A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of tirzepatide, a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors in rat plasma. Tirzepatide was extracted from rat plasma by protein precipitation using methanol. Chromatographic separation was achieved using a peptide C18 column with gradient elution of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed in positive electrospray ionization mode using multiple reaction monitoring with transitions of m/z 1204.4 → 1473.6 for tirzepatide and m/z 1029.4 → 1238.4 for the internal standard, semaglutide. The developed method exhibited good linearity over a concentration range of 1-1000 ng/mL (r > 0.99). Intra- and inter-day accuracy (-4.324-5.057 %) and precision (5.250-9.000 %) met the regulatory criteria at all quality control levels, and were stable under various plasma handling and storage conditions. The validated method was successfully applied to a pharmacokinetic study in rats following the intravenous and subcutaneous injection of tirzepatide at 0.3 mg/kg. The terminal half-lives were 10.04 h and 9.803 h after intravenous and subcutaneous administration, respectively, indicating comparable elimination profiles. The bioavailability following subcutaneous dosing was estimated to be approximately 62.38 %. These findings highlight the robustness and applicability of the developed method, suggesting its potential utility for the quantitative analysis of other peptide therapeutics with structures or mechanisms of action similar to those of tirzepatide. 2
