Separation of cis-trans phospholipid isomers using reversed phase LC with high resolution MS detection

Abstract

The increased presence of synthetic trans fatty acids into western diets has been shown to have deleterious effects on physiology and raising an individual's risk of developing metabolic disease, cardiovascular disease, and stroke. The importance of these fatty acids for health and the diversity of their (patho) physiological effects suggest that not only should the free trans fatty acids be studied but also monitoring the presence of these fats into the side chains of biological lipids, such as glycerophospholipids, is also essential. We developed a high resolution LC-MS method that quantitatively monitors the major lipid classes found in biospecimens in an efficient, sensitive, and robust manner while also characterizing individual lipid side chains through the use of high energy collisional dissociation (HCD) fragmentation and chromatographic alignment. We herein show how this previously described reversed phase method can baseline separate the cis-trans isomers of phosphatidylglycerol and phosphatidylcholine (PC) with two 18:1 side chains, in both positive and negative mode, as neat solutions and when spiked into a biological matrix. Endogenous PC (18:1/18:1)-cis and PC (18:1/18:1)-trans isomers were examined in mitochondrial and serum profiling studies, where rats were fed diets enriched in either trans 18:1 fatty acids or cis 18:1 fatty acids. In this study, we determined the cis:trans isomer ratios of PC (18:1/18:1) and related this ratio to dietary composition. This generalized LC-MS method enables the monitoring of trans fats in biological lipids in the context of a nontargeted method, allowing for relative quantitation and enhanced identification of unknown lipids in complex matrixes.

Figure 1

PG (18:1/18:1) cis-trans isomers, showing how the structure changes due to the stereochemistry around each double bond.

Figure 2

The PG (18:1/18:1) cis and trans separation for the Ascentis Express, Waters and Phalanx columns respectively. Each panel represents the XIC of m/z 773.5333 representing the [M-H]− ion of the standards.

Figure 3

Separation of cis-trans PG (18:1/18:1) isomers in a serum pool sample. Top panel shows the TIC of the serum pool and the bottom panel is the XIC of m/z 773.5338 representing the [M-H]− ion of the PG (18:1/18:1) isomers that were spiked into the pool sample.

Figure 4

Two XICs of m/z 786.5992 representing the [M+H]+ ion of PC (36:2). Panel A is a serum pool sample, and panel B shows that same sample spiked with the PC (18:1/18:1) cis and trans isomers. Peaks at 14.32 and 15.06 minutes represent the Cis and Trans isomer respectively.

Figure 5

panels A–D represents the average signal of PC (18:1/18:1) cis and PC (18:1/18:1) trans in mitochondria (A–B) and serum (C–D). Panel E shows the cis-trans ratio of PC (18:1/18:1) found in mitochondria from rats fed each of the MUFA (white bars) and Trans (black bars) fat diets. The two high glycemic index diet pairs (HGI) are followed by the medium high glycemic index diets (MHGI), the medium low (MLGI) and finally the low glycemic index diets (LGI). The bars represent the average signal for each molecule, with background subtraction, across all rats in the diet (n=8), and error bars represent the standard error of the mean. Statistical significance was assessed via t-tests without correction for multiple comparisons.

Figure 6

panel A shows the results from the PC (18:1/18:1) cis-trans biotransformation experiment and the baseline separation of all three species, the PC (18:1/18:1) cis, PC (18:1/18:1) cis-trans and PC (18:1/18:1) trans. Panels B and C show how in the mitochondrial pool analysis, only the PC (18:1/18:1) cis and PC (18:1/18:1) trans species can be accounted for, due to co-elution of the PC (18:1/18:1) cis-trans with the PC (18:0/18:2) isomer.