Development of a high throughput UHPLC-MS / MS ( SRM ) method for the quantitation of 1 endogenous glucagon from human plasma 2

13 Background: Published LC-MS/MS methods are not sensitive enough to quantify 14 endogenous levels of glucagon. Results: A UHPLC-MS/MS (SRM) method for the 15 quantitation of endogenous levels glucagon was successfully developed and qualified. A 16 novel 2D extraction procedure was used to reduce matrix suppression, background noise 17 and interferences. Glucagon levels in samples from healthy volunteers were found to agree 18 with RIA derived literature values. Bland-Altman analysis showed a concentration-dependent 19 positive bias of the LC/MS-MS assay versus an RIA. Both assays produced similar 20 pharmacokinetic profiles, both of which were feasible considering the nature of the study. 21 Conclusions: Our method is the first peer reviewed LC-MS/MS method for the quantitation 22 of endogenous levels of glucagon, and offers a viable alternative to RIA based approaches. 23