Elucidation of a highly complex fatty acid sample (cod liver oil) by using cryogenically-modulated comprehensive 2D GC-MS


Fatty acids (FAs), either of vegetable, land or marine animal origin, usually consist of straight-chain, saturated or unsaturated, even-carbon-number carboxylic acids. Generally, chain lengths do not exceed C24, while double bonds (DBs) are in the cis configuration. It is a well-known fact that odd-numbered FAs are generated by many microorganisms and are synthesized, in a limited manner, in animal tissues, such as fish oils.
Single-column GC has been the most common method used for the determination of FA profiles. To analyse these lipidic constituents, it is necessary to transform them into more-volatile and less polar analytes, such as methyl esters. There is a widespread use of 25–30 m columns, with a polyethylene glycol-type stationary phase, for the separation of the most important saturated and unsaturated FAs. However, it will be demonstrated that single column GC can fall short in the full unveiling even of simple lipid profiles.
The present investigation is focused on the use of cryogenically-modulated comprehensive 2D GC (GC×GC), combined with a rapid-scanning quadrupole mass spectrometer (qMS), for the elucidation of a highly complex fatty acid sample, namely cod liver oil.


Sample and sample preparation

The cod liver oil was purchased in a chemist located in Messina (Italy).
The sample was stored at 4°C prior to analysis.The fatty acid methyl esters (FAMEs) were prepared as follows: 1 mL of a 10% v/v solution of concentrated H2SO4 in MeOH was added to 1 mL of oil, then heated at 110°C for 2 h in a closed vial. After cooling, the FAME phase was separated from the acidic solution.


The GC×GC-qMS application was performed on a system consisting of two independent Shimadzu GC2010 gas chromatographs, and a Shimadzu QP-2010 Ultra quadrupole mass spectrometer. The two GC ovens were linked through a heated transfer line. The primary GC was equipped with an AOC-20i auto-injector and a split-splitless injector (310°C).
The primary column was an SLB-5ms [(silphenylene polymer, practically equivalent in polarity to poly(5% diphenyl/95% methylsiloxane)] of dimensions 30 m × 0.25 mm ID × 0.25 μm df; the secondary column was a Supelcowax-10 (polyethylene glycol) of dimensions 1 m × 0.10 mm ID × 0.10 μm df.
Thermal modulation was performed by using a loop-type modulator.
Data were acquired using the GCMSsolution software. Bidimensional chromatograms were generated by using the ChromSquare software v. 2.2. MS database matching was performed by using the “FAMEs Fatty Acid Methyl Esters: Mass Spectral Database”.

Operational parameters

Both columns were subjected to the same temperature program 180°C-280°C (10 min) at 3°C/min
Injection volume and mode 0.8 μL; split (150:1). Helium was used as carrier gas at constant linear velocity with an initial head pressure of 102.3 kPa
Quadrupole MS conditions Ionization mode: electron ionization (70 eV)
Interface and ion source temperatures 250 and 200°C, respectively
Mass range 40-330 m/z; scan frequency: 50 Hz.

Results and discussion

In general, the main limitations that can be encountered in the conventional GC-MS analysis of complex lipid samples are: I) if a GC-isolated FAME is introduced into the MS ion source then the identification of positional isomers can be problematic: e.g., the mass spectra of C18:3ω6 and C18:3ω3 are very similar; II) another limitation, related to the GC step, is that the number of FAMEs contained in these sample types exceeds the resolving power of a conventional polar column; III) a further issue is that many trace amount constituents, on a conventional capillary, reach the detector at very low concentrations and remain undetected. Such drawbacks can be almost entirely avoided through the use of GC×GC-qMS.

The GC×GC-qMS chromatogram of a sample of cod liver oil FAMEs is shown in Figure 1; apart from the extreme complexity, it can be observed that the FAMEs are situated within a diagonal band that widens gradually across the chromatogram.
Considering one of the advantages of GC×GC, specifically the formation of group-type patterns of homologous series of compounds, it can be seen that: 1) methyl esters are grouped in specific locations of the space plane in relation to their carbon number (the C14-C24 zones are indicated by arrows); II) FAMEs tend to align themselves along distinct bands on the basis of their double-bond (DB) content, specifically, seven bands in the DB0-6 range; 3) ω-defined methyl esters (e.g., ω3: C20:5-C20:4-C20:3) are located along descending diagonal bands. Overall, 82 compounds were identified through MS database matching and specific 2D locations (Table 1).
For example, it was rather simple to identify peak 47 as C18:2ω3 because it was located at the intersection point of the DB2 band and of the ω3 diagonal.
Finally, with regard to another GC×GC advantage, namely re-concentration through cryogenic modulation, several trace-amount FAMEs were determined. A series of unexpected, odd-numbered saturated (i.e., C9:0, C11:0, anteiso C19:0, etc.) and unsaturated (i.e., C19:4ω6, C21:4ω6, C23:3, etc.) fatty acids were determined at very low concentrations

Figure 1

Table 1. Identification of cod liver oil FAMEs. Abbreviations: i = iso; a = anteiso.