Method Development and Validation for Ritonavir with Comprehensive Impurity and Toxicity Profiling

To develop and validate the stability indicating method for Empagliflozin and Linagliptin using HPLC method

The fixed-dose combination of linagliptin and empagliflozin for the treatment of type 2 diabetes mellitus is increasingly being used in clinical practice. Consequently, there is a need for stability studies and routine quality control testing of the drug product, which requires the development of a simple, reliable, and stability-indicating analytical method. This research was aimed to develop, optimize and validate the simultaneous determination of two compounds Linagliptin and Empagliflozin in their bulk and pharmaceutical tablet dosage form using reverse phase high-performance liquid chromatography (RP-HPLC) with ICH Q2(R2) criteria. The separation was performed by HPLC on a C18 column with mobile phase methanol/0.1% orthophosphoric acid (30:70 v/v) at 0.7mL/min flow rate at 235nm UV. The developed method was validated in terms of system suitability, linearity, accuracy, precision, repeatability, sensitivity, ruggedness, robustness, assay and forced degradation studies. The linearity of both drugs was good, R² value were 0.9996 within their respective concentration ranges. The method was found to be reliable and reproducible with the recovery value of 99.72-102.17% for Linagliptin, whereas %RSD was found to be below 2% for all the precision studies which indicates the reliability of the method, and found to be below 2% for Empagliflozin which indicates recovery value of 99.29-101.20%. The assay results showed acceptable release of drug from the marketed formulation, while the stress degradation studies under acidic, alkaline, oxidative, neutral and photolytic conditions showed good separation of the drug degradation products from the analytes, proving the stability indicating nature of the method. The developed RP HPLC method is simple, rapid, selective, accurate, economical, and can be used for routine pharmaceutical quality control, stability testing and analysis of combined Linagliptin and Empagliflozin formulation.

Stability indicating Method Development and Validation of Inavolisib and its impurities by RP-HPLC Method

Aim: The present study describes about the development and validation of novel RP-HPLC analytical method for the analysis of Inavolisib and its impurities and to carry out stability studies by forced degradation to estimate the stability of drug and its impurities. The proposed method was novel for the estimation of anti cancer drug. Materials and Methods: Based on the solubility studies carried out at 25oC using variety of solvents and buffers, Acetonitrile and Ammonium formate pH-2.5/formic acid (40:60) were found to be suitable mixture of solvents for mobile phase system. The analysis of drug is accomplished by using Waters X-Terra RP-18 column (150mm x 4.6mm ID, 3.5µm) at 259 nm. Results and Discussions: The method is then validated according to the ICH guidelines. The analytical results revealed that the method developed is within the limits for all the validated parameters. The forced degradation studies revealed that 3.25% and 3.46% of Inavolisib degradation was observed in presence of peroxide and alkali conditions. Summary and Conclusion: From the results of the study, the sample recoveries were found within the limits. Hence the proposed method can be conveniently and easily adopted to the determination of Inavolisib and its impurities.

Development and Validation of a Sensitive UHPLC-MS/MS Analytical Method for Fulzerasib in Rat Plasma and its Application to Pharmacokinetics Studies.

Fulzerasib is a novel, potent, and irreversible covalent inhibitor targeting the KRAS G12C mutation, recently approved in China for advanced nonsmall cell lung cancer. This study aimed to develop and fully validate a rapid, specific and sensitive UHPLC-MS/MS method for the quantification of Fulzerasib in rat plasma. Sample preparation was streamlined using a simple protein precipitation technique with acetonitrile. Chromatographic separation was achieved within a 4.0-min run on a C18 column, with verapamil as the internal standard and detection via multiple reaction monitoring. The method was rigorously validated over a linear range of 5 to 5000 ng/mL (R2 > 0.9933), demonstrating excellent accuracy (102%-112.7%) and precision (≤ 10.3% for QCs). Stability was confirmed under various storage and processing conditions. The validated method was successfully applied in the first pharmacokinetic study of Fulzerasib in Sprague-Dawley rats following single intravenous (1 mg/kg) and oral (3 mg/kg) administration. Fulzerasib exhibited favorable pharmacokinetic properties, including low plasma clearance (5.02 mL/min/kg), moderate volume of distribution and notably high oral bioavailability of 38.6%. The established method is reliable, efficient and readily applicable, thereby providing a vital tool for accelerating the ongoing preclinical and translational research of Fulzerasib.

Creation and verification of an RP-UHPLC technique for abemaciclib and associated impurities in formulations and APIs using Box-Behnken design

Background: Strict impurity profiling is necessary to ensure the safety, effectiveness, and regulatory compliance of Abemaciclib (AbB), a selective CDK4/6 inhibitor frequently used in breast cancer treatment. However, a lack of thorough impurity separation, lengthy run times, and low sensitivity are common problems with current analytical techniques. Methodology: A novel reverse-phase ultra-high-performance liquid chromatography (RP-UHPLC) method was developed using a Quality by Design (QbD) framework. Critical method parameters, including mobile phase pH, flow rate, and column temperature, were optimized using Box-Behnken Design (BBD). Chromatographic separation was achieved on a C18 column using an isocratic mobile phase consisting of acetonitrile (40:60, v/v) and ammonium formate buffer (pH 2.7). The technique was validated in accordance with the ICH Q2(R1) requirements. Results and Discussion: The optimized method demonstrated exceptional linearity over the range of 0.02–150 μg/mL (R² > 0.999) with excellent precision (RSD < 2% for AbB and < 10% for related impurities) and showed acceptable accuracy, with mean recoveries ranging from 92.76% to 102.88% across AbB and its related impurities. The detection and quantification limits for AbB were 0.01 μg/mL and 0.02 μg/mL, respectively. Forced degradation studies that effectively distinguished degradation products in oxidative, acidic, and alkaline settings confirmed the method's stability-indicating nature. The 8-minute run time allowed for quick examination. The integration of BBD-based QbD significantly enhanced method robustness, resolution, and sensitivity compared to conventional chromatographic approaches, allowing simultaneous quantification of AbB and its related impurities. Conclusion: For routine quality control, stability investigations, and regulatory-compliant impurity profiling of Abemaciclib, the developed RP-UHPLC method is quick, sensitive, reliable, and appropriate.

Design of experiments (DoE) driven stability indicating RP-HPLC method development and validation of an anticancer drug, apatinib

A QBD BASED RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF KETOCONAZOLE

A stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative estimation of ketoconazole in bulk drug and pharmaceutical dosage forms using the Analytical Quality by Design (AQbD) framework. Critical method parameters were identified through risk assessment and optimized via Design of Experiments (DoE), ensuring robustness, reliability, and regulatory compliance. Chromatographic separation was achieved on an Agilent Zorbax SB-Aq column (250 × 4.6 mm, 5 µm) employing a mobile phase of phosphate buffer and acetonitrile (35:65, v/v) at a flow rate of 0.8 mL/min, with detection at 225 nm. Ketoconazole eluted at approximately 5.47 min within a total runtime of 15 min. Validation, performed in accordance with ICH Q2(R1) guidelines, confirmed excellent specificity, linearity, precision, accuracy, robustness, sensitivity, and reproducibility. The calibration curve was linear across 80–120 µg/mL with a correlation coefficient (R²) of 1.000. Precision studies yielded %RSD values below 2%, while recovery ranged between 99.75% and 100.07%, indicating high accuracy. Limits of detection and quantification were 0.31 µg/mL and 0.94 µg/mL, respectively. Robustness evaluation revealed negligible effects of minor variations in pH and detection wavelength. Forced degradation under acidic, alkaline, oxidative, thermal, and photolytic conditions demonstrated the method’s stability-indicating capability. Maximum degradation was observed under oxidative stress (16.14%), followed by thermal (9.34%), photolytic (8.97%), alkaline (6.71%), and acidic (6.09%) conditions, with degradation products well resolved from the analyte peak. The AQbD-based RP-HPLC method is rapid, accurate, economical, and highly robust, making it suitable for routine quality control, stability testing, and regulatory applications in ketoconazole analysis