At most of the primary metabolites are water soluble and ionic, capillary electrophoresis (CE) provides a very effective analytical tool for metabolomics. Advantages of CE over LC and GC as an analytical tool include;
Therefore, capillary electrophoresis mass spectrometry (CE-MS), is a very powerful measurement method in cases requiring high quantitative and qualitative performance, capable of building major metabolic pathway maps and discovering biomarkers.
Capillary electrophoresis is called free zone electrophoresis because it uses uncoated hollow glass fused capillary tubes with an inner diameter of 50 to 100 μm, filled with only an electrolyte solution. An electric field is generated by applying a voltage to both ends of the capillary, the direction and rate of migration of metabolite ions depend on physical properties of each metabolite. Concurrent separation of hundreds to thousands of metabolites present in assay samples is achieved through differences in mobility of ions. The rate of migration of such ions in an electric field is expressed by the following equation:
where υ is the rate of migration of ions (cm/s), μe is the ion‐specific electrophoretic mobility (cm2/Vs), and E is the strength of the electric field (V/cm). The electric field is a function of the voltage applied and the length of the capillary. The mobility is the ion specific constant. As the mobility is determined by the electric force acting on molecules, which, in turn, balances the friction force on ions (spheres) that migrate in the solvent, the mobility is expressed by the following equation:
where q is the ionic charge, η is the viscosity of the solution, γ is the ionic radius, and υ is the rate of migration of ions. The second equation shows that the mobility of a substance is defined by the ionic charge (almost equivalent to the dissociation in electrolyte solution) and the ionic radius (almost equivalent to the size of the conformation of a molecule). In other words, the higher charge and the smaller ionic radius substances have, the greater mobility they show, and the lower charge and the larger ionic radius substances have the less mobility they show. The ionic charge q is dependent on the pH of electrolytes, and basic functional groups within a molecule.
One of the important factors determining the separation capacity of capillary electrophoresis is electroosmotic flow (EOF), a phenomenon where the electrolyte solution itself flows inside the capillary. This flow of the electrolyte solution is the main driving force that pushes samples out of the detector side of the capillary. A fused-silica capillary is generally used for capillary electrophoresis, and surface charges of silanol groups (SiOH) present on the inner wall of the capillary play an important role in the formation of EOF. The silanol groups on the capillary inner wall are ionized (SiO-) and excessively negatively charged in an electrolyte solution. Opposing ions in the electrolyte solution (mainly positively charged ions) are attracted to the inner wall surface to achieve a balance of electric charges, resulting in the formation of a double-layer with ionized silanol groups. Under these conditions, a potential difference is created very close to the inner wall. The application of a voltage to both ends of the capillary attracts the positively charged ions of the diffuse double-layer to an anode. In contrast, the silanol groups cannot move due to the fixation on the wall surface and the entire electrolyte solution in the capillary is directed toward the anode with the migration of the positively charged ions, thereby generating a flow.
The great characteristic of EOF generated in a capillary is its flat profile shown on the tip of the flow. An even distribution of the driving force of the flow on the capillary inner wall prevents a pressure drop in the capillary, resulting in the same speed of the flow near the inner wall and at the center of the capillary. This flat flow profile helps to prevent the diffusion of metabolites and provides the number of theoretical plates as large as 105 to 106 /m by creating sharp signal peaks.
The high resolution ability of CE enhances the separation of compounds, key to the identification and annotation of metabolites from a metabolome pool comprising of thousands of chemical compounds. There are a lot of metabolites with similar chemical structure and physical properties, with significantly different biological functions in biological systems. For example, Pyruvate and Lactate, are important intermediates and end-product of glycolysis, the CE separation and identification of these metabolites is an essential process for pathway understanding. CE can separate many other isomeric compounds, for example, Glucose-6-phosphate and Fructose-6-phosphate, which have the same chemical formulae and molecular weights, are not be resolved by LC-MS, but can be separated and quantitated by CE-MS. The employment of CE-MS in metabolomics provides higher resolution profiling of many metabolic intermediates.
Together with high separation ability, the low sample volume in CE-MS enables accurate quantitation for a variety of metabolites. The low injection volume of samples effectively reduces the matrix effect, which is caused by a variety of ionic compounds including salt, protein and RNA derived from biological samples. The reduction of the matrix effect is important for not only the separation of peaks in CE, but also quantitation capabilities. With the injection of a sample into a MS, a composition of salt, solvent, or too many metabolites can cause ion suppression resulting in insufficient ionization of target compounds. Ion suppression will lower the intensity of detection peaks, and decrease the performance of quantitation especially for high- and low- concentration metabolites in the sample. Because metabolites exist with a large range of concentrations, a wide range of quantitation linearity is required for precise measurement for biomarker screening, relative comparisons and quantitative measurements. The CE-MS requires ~50 nano-L injection of sample solution, and therefore has a lower ion suppression and higher peak intensity compared with conventional methods such as LC-MS and GC-MS.
Because CE is suitable for the separation of ionic and hydrophilic compounds, CE-MS based metabolomics has advantages in the measurement of compounds including organic acids, amino acids, phosphorylated sugars, nucleotides etc. Clinical samples such as blood, urine and CSF includes many hydrophilic compounds secreted from cells, their profiling essential for biomarker discovery, disease progression or treatment effects. On the other hand, CE-MS is not as useful for the measurement of large neutral metabolites such as long chain fatty acids and lipids. The profiling of these hydrophobic compounds require the use of reverse phase HPLC – MS.
Based on over 10 year’s operation, we have established SOPs and management systems for CE-MS analysis. All instruments pass strict criteria for each analysis, and quality control is performed for all samples by using several internal standards to check extraction efficiencies and normalize against migration time and m/z drift. The obtained high quality data can be used for high value studies such as for clinical trial and pharmaceutical development.