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Quantitative Analytical Method for Determining the Levels of Gastric Inhibitory Polypeptides GIP1–42 and GIP3–42 in Human Plasma Using LC–MS/MS/MS

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journal contribution
posted on 07.06.2013, 00:00 by Atsushi Miyachi, Takayo Murase, Yuichiro Yamada, Takeshi Osonoi, Ken-ichi Harada
Gastric inhibitory polypeptide (GIP), an incretin, is an important subject in endocrinology. Some LC–MS assays have been proposed; however, their sensitivities are insufficient for the study of endogenous human incretin. Here, we describe a nanoflow LC hybrid triple quadrupole/linear ion trap MS assay for the simultaneous quantification of GIP1–42 and GIP3–42 from human plasma. We selected the surrogate peptide to avoid oxidative modification, and the endoproteinase Asp-N was selected for the proteolysis of GIP1–42 and GIP3–42. The phenylalanine residue at position 6 in both GIP1–42 and GIP3–42 was substituted with 13C9,15N-labeled phenylalanine, and these substituted GIPs were used as the internal standards. This facilitated accurate and precise quantification because large corrections are possible at all steps of sample pretreatment and ionization efficiency. The lower limit of quantification was 1 pM for GIP1–42 and 10 pM for GIP3–42 by using 200 μL of plasma. Quantification of GIP1–42 and GIP3–42 in plasma from patients with type 2 diabetes was possible using this method, which included protein precipitation, Asp-N proteolysis, solid-phase extraction, nanoflow LC, and positive-ion multiple reaction monitoring cubed (MRM3) for GIP1–8, and MRM for GIP3–8 to achieve accurate, precise, and quantitative analysis that can be validated to support large clinical trials.