Development Of A High Sensitivity SPE-LC-MS/MS Assay For The Quantification Of Glucagon In Human Plasma Using The ionKey/MS System

The integration of ionKey/MS technology, mixed-mode μElution solid-phase extraction (SPE), and higher m/z b or y ion MS fragments has enabled a highly selective and sensitive method for quantifying glucagon at low picogram-per-milliliter (pg/mL) concentrations in extracted plasma. By utilizing the μElution SPE format, the need for evaporation is eliminated, significantly reducing glucagon loss due to adsorption and non-specific binding. The use of a 150 μm iKey column supports the development of a low-flow, highly sensitive multiple reaction monitoring (MRM) method, which achieves a limit of detection (LOD) of 12.5 pg/mL and a dynamic range of 12.5–1,000 pg/mL. This method requires only 200 μL of plasma and offers a marked improvement in signal-to-noise ratio and sensitivity compared to traditional analytical scale methods, even with just one-fifth the sample injection volume. Notably, injecting the same volume (5 μL) of sample results in a tenfold increase in on-column sensitivity, enhancing confidence in the results.

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