Liquid chromatography and tandem mass spectrometric method for the quantification of Tepotinib in plasma samples

Arjun Siliveri; Kavitha Pingili

doi:10.7324/JAPS.2025.206883

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Abstract

A precise and accurate liquid chromatographic tandem mass spectrometric technique was developed to measure the levels of Tepotinib in plasma. Axitinib and Tepotinib were separated from the plasma sample solution using a protein precipitation method. A chromatographic isolation was carried out using a ZorbaxC18 stationary phase (2.1 × 100 mm, 3 μm) and mobile phase proportion of 0.1 % HCOOH and acetonitrile (15:85). The analytes were quantified using positive ionization methodology with electrospray ionization. Mass transitions for Tepotinib were m/z 493.23 → 296.17 and for Axitinib (IS) they were m/z 387.12 → 220.08. No interference from any components of blank plasma or additional substances was identified. Correlation between Tepotinib concentrations and the respective area of the peaks ratio to Axitinib showed a linear pattern across a range of 1.5–1200 ng/mL. The precision of Tepotinib was excellent, with an intra-day precision of ≤4.92% (n = 10) and an inter-day precision of ≤04.76% (n = 20, over three days). The measured average extraction recoveries of Tepotinib were 98.32%. Tepotinib underwent various stability tests at low and high-quality control levels. The results showed that Tepotinib remained stable and was within 95.41%–102.43%. The developed technique can be valuable for regular quantification of Tepotinib plasma samples in clinical organizations, various industries, and forensic laboratories.

Keyword: Tepotinib lung cancer plasma LC-MS/MS validation

Citation:

Pingili K, Siliveri A. Liquid chromatography and tandem mass spectrometric method for the quantification of Tepotinib in plasma samples. J Appl Pharm Sci. 2025. Online First. https://doi.org/10.7324/JAPS.2025.206883

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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14. Guodong He, Liping Mai, Xipei Wang. Development and validation of an HPLC-MS/MS method for rapid simultaneous determination of cefprozil diastereomers in human plasma. Hindawi Int J Ana Chem. 2018;6959761:1-9. https://doi.org/10.1155/2018/6959761
15. Bhatt D, Rajkamal B. A UPLC-MS/MS method development and validation for the estimation of sofosbuvir from human plasma. Int J Appl Pharm. 2017;9(1):30-6. https://doi.org/10.22159/ijap.2017v9i1.15652
16. Zhou C, Tian J, Lin P, Liu T, He A, Fang L, et al. Quantitation of fostemsavir, a mesenchymal-epithelial transition factor inhibitor by UPLC-MS/MS in rat plasma and its application to a pharmacokinetic study. Bioanalysis. 2020;12(5):285-93. https://doi.org/10.4155/bio-2020-0011
17. ICH guidelines for bioanalytical method validation and study sample analysis. Geneva, Switzerland: M10 ICH; 2022. p 1-59.
18. US FDA. Guidance for Industry Bioanalytical Method Validation, Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Rockville, MD: US FDA; 2001.
19. Glaenzel U, Jin Y, Hansen R, Schroer K, Rahmanzadeh G, Pfaar U, et al. Absorption, distribution, metabolism, and excretion of fostemsavir (INC280) in healthy male volunteers and in vitro aldehyde oxidase phenotyping of the major metabolite. D Met Disp. 2020;48(10):873-85. https://doi.org/10.1124/dmd.119.090324
20. Ravi Y, Bhikshapathi D, Shankar C, Rajkamal B. Development of fast and simple LC-ESI-MS/MS technique for the quantification of regorafenib; application to pharmacokinetics in healthy rabbits. Curr Pharm Anal. 2021;17(4):554-63. https://doi.org/10.2174/1573412916666191111144707
21. Shah JV, Shah PA, Shah PV, Sanyal M, Shrivastav PS. Fast and sensitive LC-MS/MS method for the simultaneous determination of lisinopril and hydrochlorothiazide in human plasma. J Pharm Anal. 2017;7:163-9. https://doi.org/10.1016/j.jpha.2016.11.004
22. Lolla S, Gubbiyappa KS, Shankar CH, Bhikshapathi DVRN. Validation of an LC-MS/MS method for quantitation of fostemsavir in plasma. J Pharmacol Toxicol Methods. 2023;120:107254. https://doi.org/10.1016/j.vascn.2023.107254

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1. Mathieu LN, Larkins E, Akinboro O, Roy P, Amatya AK, Fiero MH. FDA Approval summary: capmatinib and tepotinib for the treatment of metastatic NSCLC harboring MET exon 14 skipping mutations or alterations. Clin Cancer Res. 2022;8(2):249-54. https://doi.org/10.1158/1078-0432.CCR-21-1566

2. Morise M, Sakai H, Veillon R, Le X, Felip E, Garassino MC, et al. O13-4 Tepotinib safety in MET exon 14 (METex14) skipping NSCLC: updated results from the VISION trial. Ann Oncol. 2021;32:S291. https://doi.org/10.1016/j.annonc.2021.05.541

3. Abdelhameed AS, Attwa MW, Kadi AA. Identification of iminium intermediates generation in the metabolism of Tepotinib using LC-MS/MS: in silico and practical approaches to bioactivation pathway elucidation. Molecules. 2020;25(21):25215004. https://doi.org/10.3390/molecules25215004

4. FDA Approves Tepmetko as the First and Only Once-daily Oral MET Inhibitor for Patients with Metastatic NSCLC with METex14 Skipping Alterations. EMD Serono (Press release). 3 February 2021.

5. https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/214096s000lbl.pdf

6. Wu ZX, Teng QX, Cai CY, Wang JQ, Lei ZN, Yang Y, et al. Tepotinib reverses ABCB1-mediated multidrug resistance in cancer cells. Biochem Pharmacol. 2019;166:120-7. https://doi.org/10.1016/j.bcp.2019.05.015

7. Sunetha A, Sharmila D. HPLC method development and validation for the estimation of axitinibe in rabbit plasma. Brazil J Pharm Sci. 2017;53:00012. https://doi.org/10.1590/s2175-97902017000300012

8. Rini B, Rixe O, Bukowski R, Michaelson MD, Wilding G, Hudes G, et al. Amulti-target tyrosine kinase receptor inhibitor, demonstrates anti-tumor activity in a Phase 2 study of cytokine-refractory, metastatic renal cell cancer (RCC). J Clin Oncol ASCO Ann Meeting Proc. 2005;23(16S):4509. https://doi.org/10.1200/jco.2005.23.16_suppl.4509

9. Smita T Kumbhar, Pratima S Kokare, Pradip B Digge. Development of novel RP-HPLC method for estimating Tepotinib in bulk and pharmaceutical dosage form. Int J Pharm Qual. 2023;14(4):1188-93. https://doi.org/10.25258/ijpqa.14.4.56

10. Nikhil J, Parthiban C, Sudhakar M, Vijaya Sri K. Method development and validation for the estimation of Tepotinib in pharmaceutical dosage forms by RP-HPLC. Int J Pharm Pharm Res. 2022;26(1):468-77.

11. Shirwar M, Birajdar S, Garad S, Kumbhar S. Development and validation of novel UV-visible spectrophotometric method for estimation of tepotinib in bulk and in pharmaceutical formulation. Int J Pharm Pharm Sci. 2023;15(9):32-6. https://doi.org/10.22159/ijpps.2023v15i9.48431

12. Attwa MW, Mostafa GAE, AlRabiah H, Kadi AA. An LC-MS/MS analytical method for quantifying Tepotinib in human liver microsomes: application to in vitro and in silico metabolic stability estimation. Separations 2023;10:330. https://doi.org/10.3390/separations10060330

13. Saraner N, Karagoz A, Guney B, Saglam O. Determination of dasatinib in human plasma by using liquid chromatography-tandem mass spectrometry. Int J Anal Bioanal Meth. 2019;1:2. https://doi.org/10.15761/BRR.1000124

14. Guodong He, Liping Mai, Xipei Wang. Development and validation of an HPLC-MS/MS method for rapid simultaneous determination of cefprozil diastereomers in human plasma. Hindawi Int J Ana Chem. 2018;6959761:1-9. https://doi.org/10.1155/2018/6959761

15. Bhatt D, Rajkamal B. A UPLC-MS/MS method development and validation for the estimation of sofosbuvir from human plasma. Int J Appl Pharm. 2017;9(1):30-6. https://doi.org/10.22159/ijap.2017v9i1.15652

16. Zhou C, Tian J, Lin P, Liu T, He A, Fang L, et al. Quantitation of fostemsavir, a mesenchymal-epithelial transition factor inhibitor by UPLC-MS/MS in rat plasma and its application to a pharmacokinetic study. Bioanalysis. 2020;12(5):285-93. https://doi.org/10.4155/bio-2020-0011

17. ICH guidelines for bioanalytical method validation and study sample analysis. Geneva, Switzerland: M10 ICH; 2022. p 1-59.

18. US FDA. Guidance for Industry Bioanalytical Method Validation, Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Rockville, MD: US FDA; 2001.

19. Glaenzel U, Jin Y, Hansen R, Schroer K, Rahmanzadeh G, Pfaar U, et al. Absorption, distribution, metabolism, and excretion of fostemsavir (INC280) in healthy male volunteers and in vitro aldehyde oxidase phenotyping of the major metabolite. D Met Disp. 2020;48(10):873-85. https://doi.org/10.1124/dmd.119.090324

20. Ravi Y, Bhikshapathi D, Shankar C, Rajkamal B. Development of fast and simple LC-ESI-MS/MS technique for the quantification of regorafenib; application to pharmacokinetics in healthy rabbits. Curr Pharm Anal. 2021;17(4):554-63. https://doi.org/10.2174/1573412916666191111144707

21. Shah JV, Shah PA, Shah PV, Sanyal M, Shrivastav PS. Fast and sensitive LC-MS/MS method for the simultaneous determination of lisinopril and hydrochlorothiazide in human plasma. J Pharm Anal. 2017;7:163-9. https://doi.org/10.1016/j.jpha.2016.11.004

22. Lolla S, Gubbiyappa KS, Shankar CH, Bhikshapathi DVRN. Validation of an LC-MS/MS method for quantitation of fostemsavir in plasma. J Pharmacol Toxicol Methods. 2023;120:107254. https://doi.org/10.1016/j.vascn.2023.107254