Development And Validation of Bioanalytical Method for the Quantification of Finerenone in Rat Plasma by RP-HPLC
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Abstract
Introduction: Bioanalytical method development involves designing and optimizing methods to quantify drugs, metabolites, or biomarkers in biological samples (such as blood, plasma). This process is critical in various fields, including pharmacokinetics, toxicology, clinical research, and drug development. Finerenone is a selective non-steroidal mineralocorticoid receptor antagonist, is used primarily for the treatment of chronic kidney disease (CKD) in patients with type 2 diabetes, it helps to reduce the risk of kidney function by blocking the effects of aldosterone, a hormone that promotes sodium retention and contributes to CKD.
Objectives: To optimize the chromatographic condition for the selected approved protocol.To develop a suitable bioanalytical method for the assessment of Finerenone in rat plasma by RP-HPLC method. To validate the developed method as per ICH M10 guideline for intended bioanalytical application. To estimate the in vivo pharmacokinetic parameter for Finerenone in rat plasma by RP-HPLC.
Methods: Chromatographic separation was conducted using an HPLC system from Shimadzu (LC 20 AD) equipped with a PDA detector, controlled by Lab Solution software. Phenomenex Luna C18 column (250mm × 4.6mm, 5µm) was utilized for the separation process. Sample preparation was carried out using an ultra-cooling centrifuge. pH measurement was performed using an ELICO pH meter (LI 120). Solutions were degassed using a sonicator from Lifecare. Micropipettes with a capacity of 100-1000 µl were utilized for measurements. Plasma samples underwent filtration using syringe filters with a pore diameter of 0.25 μm.Finerenone was procured from Hangzhou Jinlan Pharm-Drugs Technology CO., Ltd. China. Niacinamide (IS) was procured from Dhamtec pharma pvt limited Mumbai, India. Blank plasma was collected from Animal House, KMCH College of Pharmacy, Coimbatore. All additional chemicals utilized were of high quality (HPLC grade) and were procured from Rankem Chemicals, India.
Results: The HPLC-PDA method with simple and economical protein precipitation extraction was developed and validated for the estimation of Finerenone in wistar rat plasma. The developed method has a shorter retention time (7.3 mins) using water and acetonitrile (15:85 % v/v). In this method, the validated HPLC technique was utilized to simultaneously determine finerenone levels in rats post-oral administration of 10 mg/kg. Graphical depictions illustrating mean plasma concentration over 4 hours with pharmacokinetic parameters. The maximum concentrations (Cmax) of finerenone were found to be 75 ng/mL and the time observed for maximum drug concentration (Tmax) being 75 mins. The half-lives (T1/2) of finerenone were determined to be 2.58 h. Additionally, the area under the curve for 4 hours (AUC0−t) and the elimination rate constant (Ke) were also evaluated.
Conclusions: A robust and sensitive RP-HPLC method was successfully developed for the accurate quantification of finerenone in rat plasma, ensuring selectivity and reproducibility. The developed method underwent validation according to ICH standards (M10), meeting all validation criteria within specified limits. The results indicated that the method was accurate and precise. The validated method is suitable for application in pharmacokinetic and preclinical studies of finerenone, supporting further drug development and research.