Determination of oxyntomodulin, an anorectic polypeptide, in rat plasma using 2D-LC-MS/MS coupled with ion pair...

Polypeptide therapeutics present a challenge for quantitative analysis when using immunoassays or recently, liquid chromatography-tandem mass spectrometry because of their structural similarities to endogenous proteins and peptides in plasma. In this assay, a Waters Oasis® mixed-mode anion exchange (MAX) microelution modified solid phase extraction (SPE) method coupled with two-dimensional reversed phase ion pair chromatography-tandem mass spectrometry was used for the validation and analysis of oxyntomodulin in rat plasma. Oxyntomodulin (OXM) and its isotope labeled internal standard were extracted from rat plasma and analyzed with a chromatographic run time of 8 min. Modified SPE, two-dimensional liquid chromatography coupled with 3-nitrobenzyl alcohol as a mobile phase additive, and monitoring of multiply charged SRM transitions (+7 charge state) of OXM were necessary to achieve a lower limit of quantification of 1 ng/mL. The method was validated with a linear range of 1-1000 ng/mL, with average R² of 0.992, and reversed calculated residuals between -8.6% and 6.0%. Precision and accuracy for inter- and intra-day were determined to be ±17%. Following a complete validation, the method was applied to show utility using rat plasma samples that were intravenously dosed with oxyntomodulin.