Modification and Validation of HPLC Analytical Method for Estimation of Clotrimazole in Health Care Products

Metformin hydrochloride is a diabetes medication that is used orally. Evogliptin tartrate is a dipeptidyl peptidase-4 (DPP-4) inhibitor which has been authorized as a new and powerful treatment in the process of reducing glucagon-like peptide 1 (GLP-1) deficiency while increasing insulin production and decreasing glucagon levels. The current study created a simple, highly exact, and rapid reverse phase high-performance liquid chromatography (RP-HPLC) approach for the simultaneous measurement of metformin hydrochloride and evogliptin tartrate in their combined dose form. High-performance liquid chromatography (HPLC), was performed on a phenomenex Luna C18 column with a mobile phase of water 25: acetonitrile 75 (25:75) at a 1.2 mL/min flow rate, with detection at 210 and 230 nm for metformin hydrochloride.and evogliptin tartrate. The validation method comprised of several variables like linearity, accuracy, selectivity, precision, robustness and specificity. The retention time of the metformin hydrochloride and evogliptin tartrate was 2.009 and 2.956 minutes, respectively. The method procedure linear responses of metformin hydrochloride and evogliptin tartrate was found to be 1 and 0.997, respectively. The percent relative standard deviation was less than 2% for intraday variation/precision, interday variation/precision, intermediate ruggedness, and robustness. The projected technique produced excellent results for many dependent variables. The projected method showed outstanding results with respect to different dependent and independent variables like limit of detection and limit of quantitation, suitability of system, linearity range etc., linearity, accuracy, precision, and specificity. The results were within the acceptance criteria given in ICH guideline.

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.

Evogliptin is an anti-diabetic drug, which comes under the class of gliptin derivatives for the inhibition of selective dipeptidyl peptidase-4 inhibitor. The developed ultraviolet spectrophotometric method was simple, sensitive, accurate, precise and economic for the development and validation of evogliptin in bulk and tablet dosage form. In this present study, the analytical method validation and development of evogliptin was done using the different parameters of method validation as per International Council for Harmonisation Q2(R1) guidelines. Water using as a solvent and it shows the maximum wavelength at 266 nm and then performed all the parameters of analytical method validation like accuracy, precision, linearity, range, robustness, ruggedness, limit of detection and limit of quantitation. Evogliptin showed linearity over the range of 2-48 µg/ml. The correlation coefficient value obtained was 0.996 with the regression equation y=0.0032x+0.0005. The accuracy studies was done in spiking method and the recoveries ranging from 97.07 %-106.13 %. The percentage relative standard deviation for intra-day precision was 0.44 and inter-day precision was 0.59. The limit of detection was 1.1 µg/ml and limit of quantitation was 3.33 µg/ml respectively. The method has shown good and consistent recoveries and is validated as per International Council for Harmonisation guidelines and can be used for routine quality control analysis of evogliptin in dosage form.

A robust and unified reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantitative analysis of pitavastatin calcium and fenofibrate in dual-layer tablet formulations. Although individual analytical methods for each active pharmaceutical ingredient have been reported, a single analytical procedure applicable to both assay and dissolution testing of this fixed-dose combination has not been sufficiently established. In this study, a single RP-HPLC method was optimized to support both quality control purposes, thereby improving analytical efficiency and reducing method-related variability. Chromatographic separation was achieved using a C18 column (4.6 × 150 mm, 5 µm) under isocratic conditions, with a flow rate of 1.1 mL/min, an injection volume of 40 µL, and UV detection at 245 nm. The total run time was 10 min. The method was validated in accordance with ICH guideline Q2(R1) for system suitability, specificity, linearity and range, accuracy, precision (repeatability and intermediate precision), limits of detection and quantitation, and solution stability. Validation was conducted for both assay and dissolution samples using the same chromatographic conditions. The method demonstrated excellent linearity over the validated concentration ranges for both assay and dissolution analyses (r2 ≥ 0.99). Accuracy and precision results satisfied the predefined acceptance criteria for assay (98.0–102.0%) and dissolution-related quantification (95.0–105%), with relative standard deviation values not exceeding 2.0%. The method showed adequate sensitivity, specificity, and solution stability, confirming its suitability for routine analysis. Application of the validated method to finished dual-layer tablets demonstrated reliable assay results and clearly distinguished the rapid dissolution of pitavastatin calcium from the delayed dissolution behavior of fenofibrate. Overall, the developed RP-HPLC method provides a validated analytical platform capable of supporting both assay and dissolution testing of pitavastatin–fenofibrate dual-layer tablets. Beyond routine validation, the proposed analytical framework demonstrates how a single chromatographic condition can support multiple quality attributes, offering an analytically integrated approach for supporting multiple quality attributes in complex combination drug products.

An isocratic reversed phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the determination of gemifloxacin in bulk, dosage formulations and human serum at 270 nm. Chromatographic separation was achieved on Purospher STAR C18 (250 × 4.6 mm, 5 µm) column using mobile phase methanol:water (90:10, v/v) adjusted pH 2.8 via phosphoric acid 85% having flow rate of 1.5 mL min-1 at ambient temperature. Calibration curves were linear over range of 5-100 µg mL-1 with a correlation coefficient 0.9998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.015 and 0.045 µg mL-1, respectively. Intra and inter-run precision and accuracy results were 98.73-100.12% and then correlated through student'st-test. This method was further applied for in vitro interactions of gemifloxacin with essential and trace elements.

Optimization and qualification of a quantitative reversed-phase HPLC method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays

A simple, rapid, selective, economical, accurate, reverse phase high performance liquid chromatography (RP-HPLC) was developed for simultaneous estimation of sofosbuvir, velpatasvir and voxilaprevir in its tablet dosage form. The separation was accomplished by means of a mobile phase of buffer and acetonitrile in the ratio of 50: 50 pumped at a flow rate of 1 ml/min along with 220 nm as a UV detection wavelength. The stationary phase used was column Waters C18 250 x 4.6 mm, 5m.μ. Sofosbuvir, velpatasvir and voxilaprevir were eluted at a retention times were 2.091 for sofosbuvir, 2.640 for velpatasvir and 3.349 min for voxilaprevir. Analytical method of the proposed RP-HPLC procedure was statistically validated with as per ICH guidelines by considering the parameters such as, system suitability, linearity ranges, precision, accuracy, specificity, and robustness, limit of detection and limit of quantification. The concentration ranges for linearity were 100–600, 25–150 and 25–150 ppm for sofosbuvir, velpatasvir and voxilaprevir respectively with correlation coefficients >0.999. The three drug samples were undergone degradation conditions of acidic and alkaline hydrolysis, oxidation, and photolysis, neutral and thermal degradation. The designed method proved to be stabilityindicating by resolution of the analytes from their forced-degradation products. The developed RP-HPLC method can be used for routine analysis of sofosbuvir, velpatasvir and voxilaprevir in combinational dosage form. The planned method finished by PDA as a tool for peak detection and purity confirmation.

Objective: The aim and objective of this study was to develop and validate Stability Indicating HPLC method for determination of Vilazodone Hydrochloride.Methods: The method was carried out on a Phenomenex, C18 (250x4.6 mm, 5 µm) Column using a mixture of Acetonitrile: Water (50:50v/v), pH adjusted to 3.3 with Glacial Acetic Acid for separation. The flow rate was adjusted at 1 ml/min and Detection was carried out at 240 nm.Results: The retention time of vilazodone hydrochloride was found to be 2.3 min. The calibration curve was found to be linear in the range 25-75µg/ml with a correlation coefficient (R2=0.996). The limit of detection and limit of quantitation were found to be 4.78µg/ml and 14.48µg/ml respectively. The % recovery of vilazodone hydrochloride was found to be in the range of 98.21±0.08 % to 99.07±0.64%. The proposed method was successfully applied for the estimation of vilazodone hydrochloride in marketed tablet formulation.Vilazodone Hydrochloride was subjected to forced degradation under Acidic, Alkaline, Oxidation, Dry Heat and Photolytic degradation conditions. Vilazodone hydrochloride showed 3.12% degradation under acidic condition, 4.78% under alkaline condition, 7.8% under oxidation condition, 3.53% under dry heat condition and 4.9% under photolytic condition.Acid degradation impurity was identified and characterised by LC-MS/MS was found to be 1-(4-Penten-1-yl) piperazine having molecular weight 154.253 (m/z 155.08) and Molecular Formula C9H18N2.Conclusion: A simple, precise, rapid and accurate Stability Indicating HPLC method has been developed and validated for the determination of Vilazodone Hydrochloride in presence of its degradation products as per the ICH Guidelines.

Background: Ertugliflozin is an antidiabetic medication that belongs to the class of Sodium-Glucose Cotransporter2 inhibitors. Ertugliflozin is mainly employed to reduce elevated blood glucose levels. Objective: The aim of the current study is to establish a reliable, precise, and validated bioanalytical method for quantifying Ertugliflozin in human plasma using reverse-phase high-performance liquid chromatography. Materials & Methods: Chromatographic analysis was performed at 239 nm on a Thermo C18 column (150 mm × 4.6 mm, 5 μm) using an optimized method. The flow rate was 0.9 mL/min, using a methanol:0.1 % GAA (75:25% v/v) mixture at pH 3, with 1 mL of plasma as the mobile phase. The method underwent validation for its linearity, accuracy, precision, and robustness, following the standards outlined in the ICH guidelines. The validated method was then used to determine the marketed formulation in plasma. Results: Among six trials, the optimized method showed a sharp peak. Chromatographic parameters included a retention time of 3.872 minutes, 5931 theoretical plates, a tailing factor of 0.94, and a resolution of 2.5. A linear response was observed over the concentration range of 2 to 10 µg/ml, with a correlation coefficient of 0.9992, indicating excellent linearity. Recovery studies at three concentration levels showed 99.39% at 80%, 99.25% at 100%, and 99.58% at 120%, all with RSD less than 2%. This indicated good accuracy. LOD and LOQ were 2.1782 μg/mL and 0.7188 µg/mL, respectively. The estimation of EFG in the marketed formulation was 99.25%. Conclusion: The developed RP-HPLC method was validated and is suitable for routine quantification of drugs in biological samples

Abstract: A simple and new isocratic high-performance liquid chromatography (HPLC) method was developed for the quantitative determination of Venetoclax in its tablet dosage form. The chromatographic separation was achieved on Perkin C8 (15cm x 4.6mm id, 5µ) column. The mobile phase selected was buffer (25 mm ammonium acetate) adjusted to pH 3.0 with orthophosphoric acid and acetonitrile in the ratio of (55:45) v/v at a flow rate of 0.7ml/min with column temperature maintained at 400C and 10µl injection volume. The detection was carried out at 286nm by a UV-visible detector. The retention time of Venetoclax was found to be 4.19 minutes. The proposed method was linear over the range of 5-50µg/ml with a correlation coefficient (R2) of 0.9998. The limits of detection and quantification values were found to be 0.53µg/ml and 1.62µg/ml respectively. All validation parameters were within the limits as per the ICH guidelines. The results of validation parameters indicate that the developed HPLC method was specific, accurate, precise, rapid, reliable and reproducible, therefore it can be applied for routine quality control analysis of Venetoclax in its tablet dosage form.

A simple, specific, accurate, precise and stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method is developed for estimation of montelukast in tablet dosage form. The HPLC method, C-8 Column consisting of (25mm x 4.6mm x 5um - BDS) i.d in isocratic mode, with mobile phase containing 0.01M Potassium di-hydrogen orthophosphate buffer and acetonitrile in the ratio of 35:65 v/v was used. The flow rate was 1mL min -1 and effluent was monitored at 222 nm. Retention time was found to be 3.08min, respectively. The method was validated in terms of Linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc. in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there was good linear relationship between response and concentration in the range of 10 - 100 µg/ml respectively. The LOD and LOQ values for HPLC method were found to be 0.03 and 0.09 µg/ml respectively. No chromatographic interference from tablet excipients was found. The proposed method was successfully used for estimation of montelukast in tablet dosage form.

Objective: The objective of this study was to develop a new and simple reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of riluzole (RZ) and levodopa (LD) in the marketed tablet dosage form. Materials and Methods: The chromatographic separation was achieved on a RP hypercil C18 column (250 × 4.6 mm i.d., 5 μm) using a mobile phase consisting of methanol and water (75:25 v/v) at a flow rate of 1.0 ml/min with the ultraviolet detection wavelength of 273 nm at ambient temperature. The developed method was validated as per the International Council on Harmonization guideline for linearity, accuracy, precision, robustness, ruggedness, limit of detection (LOD), limit of quantitation (LOQ), and specificity. Results: Results of validation studies were found satisfactory with % relative standard deviation values of <2% indicating good specificity, validity, and reliability of the method. The method showed good linearity over the concentration range of 20–80 μg/ml with correlation coefficient (r2) values of 0.990and 0.999 for RZ and LD, respectively. The mean percentage recoveries were between 99.46–99.80% and 100.15–101.24% for RZ and LD, respectively. The LOD and LOQ values were found to be 0.036and 0.012 μg/ml, and 0.110and 0.036 μg/ml for RZ and LD, respectively. The assay of RZ and LD in the marketed tablet formulation was found to be 99.67and 98.95%, respectively. Conclusion: The RP-HPLC method is reported to be simple, specific, accurate, and precise. The proposed method can be successfully applied for the routine analysis of RZ and LD in the bulk drugs as well as in combined pharmaceutical dosage forms.

Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM

A simple reversed phase high-performance liquid chromatographic (RP-HPLC) method coupled with a photodiode array detector (PAD) has been developed and validated for the analysis of hederacoside C, the marker of ivy plant, in Ivy-Thyme cough syrup. Separation of hederacoside C was achieved using a Phenomenex-Gemini C18 column isothermally at 40°C. A mobile phase system constituted of solvent A (water: acetonitrile: orthophosphoric acid (85%), 860 : 140 : 2 v/v) and solvent B (acetonitrile: orthophosphoric acid (85%), 998 : 2 v/v) was used, at gradient conditions, at a flow rate of 1.5 mL/min. Analysis was performed using UV-detection (205 nm). The method was linear over the range (0.03–0.15) mg/mL of hederacoside C (r = 0.9992). Repeatability and intermediate precision were acceptable (RSD <2%). Limits of detection (LOD) and quantitation (LOQ) were 0.011 and 0.032 mg/mL, respectively. Percentage recovery was found to lie between 99.69% and 100.90% (RSD <2%). The method was also proved to be specific (peak-purity coefficient = 0.996).

A scientifically rigorous and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed using a Quality by Design (QbD) approach for the simultaneous estimation of Voglibose and Metformin in fixed-dose combination formulations. The method was systematically optimized through Design of Experiments (DoE), evaluating critical parameters such as flow rate, buffer pH, and organic solvent ratio for their impact on chromatographic responses like resolution and tailing factor. Statistical analysis via ANOVA confirmed the significance of the quadratic model in achieving optimal separation. The final method employed a Zorbax C18 column with phosphate buffer (pH 4.5) and acetonitrile (45:55 v/v) as the mobile phase, operating at a flow rate of 1.2 mL/min and detection at 225 nm. This setup yielded excellent peak separation with a resolution of 10.10 between the analytes. Validation was conducted in accordance with ICH Q2(R1) guidelines, demonstrating outstanding linearity for Metformin (25–125 µg/mL) and Voglibose (0.1–0.5µg/mL), with correlation coefficients of 0.9998 and 0.9994, respectively. Precision studies confirmed repeatability and intermediate precision (%RSD &lt; 2%), while accuracy was validated through high recovery rates (99.4% for Voglibose and 99.6% for Metformin). Sensitivity was affirmed with acceptable LOD and LOQ values. This QbD-driven RP-HPLC method provides a robust, validated, and regulatory-compliant solution for simultaneous estimation of Voglibose and Metformin in pharmaceutical applications