The Role of Automated Sample Preparation in Metabolomics
Precise replication of sample preparation steps in metabolomics is critical in obtaining analytically precise results to determine true, often small differences between the treated and control samples.
Sample preparation consists of:
- Sampling a representative portion of the sample (blood, cell culture, plant, soil, headspace).
- Stabilising the sample using cryogenics, freeze-drying or buffering.
- Extracting the sample.
- Analysing it.
Depending on the analysis done, filtration, centrifugation, fractionation, pre-concentration and derivatization steps may also be required.
Ultimately precision is dependent upon sample preparation being as simple as possible, robust and being able to maintain the integrity of the sample; Automation really helps by eliminating human errors and variations in technique. If on-line automation is used, then samples can be prepared individually in a just-in-time fashion. In this way, every sample is guaranteed to be treated in exactly the same way.
The GERSTEL MultiPurpose Sampler (MPS) and MAESTRO software have advanced PrepAhead functions that facilitate this way of working.
The GERSTEL MPS can be used for on-line and off-line automation of sample preparation for both GC-MS and LC-MS.
Derivatization is commonly used to make involatile analytes amenable to gas chromatographic analysis. It can also be used to selectively derivatise functional groups for the selective isolation of specific metabolites or to assist in compound identification.
The most commonly used derivatization in metabolomics is the two step methyloximation/silylation reaction. This reaction is non-selective, reacting with a wide range of functional groups (thiols, alcohols, carboxylic acids, aldehydes, amines, keto-acids) and makes it possible to capture a profile of the polar metabolome.
Derivatization is really useful, but does have its limitations.
The derivatives of some metabolites are unstable, inter-conversion of metabolites on derivatization can occur and mixed reaction products are formed for some metabolites. These limitations are not a problem if they are well managed, by carrying out reactions at optimal temperatures and by precisely controlling reaction temperatures and times; again, automated sample preparation helps to obtain precise results and preparing samples just-in-time, eliminates imprecision due to sample instability.
So what is Metabolomics?
Metabolomics analyses measure metabolites produced by all kinds of organism in order to determine the effect of an input (mutation, treatment, stress, drug, disease) to a metabolic system on the metabolic pathways within that system.
Two distinct approaches exist:
- Targeted metabolic profiling, where a subset of metabolites (e.g., fatty acids) are measured.
- Metabolomics where as many metabolites as possible are determined simultaneously.
A variety of analytical techniques are used, not just GC and LC-MS but also nuclear magnetic resonance (NMR) and flow injection analysis-mass spectrometry (FIA-MS or DI-MS).
Metabolomics analyses compare treatment and control samples and use powerful statistical techniques to identify and classify the differences between samples.
Good quality (precise) data is essential for these techniques to work.
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