Metabolomic Analysis an new technology-------------

Metabolomic Analysis an new technology- what is that?

Metabolomic Analysis is a fairly new technology. Some scientist call it yust Metabolomics. Speaking about, it is the study of chemical processes that involve metabolites.

Metabolites are small molecules present in the blood, tissues and urine. Metabolomics is about the pertains to the study the unique chemical fingerprints left behind by cellular processes so as to understand the health /disease state of an organisms. Indeed, metabolomics is an bio analytical experimental technique. Its purpose is to elucidate better understanding in medicine, biology, environmental and food science

Metabolomic Analysis an new technology is defined as the quantitative measurement of all low molecular mass metabolites in an organism’s cell at a specific time under specific environmental conditions.

The metabolome represents the complete set of small metabolites. This are the intermediates and products of metabolism, found in a biological cell, tissue, organ or organism.

In general, for metabolomics a metabolite is usually defined as a molecule of less than 1 kDa in size.

What techniques are used in Metabolomic Analysis

Targeted Metabolomic Analysis:

The compounds in a given bio-fluid or tissue extract are identified and quantified by comparing the spectrum of interest to a library of reference spectra. This is generally used for the determination of a few specific known metabolites

Metabolomic Profiling:

Metabolomic Analysis an new technology. Indeed the technology involves a qualitative and quantitative determination of a particular class of metabolites or compounds from a specific metabolomic pathway

Metabolomic Fingerprinting:

This compares the pattern of metabolites and help to distinguish between samples based on the metabolomites characterised

Application Areas of Metabolomic Analysis an new technology

General

Today Metabolomics analysis is conducted world-wide in Universities, University hospitals Large public research organization and in the laboratories of very large multinational enterprises that are active in Biomarkers Discovery, Drug Discovery, Toxicology Testing, Nutrigenomics and Clinical & Preclinical Studies. Of particular interest are complex indication such as Oncology, Neurology and Cardiology

Clinical Metabolomics:

This is of interest to the medical science as it may lead to improvements in the diagnosis and treatment of human diseases. Finding unique paterns of metabolites could aid the identification of a target enzyme or protein (Biomarker) for the disease, resulting in faster drug development.

Drug Discovery

Important for drug target identification & validation and optimization & prioritization of diagnostic approaches for oncology research

Toxicology Metabolomics:

Profiling of urine and blood samples can detect physiological changes caused by toxic chemicals. This is used in the pharmaceutical industry for toxicity testing of potential drug candidates.

Nutrition Metabolomics

Can be used for physiological monitoring in food intervention or diet challenges studies. Included in Nutrigenomics
Food Metabolomics
Is used as an aid to developing high performing crop varieties, e. g. it provides biomarkers of flavour in tomatoes and other fruit and vegetables. It is also used in food quality testing and the detection of food adulteration

Environmental Metabolomic

Is used for used to study the interaction of organisms with the environment and has applications in the field of ecology and ecophysiology.

Separation and Detection Methods in Metabolomic Analysis an new technology

Separation methods

Gas chromatography, (GC) especially when interfaced with mass spectrometry (GC-MS), is one of the most widely used and powerful methods. It offers very high chromatographic resolution, but requires chemical derivatisation for many biomolecules: only volatile chemicals can be analysed without derivatisation. Some modern instruments allow ‘2D’ chromatography, using a short polar column after the main analytical column, which increases the resolution still further.

High performance liquid chromatography (HPLC).
Compared to GC, HPLC has lower chromatographic resolution, but it does have the advantage that a much wider range of analytes can potentially be measured.

Capillary electrophoresis (CE).
CE has a higher theoretical separation efficiency than HPLC, and is suitable for use with a wider range of metabolite classes than is GC. As for all electrophoretic techniques, it is most appropriate for charged analytes.

Detection methods

Nuclear Magnetic Resonance
To elucidate chemical structure of the target compound

Mass spectrometry (MS)
Is used to identify and to quantify metabolites after separation by GC, HPLC (LC-MS), or CE. GC-MS is the most ‘natural’ combination of the three, and was the first to be developed. In addition, mass spectral fingerprint libraries exist or can be developed that allow identification of a metabolite according to its fragmentation pattern. MS is both sensitive (although, particularly for HPLC-MS, sensitivity is more of an issue as it is affected by the charge on the metabolite, and can be subject to ion suppression artifacts) and can be very specific.

Direct injection MS
may be used to obtain metabolite mass profiles without any chromatographic separation. Using electrospray ionisation (ESI), mainly protonated or deprotonated ions are formed for each species, with very little fragmentation. A fingerprint spectrum of metabolites is obtained with metabolites being separated be accurate molecular masses

Surface Base Mass Analysis

Strategies in Metabolomic Analysis an new technology

Industry evolution

Metabolomics is a fairly young science that started around 2000.In the early phase of a new technology standard techniques are employed. Standard techniques include gas chromatography, high performance liquid chromatography (HPLC), ultra performance liquid chromatography, and capillary electrophoresis. HPLC holds the dominant share in the metabolomics market. The following detection technologies are being employed

Nuclear Magnetic Resonance, Mass Spectrometry and Surface Base Mass Analysis

As times goes by techniques need to be fine tuned and so special more intelligent equipment and tools emerge. Nobody can predict what type of equipment and tools emerge because application companies work together with their preferred partners under confidential condition. After a certain time the new technology is emerging and sold to other potential application companies. Some of the emerging technologies suddenly increase productivity manifold.

We have participated in the evolution and fine tuning of a number of advanced technologies together with partners. Partner selection in large research companies often starts at an early state starts by inviting the well known large system suppliers. They will make sure that the application company pays part of the technology development by relevant contracts. The application partner will also out source some research to Universities. Regularly a insider in the application lab evolve his own new technology in competition with the chosen large system supplier. In a few cases the insider is financed by his employer with the idea that if the new technology becomes a success the Application company is able to participate in the commercialisation of the new technology.

In the meantime some additional start up or service companies to offer additional ideas. The relation of large company collaborating with a small innovative enterprise is often a difficult construct since both have totally requirements.

Multivariate Analysis

Due to the complex nature of the metabolome, it is difficult to make a visual comparison of the large numbers of spectra and chromatograms. As a result, data analysis and interpretation is often accomplished using a chemometric approach, including using principle component analysis

Columns currently used

GC Columns you find here
RPC Columns you find here
HILIC Columns you find here

Speciality Columns

Innovative Tools for Metabolomics

Reference

Metabolomics, Amino Acids, and Polar Small Solutes

Underivatized amino acids and other small, polar solutes are poorly retained in reversed-phase HPLC but well-retained in HILIC. Volatile mobile phases can be used for HILIC-ESI-MS. HILIC-MS/MS permits the quantitative analysis of small polar solutes even in crude extracts such as seeds, leaves, and whole serum or plasma. Generally, the best material for this purpose is PolyHYDROXYETHYL Aspartamide™ with a pore diameter of 60- or 100-Å, with either 3- or 5-µm particle diameter. The 3-µm, 100-Å material has yielded especially good results.
Order PolyHYDROXYETHYL A™ now.

Metabolomics: The following two chromatograms show an extract of whole Arabidopsis leaf [Top], eluted with a decreasing ACN gradient from a 150×0.32-mm capillary of PolyHYDROXYETHYL A™ (item# 150.32HY0301) or [Bottom] from a reversed phase capillary. The order of elution is largely inverted.

Detection is via ESI-MS. Peaks correspond to such solutes as amino acids, anthocyanins, oligosaccharides, glucosinolates, glycolipids, etc., as shown.

Amino Acids: Most amino acids can be resolved using routine HILIC conditions [BELOW]

Alternatively, amino acids and other small solutes can conveniently be measured quantitatively with a PolyHYDROXYETHYL A™ column (100-Å) using HILIC-MS/MS under isocratic conditions. In his poster from ASMS ’99, Robert Croes (DuPont Biotech) demonstrated the measurement of amino acids in individual seeds. Analysis of the 20 common amino acids is conveniently performed with 70% MeOH in an ammonium formate buffer. If some metabolites of amino acids are also to be measured [BELOW], then 60% ACN is used in place of the MeOH. This reduces MS sensitivity but affords better chromatographic separation. This is important with some metabolite pairs (e.g., lysine and pipecolic acid) that interfere with each other’s detection in MS/MS.

Salt Concentrations and Extremely Polar Small Solutes: To obtain sharp, symmetrical peaks in HILIC for small polar solutes, the following salt levels are required in the mobile phase:

a) 10-15 mM: Typical polar solute, such as a neutral amino acid.
b) 40 mM: Solutes with a higher charge-to-mass ratio, such as
c) > 120 mM: Solutes with an extremely high charge, such as
ATP or aminoglycoside antibiotics.

Fortunately, ESI-MS can tolerate up to 400 mM ammonium acetate in the mobile phase.

A plausible explanation is that the retention of highly-charged solutes is strongly influenced by the polarity of their counterions. A high concentration of salt insures that all the charged groups have the same counterions. If this is not the case, then the peak can be skewed. In an extreme case, two well-separated peaks can be obtained for a single pure solute, corresponding to the same solute with different counterions, with a continuum between them corresponding to molecules that exchanged their original counterions for those in the mobile phase during their migration through the column. This rate of exchange is slow on the time scale of HPLC if the salt level in the mobile phase is too low.

Normally, HILIC of small solutes is best done with a column with a high surface area; typically, 60- or 100-Å pore diameter. However, with extremely polar solutes such as aminoglycoside antibiotics, use of a 1000-Å material with a low surface area facilitates elution using convenient mobile phases, as in the following example:

                   COLUMN: PolyHYDROXYETHYL A™ (Item # 204HY0510)
                   MOBILE PHASE: 250mM NH₄OAc, pH 4, with ACN
                   GRADIENT: 0-5′: 80-25% ACN; 5-15′: 25% ACN. FLOW: 1 ml/min

Small Solutes in Serum or Plasma: Addition of 2+ vol. of ACN to plasma or serum causes the precipitation of > 98% of the protein while leaving most small solutes in solution. The supernatant can be concentrated and analyzed via HILIC-MS/MS, often isocratically. This permits high-throughput analysis of picomolar levels of drugs, metabolites, etc. in serum or plasma. The following analysis of folates, methotrexate etc. is from S.D. Garbis et al., Anal. Chem. 73 (2001) 5358-64:

Literature General

http://www.polylc.com/downloads/Metabolomics_brochure_2004.pdf