Development of a High Sensitivity SPE-LC-MS/MS Assay for the Quantification of Glucagon in Human Plasma Using the ionKey/MS System,

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Mary E. Lame, Erin E. Chambers, Sukhdev S. Bangar, and Kenneth J. Fountain
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Protein Quantification

The combination of ionKey/MS, mixed-mode μElution SPE, and higher m/z b or y ion MS fragments provided the level of selectivity and sensitivity necessary to accurately quantify low pg/mL concentrations of glucagon in extracted plasma. Use of μElution format SPE eliminated the need for evaporation, reducing glucagon losses due to adsorption and non-specific binding. The 150 μm iKey enabled the development of a highly sensitive, low flow quantitative MRM method for glucagon with an LOD of 12.5 pg/mL and a dynamic range from 12.5–1,000 pg/mL. The current analysis uses 200 μL of plasma and provides a significant improvement in sensitivity and S:N over the analytical scale (2.1 mm I.D.) analysis using 1/5th the sample injection volume. Furthermore, an injection of the same volume (5 μL) of sample corresponded to a 10X increase in on-column sensitivity allowing for greater confidence in results, as compared to the traditional analytical method for this peptide. In addition, ionKey/MS reduces solvent and sample consumption, thereby reducing cost and allowing for multiple injections of samples for improved accuracy or to meet the guidelines for incurred sample reanalysis (ISR).

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