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

Abstract

While the presence of multiple unsaturated fatty acids tends to slightly decrease the slopes of the calibration plots, the data suggest that the sensitivities are sufficient for performing positional analysis of most triglyceride systems. However, the presence of unsaturated fatty acids that contain double bonds close to the carbonyl group, such as arachidonic acid, tends to dramatically decrease positional sensitivity.

Impact of the complexing cation on the sensitivity of collision‐induced dissociation spectra to fatty acid position for a set of YXY/YYX‐type triglycerides

“…In addition, the presence of fatty acids that contain double bonds relatively close to the carbonyl (4 th , 5 th and 6 th position) dramatically degrades the sensitivity of the CID spectra to fatty acid position, making positional analysis by this method impractical for many of these TAG systems. We refer to this phenomenon as “the arachidonic acid effect”, as we have seen this consistently with TAGs containing arachidonic acid in our previous publications . While the positional sensitivity issues with some of the ammonium adducts limit its utility significantly, the large variability in the relative intensities of the DAG product ions in the CID spectra of the ammoniated adducts has also impeded progress on the development of an accurate model for predicting the CID spectra of positionally pure TAG species.…”

“…The relative compositions of the samples were taken to be 0.00 for the samples from the YXY synthesis and 1.00 for samples from the YYX syntheses. In our previous publication we carried out a third type of synthesis using fatty acid chlorides and glycerol to give sample that contained a 2:1 mixture of YYX and YXY . While this approach works well to provide a third point in the calibration plot, it was deemed unnecessary for the purposes of this work.…”

“…In our previous work, we have shown that for ammonium adducts the ratio of the DAG product ions in its CID spectra is highly dependent on the fatty acid composition of the TAG . In addition, the sensitivity of the relative intensities of the DAG product ions to fatty acid position (the slope of the calibration plot) is also highly dependent on fatty acid composition of the TAG . In general, the CID spectra of ammonium adducts for TAGs that contain multiple polyunsaturated fatty acids show lower sensitivity to fatty acid position than other TAGs.…”

“…Reversed‐phase high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (RP‐HPLC/ESI‐MS/MS) is a powerful technique for the analysis of complex mixtures of triglycerides (TAGs) . RP‐HPLC provides adequate fractionation of complex TAG mixtures, and MS/MS analysis yields information about molecular weight and fatty acid composition and position of TAG species present in the mixture.…”

“…Since positional isomers generally co‐elute in traditional RP‐HPLC, an average collision‐induced dissociation (CID) spectrum across the chromatographic peak corresponding to a given system of positional isomers is a composite spectrum based on the relative abundances of the positional isomers in the sample. The relative abundance data from this composite CID spectrum can be obtained from calibration plots of the relative ion intensity ratio of the DAG products as a function of the relative abundance of the positional isomers …”