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Development of a UHPLC–MS/MS (SRM) method for the quantitation of endogenous glucagon and dosed GLP-1 from human plasma

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Aim: The performance of glucagon and GLP1 immunoassays is often poor, but few sensitive LC–MS/MS methods exist as alternatives.

Experimental: A multiplexed LC–MS/MS method using a 2D extraction technique was developed.

Results: The method was established for the quantitation of endogenous glucagon (LLOQ: 15 pg/ml) and dosed GLP1 (LLOQ: 25 pg/ml) in human plasma, and is the first such method avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier SRM. Endogenous glucagon concentrations were within the expected range, and showed good reproducibility after extended sample storage. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between methods.

Conclusion: The LC–MS/MS method offers a viable alternative to immunoassays for quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP1.

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