WARWICK DUNN: In the previous steps, we discussed Direct Infusion Mass Spectrometry. This is the simplest method to determine the metabolite composition of biological samples applying mass spectrometry. There are, however, other methods available. One of the most common to analyse biological samples involves a chromatographic process prior to detection of metabolites by a mass spectrometer. Chromatography provides the partial or complete separation of metabolites prior to analysis by a mass spectrometer, and there are a number of reasons why chromatography is employed, which we will discuss later. Chromatography operates by separation of metabolites in a chromatography column. Each column contains a stationary phase and a liquid or gas continuously flows through the column.
When a sample is injected on to the start of a column, the sample components absorb to the stationary phase at the start of the column. And after time, they will then desorb from the stationary phase and be driven further down the column where the components will again absorb on to and then desorb from the stationary phase. This process continues along the column for each component. The time each component is absorbed to stationary phase before it’s desorbed is dependent on chemistry, specifically the chemical composition and structure of the stationary phase and the chemical properties of the specific metabolite or metabolites.
Some metabolites will absorb more strongly to the stationary phase and stay absorbed longer, while other metabolites will absorb less strongly and will be absorbed for a shorter time or elute from the column much quicker. Normally, metabolites with a similar chemical structure to stationary phase will absorb more strongly, simply like prefers like. The process of absorption and desorption controls the time taken for metabolite to pass through the column and provides separation of metabolites. Metabolites with a greater diversity in structure and composition will be separated more than metabolites with a similar structure and composition. However, even metabolites with a similar structure can be separated with an appropriately developed instrument method.
There are two types of chromatography commonly applied in metabolomics– gas chromatography, which uses a flow of helium or nitrogen gas to push metabolites through the column, and liquid chromatography, which uses a liquid solvent to push metabolites through the column. So as an example, how does liquid chromatography-mass spectrometry operate? The chromatography column them is normally a hollow, stainless steel tube with a diameter of 1 to 4 millimetres and a length of 5 to 25 centimetres. The column is packed with small spherical particles, normally of diameters between 2 and 5 microns, onto which the stationary phase is chemically bound. A liquid mobile phase flows through the column at a flow rate of between 0.1 and 1 millilitre per minute.
The liquid flow exiting the end of the column is introduced to the mass spectrometer, typically to an electrospray ionisation source, as was described for Direct Infusion Mass Spectrometry. When the eluent is introduced into the mass spectrometer, ions are created in the electrospray ionisation source operating at atmospheric pressure and are then extracted by electrical potentials into the vacuum region of the mass spectrometer. Ions are separated according into the mass-to-charge ratio and detected. Normally the mass spectrometer collects data on specific mass-to-charge ratios in a targeted assay, or cross a wide mass-to-charge ratio range for untargeted studies.
This creates a mass spectrum of mass-to-charge versus ion intensity for each scan of the mass-to-charge ratio range. Multiple mass spectra are collected each second, and hundreds to thousands of mass spectra are collected across a 15 minute analysis time. This provides a three-dimensional data set of time versus mass-to-charge ratio versus response. The methods that we use to process this complex data will be discussed next week. The time between injection of a sample and detection of a metabolite is called the retention time. Metabolites which absorb more strongly to the stationary phase have larger retention times than metabolites which only weakly absorbed to the stationary phase.
In liquid chromatography, there are two commonly used stationary phase types– reversed phase and Hydrophilic Interaction liquid chromatography. Reversed phase liquid chromatography applies long alkyl chains as the stationary phase, normally chains containing 8 or 18 carbons. These stationary phases will absorb metabolites soluble in organic solvents such as chloroform, whereas metabolites that are soluble in water will be poorly absorbed to the column and elute in the liquid phase first. So we would apply this technique to study lipids such as fatty acids and cholesterol in preference to water-soluble metabolites such as carbohydrates. The other common stationary phase is called Hydrophilic Interaction Liquid Chromatography, or abbreviated to HILIC. The technique is complementary to reversed phase liquid chromatography.
The stationary phase in HILIC is water absorbed onto a hydrophilic silica-containing surface. This provides a stable water layer which metabolites can be absorbed into and then released. This allows water-soluble metabolites to be retained more strongly than lipids, and so is applied to study water-soluble metabolites. So here we have two complementary liquid chromatography techniques to study a wide diversity of metabolites, both water-soluble metabolites such as sugars, and also lipid metabolites like fatty acids. The application of liquid chromatography provides a number of advantages. Biological samples can be very complex, composed of hundreds of thousands of different metabolites. Sometimes the separation of metabolites before they enter the mass spectrometer is a preferred option.
This approach can provide the separation of metabolites that have the same mass, and therefore in direct infusion mass spectrometry would be detected as a single signal, but if separated, can be detected as two separate signals. For example, the two amino acids leucine and isoleucine have the same mass, and without chromatographic separation, will be detected as a single signal in a mass spectrometer. The complexity of samples is also reduced when coupled to liquid chromatography. The sample entering the mass spectrometer is fractionated into a larger number of chemically less complex samples, and the data collected for each of the fractions is simpler.
The introduction of a very complex sample can cause ionisation suppression, where the ionisation of a metabolite present at high concentration reduces ionisation efficiency of metabolites present at low concentrations. This reduces the sensitivity of the method and decreases the number of metabolites detected. Separation applied liquid chromatography reduces ionisation suppression and so increases the number of metabolites detected. Finally, the retention of a metabolite, or the retention time, can also provide information on the metabolite for its chemical identification. We’ll explain the importance of this next week.