A liquid chromatography-mass spectrometry assay for quantification of Exendin[9-39] in human plasma

•We developed and validated a LC–MS/MS method for quantification of Exendin[9-39] in human plasma.•The assay required only 100 μL of human plasma.•Linearity of the standard curve was 15–1260 ng/mL.•The assay was used to quantify Exendin[9-39] concentration in plasma from subjects participating in a pilot clinical study.•Storage stability at −80 C was good after a 12 month sample storage period.

Exendin[9-39] is a glucagon-like peptide-1 receptor (GLP-R) antagonist and a potential therapeutic drug for treatment of congenital hyperinsulism by lowering insulin concentration in plasma. A specific and sensitive LC–MS/MS method was validated for quantification of Exendin[9-39] in human plasma. Exendin[9-39] and the stable isopically labeled internal standard eluted at 9.2 min and were analyzed by single reaction monitoring (SRM) of the transitions m/z 842.9 → 991.8 and 848.2 → 998.8, respectively. The calibration curve was linear in the range 15–1260 ng/mL with a limit of detection of 1.3 ng/mL. The CVs of the standards were 2.7–13.1% within-run and 3.1–13.2% between-run. The matrix effect was >100% and the SPE recovery was 98.4 ± 12.9%. In absence of protease inhibitors, short-term stability at room temperature was only one hour. Accordingly, samples were kept on ice and sample processing was kept below 1 h. Human plasma samples from a clinical pilot study in which Exendin[9-39] was administered intravenously were analyzed and concentrations up to 600 ng/mL were reported Plasma samples from the study were stored at −80 °C with internal standard and successfully reanalyzed after 12 months.