The Synthesis and Characterisation of Reactive MALDI Matrices for Mass Spectrometry Imaging of Steroids

Abstract

Matrix assisted laser desorption ionisation (MALDI) mass spectrometry imaging (MSI) is an analytical technique for molecular spatial distribution imaging which presents unique advantages over existing 3D imagine techniques and offers novel perspectives for understanding the spatial distribution of biological processes. It is a well established technique in the analysis of large molecules however, small molecule analysis has been challenging due to matrix ion spectral interference (Chacon et al. 2011). Another obstacle for many endogenous steroid molecules is their poor ability to ionise. The purpose of this study was to design, synthesise and characterise novel matrices that would react with target functional groups on endogenous steroids. This study was limited to MALDI MS 2D analysis of the matrices to attain function and behaviour data, matrices were not tested directly on tissue samples.

The matrices designed in this project all have three essential components: 1) the reactive moiety, for steroids this is a carboxylic acid that can react with a hydroxyl or ketone on the target molecule, for vitamin D the reactive component was a triazolinedione which can react, in a Diels Alder reaction with the diene on the target molecule; 2) the absorbing core, a conjugated pi system that can absorb the laser energy and 3) a basic functional group for enhanced ionisation efficiency. A library of matrices targeting steroids was developed, this was aimed at optimising the molar absorption coefficient at the laser wavelength of 337 nm and enhancing ionisation efficiency.

Seven matrices designed to react with hydroxyl and ketone containing steroids were successfully synthesised and characterised, these matrices were involved in a simulation reaction in solution with an alcohol and a coupling agent in basic conditions. The reaction was directed at activating the carboxylic acid to form an ester in an addition reaction with the alcohol. However, the esterification did not take place. Investigations towards synthesising a reactive matrix targeted to analyse vitamin D metabolites were undertaken. A variety of synthetic pathways were investigated and great strides in the groundwork towards finalising this matrix have been made. However further efforts to synthesise the stable matrix are required.

This thesis reports the design, synthesis and characterisation of a number of novel reactive MALDI matrices, optimisation analysis and activation reaction assays of the matrices. Although further work is required in developing the correct activation conditions for these matrices the development of a library of novel reactive matrices that can significantly enhance ionisation of small molecules in one application step is integral to overcoming adversities which hinder the use of MALDI MSI’s for small molecule analysis.