Solid-phase microextraction as sample preparation method for metabolomics

Abstract

The main objective of the emerging field of metabolomics is the analysis of all small molecule metabolites present in a particular living system in order to provide better understanding of dynamic processes occurring in living systems. This type of studies is of interest in various fields including systems biology, medicine and drug discovery. The main requirements for sample preparation methods used in global metabolomic studies are lack of selectivity, incorporation of a metabolism quenching step and good reproducibility. The efficiency of metabolism quenching and stability of analytes in selected biofluid or tissue dictate how accurately the analytical results represent true metabolome composition at the time of sampling. However, complete quenching of metabolism is not easily accomplished, so sample preparation can significantly affect metabolome's composition and the quality of acquired metabolomics data. In this research, the feasibility of the use of solid-phase microextraction (SPME) in direct extraction mode for global metabolomic studies of biological fluids based on liquid chromatography-mass spectrometry (LC-MS) was investigated for the first time. 
\nInitial research presented in this thesis focused on resolving several outstanding issues regarding the use of SPME for the analysis of biological fluids. SPME was not simultaneously capable to provide high-sample throughput and high degree of automation when coupled to LC-MS. This was successfully addressed through the development and evaluation of a new robotic station based on a 96-well plate format and an array of 96 SPME fibres. The parallel format of extraction and desorption allowed increased sample throughput of >1000 samples/day which represents the highest throughput of any SPME technique to date. This exceeds sample throughput requirements for a typical metabolomics study whereby ~100 samples/day are processed. 
\nSPME can also be used for direct in vivo sampling of flowing blood of an animal without the need to isolate a defined sample volume. This format of SPME is particularly attractive for metabolomic studies as it decreases the overall number of steps and also eliminates the need for metabolism quenching step because only small molecular weight species are extracted by the device, whereas large biological macromolecules such as proteins are not extracted by the coating. In current work, in vivo SPME sampling was successfully applied for sampling of mice for the first time. The proposed sampling procedure was fully validated against traditional terminal and serial sampling approaches for a pharmacokinetic study of carbamazepine and its metabolite. Excellent agreement of pharmacokinetic parameters such as systemic clearance, steady-state volume of distribution and terminal half-life was found for all three methods, with no statistically significant differences (p>0.05). The performance of new prototype commercial SPME devices based on hypodermic needle was also evaluated within the context of the study. The availability of such single-use devices with excellent inter-fibre reproducibility (<10% RSD) presents an important step forward in order to gain wider acceptance of in vivo SPME sampling.
\nFinally, existing SPME coatings were not suitable for the simultaneous direct extraction of both hydrophilic and hydrophobic species, which is one of the requirements for a successful global metabolomics study. To address this issue, a systematic study of 40 types of commercially available sorbents was carried out using a metabolite standard test mixture spanning a wide molecular weight (80-777 Da) and polarity range (log P range of -5 to 7.4). The best performance for balanced extraction of species of varying polarity was achieved by (i) mixed-mode coating containing octadecyl or octyl group and benzenesulfonic acid ion exchange group, (ii) polar-enhanced polystyrene-divinylbenzene polymeric coatings and (iii) phenylboronic acid coatings. &#