A key aim of using a metabolomics approach is to obtain (ultimately) an answer to a well-defined biological question. To this end, state-of-the-art analytical separation techniques are currently used for the reliable profiling of (endogenous) metabolites in a wide range of biological samples. Within metabolomics, capillary electrophoresis–mass spectrometry (CE-MS) has become a very strong analytical tool for the selective profiling of polar and charged metabolites. In CE, compounds are separated according to their charge-to-size ratio and, therefore, the separation mechanism of this approach is fundamentally different from chromatographic-based separation techniques. As such, CE-MS provides complementary information on the metabolic composition of biological samples. In this chapter, the utility of CE and CE-MS for the analysis of (highly) polar and charged metabolites is described from a historical perspective. Attention is devoted to some research works from a few decades ago in which the value of CE for the selective analysis of a few (endogenous) metabolites in human body fluids was demonstrated. Then, seminal research works on the development of CE-MS methods for targeted and non-targeted metabolomics studies are discussed. Finally, the current situation of CE-MS in metabolomics is considered and a view on where this approach may head to is provided.
1.1Introduction
One of the major goals of using a metabolomics approach is to obtain an answer to a specific biological/clinical question.1 For this purpose, advanced analytical separation techniques are employed for the reliable profiling of (endogenous) metabolites in various biological samples. The most recent version of the Human Metabolome Database (HMDB version 4.0) contains more than 100 000 metabolite entries including both water-soluble and lipid-soluble metabolites, as well as metabolites that would be considered as either abundant (>1 ”M) or low-abundant (<1 nM).2 This number of metabolites and their concentration range clearly indicate that various analytical separation techniques with complementary separation mechanisms, like nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) hyphenated to gas chromatography (GC), liquid chromatography (LC) and/or capillary electrophoresis (CE), are needed in order to analyze a wide range of (endogenous) metabolites in a given biological sample.3 For example, for the characterization of (endogenous) metabolites in human serum, a number of analytical separation techniques with different separation mechanisms have been used, and this strategy allowed the detection of more than 4000 metabolites at concentration levels from the low nM- to the high mM-range.4
Within the metabolomics field, two analytical strategies can be distinguished, i.e. the non-targeted approach, also known as global metabolic profiling, and the targeted approach.5â8 In non-targeted metabolomics, the aim is to analyze as many (endogenous) metabolites as possible in a given biological sample without having a priori knowledge on the nature and identity of the measured compounds. In targeted metabolomics, the analysis is often focused on the generation of quantitative data for pre-selected metabolites. Both non-targeted and targeted approaches can be employed within a single metabolomics study, where the first approach is generally used for the screening of differential metabolites (or metabolic indicators) for diseases. In the second approach, the statistically relevant differential metabolites may then be quantified and confirmed, preferably employing standardized operating procedures.9 Reproducibility and validation of confirmed differential metabolites for a certain disease require further assessment in large sample cohorts, preferably at multiple sites, in order to determine whether these compounds can be used as biomarkers.10
At present, the analytical techniques most often used for global metabolic profiling studies are 1H NMR spectroscopy, reversed-phase ultra performance (RP-UP)LC-MS and GC-MS.11â16 Notably, RP-UPLC-MS methods using columns with sub-2 ”m porous particles or coreâshell silica particles have received a lot of attention for the efficient and fast profiling of metabolites in complex samples.17â19 NMR spectroscopy can still be considered the most robust and reliable analytical tool for high-throughput and reproducible metabolic profiling of body fluids (such as for example serum and urine) using minimal sample pretreatment. Typically, this approach provides quantitative data for metabolites present in the ”M to mM concentration range employing a data acquisition time of 5 min per sample.11 However, evaluation of recorded NMR data may be considered as a challenging task due to the complex spectra and superimposition of signals at certain chemical shift regions. GC-MS has a well-known track record for the profiling of (endogenous) metabolites in various body fluids in a clinical setting due to its high separation efficiency and sensitivity.20,21 Also, the reliable identification of compounds by GC-MS is a very strong asset for metabolomics studies. However, this approach is not suitable for the analysis of non-volatile, thermolabile, and/or highly polar compounds and, therefore, derivatization is usually needed to yield volatile and thermostable compounds. Derivatization is generally a time-consuming procedure, thereby potentially limiting the high-throughput capacity of GC-MS for metabolic profiling of large sets of clinical samples.
Both LC-MS and CE-MS can be used for the analysis of polar and non-volatile metabolites in biological samples without using derivatization and laborious sample pretreatment procedures. As indicated above, RP-UPLC-MS has become a key technique for global metabolic profiling studies.22â24 A broad array of metabolites can be analyzed by RP-LC approaches; however, the hydrophobic stationary phases generally used do not provide sufficient retention and selectivity for (highly) polar and charged metabolites. To enable the analysis of such compounds by RP-LC-MS, ion-pairing agents like tributylamine or hexylamine can be added to the mobile phase.25â28 However, the use of ion-pair agents in RPLC-MS may result in severe ion suppression and it may contaminate the ion source and ion optics. Furthermore, stability and re-equilibration of the column prior to the next injection may also be considered an issue in ion-pair RP-LC-MS.8 An emerging liquid chromatographic separation tool for metabolomics is hydrophilic interaction LC (HILIC), in which a polar stationary phase is used in combination with aqueous organic eluents. This approach is increasingly employed in combination with RP-LC-MS for global metabolic profiling studies.29
In this chapter, attention will be devoted to the potential of CE and especially CE-MS for the profiling of (endogenous) metabolites in biological samples. In comparison to chromatographic-based separation techniques, CE-MS is not as often applied in bioanalysis and metabolomics. However, this approach has some unique analytical characteristics for metabolomics studies.30,31 In CE, referring in this context to capillary zone electrophoresis (CZE), compounds are separated on the basis of differences in their charge-to-size ratio. Therefore, the separation mechanism of CE is fundamentally different from that of chromatographic-based separation techniques. An important feature of CE is the intrinsically high separation efficiency as a result of the flat flow profile of the electro-osmotic flow (EOF), making this technique well-suited for the selective analysis of polar and charged metabolites in biological samples. Moreover, the main source contributing to band broadening is longitudinal diffusion as there is no mass transfer in CE separations. As only small sample volumes are needed (âŒ10â50 nL), CE is highly suited for the analysis of size-limited biological samples, thereby enabling volume-restricted metabolomics studies.
Concerning the analysis of polar and charged metabolites, both CE and HILIC may be considered as useful analytical tools for this purpose. However, small injection volumes are typically used in CE, resulting in compromised concentration sensitivities for global metabolic profiling studies by CE-MS. The concentration sensitivity of CE can be improved by the use of electrokinetic- or chromatographic-based preconcentration techniques, as outlined in Chapters 5 and 6, respectively. Though both CE and HILIC are useful analytical separation techniques for the analysis of polar and ionic metabolites, recent studies have indicated a significant degree of orthogonality between HILIC and CE for metabolomics.32,33 A comparison of CE-MS with other analytical separation techniques for metabolomics is provided in Chapter 8.
Here, CE and CE-MS approaches developed for metabolite analysis and global metabolic profiling are discussed. The chapter starts by providing a summary of the various CE separation modes used in CE and CE-MS for metabolomics. Special attention will be devoted to research work that eventually resulted in the development of CE-MS systems for global metabolic profiling of biological samples. Finally, the current situation of CE-MS in metabolomics is considered and some thoughts on where this approach may head to is provided.