The development and application of liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods for the quantification of the gut hormones peptide YY, glucagon-like peptide-1 and oxyntomodulin

Abstract

Our current understanding of gut hormones is limited by our inability to measure their concentration with sufficient specificity and sensitivity. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has already been applied to glucagon quantification and offers a potential solution. This PhD aimed to develop LC-MS/MS methods for the quantification of five peptide gut hormones: glucagon-like peptide-1 (GLP-1) 7-36 amide, GLP-1 9-36 amide, oxyntomodulin, peptide YY (PYY) 1-36 and PYY 3-36. The optimised extraction of the gut hormones from plasma comprised protein precipitation, followed by solid phase extraction using the Oasis MAX µElution plate. Sample extraction was customised to reduce protein and lipid interferents, with recoveries of 21-32%. Extraction was into a surrogate matrix of 20µg/mL bovine serum albumin, 20% acetic acid and 10% methanol developed to minimise non-specific adsorption. Stable-isotopically labelled internal standards for each gut hormone were used. The ultra performance liquid chromatography method on a Waters Peptide BEH C18 130Å column demonstrated good separation of the gut hormones from each other. The mobile phases were water (A), and acetonitrile (B), both with 0.1% formic acid. The flow rate was 0.35mL/min and the elution gradient 80-65% A over 9 minutes. A Waters Xevo TQ-S triple quadrupole mass spectrometer was used in multiple reaction monitoring mode. Ionisation was in positive mode using atmospheric pressure electrospray ionisation. Specific precursor and product ions were measured for each gut hormone and internal standard. Spiked plasma samples for calibration line standards and quality control samples were measured. The method was imprecise with coefficients of variation >15%. The method could not be validated. Non-specific adsorption of the peptide gut hormones occurred despite optimisation of container type, temperature, solvent and sample handling. Specificity was improved in comparison to immunoassay. Future work will concentrate on PYY species and aim to improve the sample extraction recovery and precision.