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

Positional analysis of intact triglycerides could provide greater insights into the link between fatty acid position and lipotoxic diseases. However, this methodology has been impeded by lack of commercial availability of positionally pure triglycerides. This work reports on a strategy for defining calibration plots for YXY/YYX triglyceride systems based on the product ion intensities in the collision-induced dissociation spectra of ammoniated precursor ions.A set of triglycerides were synthesized and analyzed by electrospray ionization tandem mass spectrometry using an ion trap mass spectrometer. The product ion spectra of the ammoniated precursor ions were collected for 42 triglyceride systems of the form YXY/YYX, where Y represents C16:0 , C18:1(c-9) and C20:4(cccc-5,8,11,14) . Three-point calibration plots were prepared by plotting the relative abundance of the YY+ product ion vs. the relative abundance of the YYX positional isomer.The calibration plots were shown to give relative abundances of positional isomers accurate to within ±0.02 for most systems. Using an ion trap, under a controlled set of collision parameters, the slopes of the calibration plots can be used to compare the sensitivities of the product ion intensities to fatty acid position for various triglyceride systems. The average slopes of the calibration plots for the C16:0 , C18:1(c-9) and C20:4(cccc-5,8,11,14) systems were 0.29 ± 0.05, 0.21 ± 0.05 and 0.045 ± 0.005, respectively.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.