posted on 2018-04-06, 08:12authored byJames W. Howard
The aim of this research was the development of mass spectrometry-based methodologies for the high-throughput quantitation of peptides in biological matrices.
Glucagon and GLP-1, which are of interest as biomarkers and in the development of therapeutics, were chosen as model peptides. Immunoassays that are traditionally used to quantify these often perform poorly; therefore, necessitating the development of alternative methodologies. Application of mass spectrometry-based methodologies to these analytes has, however, been limited, primarily due to sensitivity challenges, but also due to analytical challenges associated with their endogenous nature and instability in biological matrices.
Chapter 2 describes the development and qualification of the first liquid-chromatography coupled tandem mass spectrometry (LC-MS/MS) method for the quantitation of endogenous glucagon from human plasma. A novel 2D extraction procedure was developed to ensure robustness and sensitivity, whilst a novel surrogate matrix quantitation strategy took into account the endogenous nature of the analyte. A lower limit of quantitation (LLOQ) of 25 pg/mL was qualified, which was a considerable improvement over that previously reported in the literature (250 pg/mL) for a LC-MS/MS method. Clinical samples were cross-validated against a conventional radioimmunoassay (RIA), and similar pharmacokinetic (PK) profiles resulted, demonstrating that the methods were complementary.
In Chapter 2 glucagon instability in biological matrix was noted. To characterise this further, in Chapter 3 in vitro glucagon metabolites were identified using high-resolution mass spectrometry (HRMS). Metabolites observed by others (glucagon19-29, glucagon3 29 and [pGlu]3glucagon3 29) in alternative matrices were identified, alongside novel metabolites (glucagon20-29 and glucagon21-29). Cross-interference of these metabolites in immunoassays may help to explain their poor performance, whilst knowledge of metabolism may also aid the development of future stabilisation strategies.
The method developed in Chapter 2 was refined in Chapter 4 to improve sensitivity, robustness and throughput, and to add GLP-1 as a secondary analyte. The sensitivity achieved (glucagon: 15 pg/mL LLOQ, GLP-1: 25 pg/mL LLOQ) is the highest reported for both peptides for an extraction avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier transition. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between the methods. Differences between the immunoassay results exemplified the need to develop alternative methodologies. The resulting LC-MS/MS method is considered a viable alternative to immunoassays, for the quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1 in human plasma.
This work is made available according to the conditions of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Full details of this licence are available at: https://creativecommons.org/licenses/by-nc-nd/4.0/
Publication date
2018
Notes
A Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.