2. Analysis of Metabolites

Stationary phase, mobile phase, different polarities

Analytes (components of the mixture) have different affinities (depends on size, charge, etc. physical-chemical properties) to the stationary phase and therefore different retention times —> separation of analytes.

Analyte: compound(s) of interest, substance mixture

Eluent: mobile phase (liquid/gas)

Whole lipids cannot be analyzed by GC/MS because they are often not volatile + bigger than 600 kDa

Transmethylation (cleaving fatty acid from head group of the lipid and esterifying with methyl group instead): enhances stability and thermostability of the compound, helps detection (increased sensitivity), increases volatility.

DMOX: helps determining location of double bonds (promotes equal fragmentation of fatty acids, DBs are located where the difference between two peaks is 12 instead of 14).

Trimethylsinylation: important to make polar compounds suitable for GC-MS (decreases polarity, increases volatility).

Mobile phase: inert carrier gas (N2, Ar, He)

Stationary phase: long thin column coated with silica gel

Advantages: retention times are reproducible across labs (when working with a column of the same size and coating), no packing problems as in LC, high resolution.

Disadvantage: compounds must be thermostable, mostly unpolar and smaller than 600 kDa.

For non-targeted approaches, LC is more suitable because it can handle compounds bigger than 600 kDa.

One of the main advantages of GC is the retention time stability.

FID (flame ionization detection)

MS (mass spectrometry)

After separation, a compound is burned in a hydrogen flame, leading to the formation of ions. They are detected by electrodes and the signal is amplified. (The resulting current is proportional to the number of C atoms.) To characterize a compound, you need a standard to compare your results to. FID does not provide structural information like MS or NMR, but is much more sensitive and suitable for quantification (FID: accurate over seven orders of magnitude, MS: only 5 orders of magnitude).

High-Performance Liquid Chromatography

HPLC uses high backpressure to pump analytes/mobile phase through column, the column is packed with silica (in normal-phase HPLC). HPLC has very high chromatographic resolution and uses particles of sizes around 3.5µm (UHPLC: 1.7µm, even more precise signal).

“Shorter”/”steeper” gradient of the mobile phase leads to shorter retention times.

Tight, precise packing leads to higher separation.

Difference: PRESSURE (compared to GC and other LCs), liquid mobile phase (compared to GC), suitable for a broader range of compounds (compared to GC which has restrictions in size and polarity), lower temperature needed (than GC).

Normal: unmodified silica (polar) as stationary phase, solvent gradient from unpolar to polar.

Reverse-phase: unpolar stationary phase (e.g., silica esterified with acyl groups), mobile phase starts with polar solvent.

Normal phase: most unpolar

Reversed phase: most polar

Mobile phase: inert gas for GC, solvents for LC

Stationary phase: GC columns are coated with silica, LC columns are packed with silica

Gradient: GC uses a temperature gradient, LC a solvent gradient for elution.

Ion source: creates detectable ions (in the gas phase!) from formerly uncharged molecules, potentially fragmentation (depending on ionization technique)

Mass analyzer: separation of ions based on their m/z value.

Detector: detection of ions and their m/z value (duh)

EI and ESI:

EI is combined with GC

-        EI produces M+ radicals

ESI is combined with LC (you need a liquid to produce the ions)

-        ESI produces mostly adducts à complicated analysis for unknown compounds

-        ESI can be used in positive or negative mode, ions are either [M+H]+, [M+Na]+,… or  [M-H+]-, [M+formate]-, [M+acetate]- …

-        Some compounds can be ionized in both modes, some only in one – therefore, it is important to do both modes to detect everything (in non-targeted approach)

EI (electron ray shot at molecule) causes fragmentation, ESI does not. ESI produces adducts, EI does not.

Total ion chromatogram: all counts for all time points for all m/z

Extracted ion chromatogram: counts for all time points, but only for one m/z

Mass spectrum: all m/z for one time point

Possible exam question: Several mass spectra of different time points are given, TIC and EIC must be developed from the mass spectra (addition of counts per minute for each peak and time point for TIC, addition of counts per minute for one m/z for each time point).

A QqQ consists of two mass analyzers with a collision cell in between. Both mass analyzers can operate in either scan or select mode; a scan provides separation of all analytes according to their m/z, select results in the selection of a compound of one specific m/z. In the collision cell, the compound(s) is/are fragmented.

Depending on how scan/select modes of the two mass analyzers are combined, different kinds of analyses can be done:

1)      The m/z of a compound of interest is known but its structure is unknown. Product ion scan can be used to deduce the structure (e.g. which fatty acids are part of a specific lipid?). It can also be used to generate tables for 4).

2)      Precursor ion scan can be used to find compounds with a specific precursor in different kinds of metabolites, for example all lipids containing PC. In Q1, the different metabolites are separated, Q2 leads to fragmentation of the metabolites (in this example, leading to PC fragments), Q3 detects the fragments of interest (PC). Since Q1 and Q3 are interconnected, it is possible to find out which m/z values resulting from Q1 exhibit a PC peak in Q3.

3)      Similar research question as in 2) (?), but different approach: The fragment of interest is indicative for a specific lipid class (e.g., PC). To obtain knowledge about how many precursors (e.g., PCs) are in the mixture, Q1 is in scan mode. In Q3, the loss of a specific fragment is searched for, not the fragment itself. Afterwards, you can deduce which of the scanned compounds in Q1 showed the specific fragment you are interested in.

4)      Scenario: You have a mixture of lipids and want to find out what is in it. You need a reference table with masses of the lipids you want to detect (!). For every lipid, you run the SRM, selecting for a certain m/z and confirming the identity with the select run. You are “blind” for all the lipids that are not in your table. Anyway, this is a very fast and sensitive method.

ESI-TOF-MS principle:

• ESI is a soft ionization technique that does not cause fragmentation.

• TOF (time of flight) is a separation technique that involves a long tube in which the ions are separated by the time they require to pass through (the longer the tube the better the resolution).

• MS: mass analysis

Benefits:

• High resolution and sensitivity

• High mass accuracy

• Determines wide mass range

Limitations:

• Complex samples: difficult to identify individual components

• Expensive

• Detector saturation for too high concentrations

Total ion chromatogram

Extracted ion chromatograms