(T1430-02-07) Analysis of Glucagon-Like Peptide-1 (GLP-1) Analog in Rat Plasma Using an LC-MS/MS Workflow

Purpose: There has been an increased attention towards GLP-1 analogs as they aid in the regulation of systemic homeostasis. GLP-1 analogs, such as orally available semglutide, is clinically approved and used to treat type 2 diabetes. Certain GLP-1 analogs have low oral availability and are administered via subcutaneous injection in a frequent dosing regimen which causes a treatment burden on patients. In such cases, challenges arise in the development of alternatiive formulations, including, low bioavailability and limited sample availability impacting the quantitative performance of assays. Sensitive methods are needed to effectively detect and quantify GLP-1 analogs in biological matrices. The presented method demonstrates a quantitative method for a commercially available GLP-1 analog, semaglutide, in rat plasma.

Methods: Semaglutide was spiked into rat plasma at concentrations ranging from 0.2 to 200 ng/mL. Liraglutide was spiked at 10 ng as an internal standard. Plasma proteins were precipitated using methanol and the samples were vortexed and centrifuged at room temperature. Solid phase extraction was performed using the supernatant on an anion exchange microelution plate. The final eluent was used for LC-MS/MS based analysis. Separation was perfomed using a Phenomenex Kinetex C18 column (2.1 x 50 mm, 2.6 µm, 100 Å). The LC system was operated at a flow rate of 0.4 mL/min. Analysis was performed on a triple quadrupole system in positive ion mode. The MS and source parameters were optimized to achieve sensitive LC-MS quantitation.

Results: The calibration curve was prepared across concentrations ranging from 0.2 to 200 ng/mL. Measurements at each concentration were performed in triplicate. A lower limit of quantitation (LLOQ) for semaglutide was achieved at 0.2 ng/mL. No interferences were observed in the rat plasma matrix. Linearity was achieved across the concentration range with a r² >0.99. The linear dynamic range spanned 3 orders of magnitude. Analytical performance was evaluated for accuracy and precision. The accuracy of the calculated mean was expected to be between 80% and 120% with a %CV < 20% at the LLOQ. At higher concentrations, the accuracy of the calculated mean was expected to be between 85% and 115% with a %CV < 15%. Based on the experiment, the %CV was < 13% and accuracy was within ±11% of the nominal concentration. Therefore, the calculated accuracy and %CV values met the acceptance criteria at each concentration level.

Conclusion: Development of a highly sensitive method for low-ng/mL quantitation of GLP-1 analog, semaglutide, in rat plasma using an LC-MS/MS method.