Quantitative Analytical Method for Determining the Levels of Gastric Inhibitory Polypeptides GIP<sub>1–42</sub> and GIP<sub>3–42</sub> in Human Plasma Using LC–MS/MS/MS Atsushi Miyachi Takayo Murase Yuichiro Yamada Takeshi Osonoi Ken-ichi Harada 10.1021/pr400069f.s004 https://acs.figshare.com/articles/journal_contribution/Quantitative_Analytical_Method_for_Determining_the_Levels_of_Gastric_Inhibitory_Polypeptides_GIP_sub_1_42_sub_and_GIP_sub_3_42_sub_in_Human_Plasma_Using_LC_MS_MS_MS/2408656 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 GIP<sub>1–42</sub> and GIP<sub>3–42</sub> from human plasma. We selected the surrogate peptide to avoid oxidative modification, and the endoproteinase Asp-N was selected for the proteolysis of GIP<sub>1–42</sub> and GIP<sub>3–42</sub>. The phenylalanine residue at position 6 in both GIP<sub>1–42</sub> and GIP<sub>3–42</sub> was substituted with <sup>13</sup>C<sub>9</sub>,<sup>15</sup>N-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 GIP<sub>1–42</sub> and 10 pM for GIP<sub>3–42</sub> by using 200 μL of plasma. Quantification of GIP<sub>1–42</sub> and GIP<sub>3–42</sub> 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 (MRM<sup>3</sup>) for GIP<sub>1–8</sub>, and MRM for GIP<sub>3–8</sub> to achieve accurate, precise, and quantitative analysis that can be validated to support large clinical trials. 2013-06-07 00:00:00 GIP MRM Quantitative Analytical Method type 2 diabetes MS nanoflow LC 200 μ L