A Cost-Effective RP-HPLC Bioanalytical Technique for Simultaneous Estimation of Nintedanib and Naringenin in Mice Plasma: Development, Validation, and Stability Assessment

Nintedanib (NTB), a first-line tyrosine kinase inhibitor used for Pulmonary Idiopathic fibrosis and lung cancer, found promising effects when combined with bioenhancer Naringenin (NGN) for increased bioavailability.The purpose of the study is to developed a bioanalytical technique for concurrently estimating NTB and NGN in mice plasma using RP-HPLC using Alectinib (ALB) as internal standard.The Gradient elution technique was developed to separate NTB, NGN, and ALB within 15 minutes of total run time.The mobile phase consisted of acetate buffer pH 5.2 and Acetonitrile in a Linear gradient pattern.The flow rate of 1.0 mL/min, and the injection volume of 50 L.Detection was achieved at a wavelength of 290 nm, and the retention times for NTB, NGN, and ALB were found to be 4.5, 6.2, and 10.2 minutes, respectively.Mice Plasma samples were processed using a protein precipitation technique to extract the analytes.The method demonstrated linearity with R of 0.9953 for NTB and 0.9978 for NGN, over a concentration of 50-1000 ng/mL and 75-1050 ng/mL, respectively.The results of all the validation parameters performed as per ICH guidelines M10 were within the accepted limits with less than 3 %RSD.The % recovery from plasma was more than 95%.Freeze-thaw, Bench-top, short-term, and long-term stability studies were also achieved at LQC, MQC, and HQC levels.The established bioanalytical technique can simultaneously estimate the NTB and NGN in plasma samples and is suitable for further pharmacokinetic studies.