(M1230-01-06) Quantification of Tirzepatide in Rat Blood by a Sensitive and Robust UPLC-MS/MS Method: Development and Validation

Purpose: irzepatide (TRZ) is the first dual agonist of both GIP and GLP-1 receptors approved by FDA for the treatment of type 2 diabetes mellitus and chronic weight management [1]. Research and development of TRZ for cancer prevention and heart failure therapies has significantly increased [2, 3], creating a growing need for a sensitive and selective analytical method for its quantification, which was not available. Thus, this study is to develop and validate a sensitive and robust UPLC-MS/MS method for the quantification of TRZ in rat blood, following FDA guidelines, to facilitate accurate evaluation of TRZ in pharmacokinetic and pharmacological studies.

Methods: Different sample preparation method (protein precipitation, SPE), different columns (C18, peptide column), and detection equipment (5500, 7500) were tested during the method development. The optimized method was: 50 µL blood samples were processed using methanol-based protein precipitation followed by solid-phase extraction (SPE); chromatographic separation was performed on an AcQuity Premier Peptide BEH C18 column (2.1 × 50 mm, 1.7 µm) with a 3-minute gradient elution using 0.1% formic acid in acetonitrile and 0.1% formic acid in water at a flow rate of 0.4 mL/min. Detection was conducted on an AB SCIEX 7500 triple quadrupole mass spectrometer with an ESI source operating in positive ion MRM mode. ¹³C, ¹⁵N-labeled TRZ was used as the internal standard (IS). Pharmacokinetic studies of TRZ in rats are currently ongoing.

Results: The method showed excellent linearity over the concentration range of 0.42-207.7 nM with correlation coefficients (r) > 0.99 on the SCIEX 7500 system. Matrix effect values were between 86.8 ± 13.2 to 110 ± 2.42% across 3 QC levels (10, 50, and 800 ng/mL), indicating negligible matrix effects. Recovery values were between 85.9 ± 8.04 to 98.5 ± 2.52%, demonstrating high method recovery. Intra- and inter-day accuracy (RE) and precision (RSD) were all within ±15% across 4 QC levels (2, 10, 50, and 800 ng/mL). Dilution integrity was confirmed, with 1000 ng/mL samples remaining within ±15% accuracy and precision after 5- and 10-fold dilutions. TRZ remained stable under all tested sample preparation conditions, and after one-week storage at -80°C.

Conclusion: We have developed and fully validated a simple, sensitive, and reliable UPLC-MS/MS method for quantifying TRZ in rat blood.

References: [1]. Jastreboff, Ania M et al. “Tirzepatide for Obesity Treatment and Diabetes Prevention.” The New England journal of medicine vol. 392,10 (2025): 958-971. doi:10.1056/NEJMoa2410819;
[2]. Kamrul-Hasan, A B M et al. “Tirzepatide and Cancer Risk in Individuals with and without Diabetes: A Systematic Review and Meta-Analysis.” Endocrinology and metabolism (Seoul, Korea) vol. 40,1 (2025): 112-124. doi:10.3803/EnM.2024.2164;
[3]. Nicholls, Stephen J et al. “Comparison of tirzepatide and dulaglutide on major adverse cardiovascular events in participants with type 2 diabetes and atherosclerotic cardiovascular disease: SURPASS-CVOT design and baseline characteristics.” American heart journal vol. 267 (2024): 1-11. doi:10.1016/j.ahj.2023.09.007.