(T1230-09-53) Novel LC-MS/MS Analyses for Glucagon-Like Peptide-1 (GLP-1) Analogs, Liraglutide, Lixisenatide, and Semaglutide, and Pharmacokinetic Evaluation in Rats

Purpose: Glucagon-like peptide-1 (GLP-1) analogs have been developed as a new therapeutic option for the treatment of type 2 diabetes mellitus. Nevertheless, in vivo pharmacokinetic information on these GLP-1 analogs is still limited. This study aimed to develop novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and characterize the pharmacokinetics of three GLP-1 analogs, i.e., liraglutide, lixisenatide, and semaglutide, in rats.

Methods: Liraglutide, lixisenatide, and semaglutide in the rat plasma were extracted by simple protein precipitation using methanol as a precipitation solvent. Gradient elution profiles were optimized to achieve the maximum peak response and minimal endogenous interference. The electrospray ionization (ESI) source was operated in the positive mode. Sprague-Dawley rats were given liraglutide, lixisenatide, and semaglutide separately by intravenous (IV) or subcutaneous (SC) injection (n=5, each). The plasma concentrations of GLP-1 analogs were determined by the developed LC-MS/MS methods.

Results: The assays resulted in the lower limit of quantification (LLOQ) of 5 ng/mL for liraglutide and 10 ng/mL for lixisenatide and semaglutide in the rat plasma. The assays were fully validated according to the US FDA guidance. The intra- and inter-day accuracy was 96.18 – 113.21% for liraglutide, 97.63 – 112.28% for lixisenatide, and 99.39 – 114.23% for semaglutide. The precisions were all within 14.93%. Following IV injections, the plasma concentrations of liraglutide, lixisenatide, and semaglutide showed a multi-exponential decline with an average terminal half-life of 3.28 h, 0.37 h, and 8.18 h, respectively. The estimated subcutaneous bioavailability was 14.02% for liraglutide, 2.17% for lixisenatide, and 77.39% for semaglutide.

Conclusion: Simple and sensitive LC-MS/MS methods to quantitate the concentrations of three GLP-1 analogs, including liraglutide, lixisenatide, and semaglutide in the rat plasma, have been developed and validated. The present LC-MS/MS methods and the pharmacokinetics of the GLP-1 analogs may be useful for the further development of novel GLP-1 analogs as promising anti-diabetic agents.

Acknowledgments: This work was supported by the Ministry of Trade, Industry and Energy of Korea Grant no. 20008909.