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

Parin Chokshi*, Rajashree Mashru, Preeti Jadeja and Apeksha Kadam

Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, India

*Corresponding Author: Parin Chokshi, Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, India.

Received: August 14, 2025; Published: August 28, 2025

Abstract

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 μL. 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 R² 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.

Keywords: Nintedanib; Naringenin; Simultaneous Estimation Bioanalytical Method; RP-HPLC; Mice Plasma; Stability Studies; Bioanalytical Method Validation

References

1. Yin Zhiqiang., et al. "Second Primary Lung Cancer After Breast Cancer: A Population-Based Study of 6,269 Women”. Frontiers in Oncology (2018): 1-11.
2. Benet Leslie Z. "The role of BCS (biopharmaceutics classification system) and BDDCS (biopharmaceutics drug disposition classification system) in drug development”. Journal of Pharmaceutical Sciences 1 (2013): 34-42.
3. Balasubramanian Hari Baskar., et al. "Affinity of Nintedanib Towards New Candidate Target for Idiopathic Pulmonary Fibrosis”. Borneo Journal of Pharmacy3 (2024): 264-277.
4. Joshi Ruthvika., et al. "Pharmacokinetic, pharmacodynamic and formulations aspects of Naringenin: An update”. Life Sciences 215 (2018): 43-56.
5. Sudarshana B., et al. "Review on naringin: Method of isolation, analytical development, and its recent pharmacological activities”. International Journal of Pharmaceutical Sciences and Research4 (2023): 1622-1629.
6. Velagacherla Varalakshmi., et al. "A stability indicating method development and validation of a rapid and sensitive RP-HPLC method for Nintedanib and its application in quantification of nanostructured lipid carriers”. F1000 Research 12 (2024): 1389.
7. Xu Dan., et al. "A fast, sensitive, and high throughput method for the determination of nintedanib in mouse plasma by UPLC-MS/MS”. Analytical Methods16 (2015): 6561-6565.
8. Chowdhury N S. "A Review on Specific and Sensitive Analytical Method Development and Validation”. International Journal of Pharmaceutical Chemistry and Analysis 10 (2023): 28-30.
9. Ni Hui., et al. "Development and evaluation of simultaneous quantification of naringin, prunin, naringenin, and limonin in citrus juice”. Food Science and Biotechnology4 (2015): 1239-1247.
10. Ishii Kazuo., et al. "Determination of naringin and naringenin in human plasma by high-performance liquid chromatography”. Journal of Chromatography B: Biomedical Sciences and Applications2 (1996): 225-229.
11. Lei Yating., et al. "Quantitative analysis of multicomponents by single marker combined with HPLC fingerprint qualitative analyses for comprehensive evaluation of Aurantii Fructus”. Journal of Separation Science 7 (2020): 1382-1392.
12. ICH M10 on bioanalytical method validation - Scientific guideline, European Medicines Agency (2019).
13. Wollin Lutz., et al. "Effects of nintedanib in an animal model of liver fibrosis”. BioMed Research International 1 (2020): 3867198.

Citation

Citation: Parin Chokshi., et al. “A Cost-Effective RP-HPLC Bioanalytical Technique for Simultaneous Estimation of Nintedanib and Naringenin in Mice Plasma: Development, Validation, and Stability Assessment". Acta Scientific Pharmaceutical Sciences 9.9 (2025): 50-63.

Copyright

Copyright: © 2025 Parin Chokshi., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.