CID detects triglyceride positional isomers

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  • Published: Nov 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: CID detects triglyceride positional isomers

New methods needed to distinguish triglyceride positional isomers

The exact position of particular fatty acids on the glycerol portion of triglycerides (whether on the central or outer carbons of glycerol, HOCH2-CHOH-CH2OH) is known to affect a number diseases. Current methods of distinguishing positional isomers of triglycerides, such as YYX or YXY (where X and Y are different fatty acid chains), are awkward and not generally applicable. Many of these isomers are either expensive or not commercially available, making research into new methods difficult. They are not typically separated by standard HPLC methods.

The Boston researchers decided to use mass spectrometry to distinguish between positional isomers. Low-energy collision-induced dissociation (CID) with an ion trap was employed; this technique involves fragmentation induced by collisions with neutral gas molecules, giving diglyceride ions when employed on triglycerides. It was possible to produce calibration curves of the relative ion intensity of the diglyceride fragments against the relative amounts of positional isomers.

CID MS used to identify lipid isomers

Derivatives of arachidonate, oleate, palmitate and linoleate of the forms YYX and YXY were synthesized by adding acid chlorides to a suitable mono- or diglyceride. Statistical mixtures of triglycerides were also produced by adding an excess of two acid chlorides, X-Cl and Y-Cl, to glycerol. In such mixtures, there was a 2:1 ratio of XXY to XYX and also the same ratio of YYX to YXY (along with some XXX and YYY).

Direct injection mass spectrometry was carried out using a syringe and capillary; the latter fed into the electrospray (ESI) source of a Thermo Fisher LCQ Advantage ion trap instrument through a metal sheath. The CID parameters were carefully optimised in order to obtain good conversion of triglyceride to diglyceride ions. Averages of at least 100 spectra were used in order to obtain high quality data. The authors stated that the use of direct injection allowed for the collection of more scans than would be the case with LC/MS, thus lessening potential errors.

Calibration plots were produced, showing the relative ion intensity of YY+ (diglyceride ions) against the relative composition of YYX. The latter was taken as 0 for YXY triglycerides (since no YY+ can form), 0.67 for the 2:1 mixtures of YYX : YXY and 1.00 for YYX triglycerides. Thus, three point calibration graphs were produced where Y was palmitate, oleate and arachidonate, X being linoleate in all three cases. These three graphs were all straight lines, so could be used for calibration, but since the slope of the arachidonate/ linoleate compounds was not very steep, it would be difficult to use it in practice. The highly unsaturated arachidonate triglycerides were not easily distinguished by this method. Two point calibration plots were constructed for 42 triglycerides; such a simplified method was considered to be satisfactory, since all the three point graphs gave straight lines, suggesting that only two points were required. The aim was to apply the calibration graphs to determine the positional isomer composition of triglycerides from a variety of sources after LC/MS.

Triglyceride positional isomers ratios determined by CID MS

The calibration graphs produced should be useful for determining the positional isomer ratios for triglycerides, with the exception of a few unsaturated chains, such as arachidonate. It would be interesting to see the method applied to natural triglycerides. The work was hampered by the lack of availability of many of the species examined, with mixtures being used in a number of cases. It should be possible to synthesise these triglycerides relatively cheaply by the effective use of modern synthetic techniques, such as protecting groups and selective reactions.

Related Links

Rapid Communications in Mass Spectrometry, 2017, 31, 1690-1698. Judge et al. A simple and economical strategy for obtaining calibration plots for relative quantification of positional isomers of YYX/YXY triglycerides using high-performance liquid chromatography/tandem mass spectrometry.

Lipids, 2004, 39, 273-284. Malone et al. Determining the relative amounts of positional isomers in complex mixtures of triglycerides using reversed-phase high-performance liquid chromatography-tandem mass spectrometry.

Wikipedia, Collision-Induced Dissociation

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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