ability to differentiate the drug substance from its degradation products was confirmed through forced degradation study. The environmental sustainability of the developed method was assessed using multiple Green Analytical Chemistry (GAC) tools. The National Environmental Methods Index (NEMI) showed two green-shaded quadrants, and the Green Certificate modified Eco-scale awarded a score of 70. The Complementary Green Analytical Procedure Index (Complex GAPI) confirmed the method environmentally friendly profile. Additionally, The AGREE tool generated a score of 0.77, indicating strong overall greenness, while the AGREEprep tool, focused on sample preparation, yielded a score of 0.54. Other tools such as the Sample Preparation Metric of Sustainability (SPMS) and the HPLC Environmental Assessment Tool (HPLC-EAT) scored 8.42 and 569, respectively, further demonstrating the method green credentials. This robust and environmentally conscious analytical framework offers significant potential for application in quality control of BEM.

Validated RP-HPLC method for estimation of azilsartan medoxomil in bulk and tablet dosage form: A green analytical approach

Validated RP-HPLC method for estimation of azilsartan medoxomil in bulk and tablet dosage form: A green analytical approach

Method development and validation on RP-HPLC method for estimation of xanthohumol in nanostructured lipid carriers drug delivery systems

Method development and validation on RP-HPLC method for estimation of xanthohumol in nanostructured lipid carriers drug delivery systems

Development, validation and greenness assessment of stability indicating RP-HPLC method for simultaneous quantification of dapagliflozin and metoprolol succinate in synthetic mixture

Development, validation and greenness assessment of stability indicating RP-HPLC method for simultaneous quantification of dapagliflozin and metoprolol succinate in synthetic mixture