(T1130-02-07) Revealing More of the Bioactive Phospholipidome in Plasma Using the High Performance Surface LC/MS/MS

Purpose: Phospholipids (PLs) are the main components of cell membranes and perform many important biological functions. The PA and PS classes tend to elute as broad peaks under the RPLC conditions generally used in lipidomics studies and this can, at least in part, be attributed to the metal-analyte interactions of these lipids, particularly the phosphate moieties, with the metal surfaces of the LC system and the column. The use of hybrid surface technology (HST), applied to the metal surfaces of an UHPLC system and column, designed to mitigate the chelation, poor peak shape and analyte loss seen with acidic phospholipids was investigated.

Methods: LPA(16:0/0:0), PA(16:0/18:1), LPS(18:1/0:0) and PS(16:0/18:1) lipid standards, egg chicken PA and brain porcine PS extracts were obtained from Avanti Polar Lipids. Chromatographic separations were performed using two seperate, but similar, chromatography systems. Either a conventional ACQUITY UPLC I-Class system with CSH C18 column or an ACQUITY PREMIER UPLC system with CSH C18 column (2.1x100mm, 1.7µm) constructed with HST. The chromatography columns were operated at a flow rate of 0.4 mL/min and eluted using a multi-phase gradient where the initial conditions were 1:1 A:B the mobile phase composition B was then gradual increased over 11 minutes using the following profile; 50-53% B from 0-0.5 min, 53-55% B 0.5-4 min, 55-65% B 4-7 min, held at 80% B 7-7.5 min, 80-99% B 7.5-10 min, held at 99% B 10-11min, followed by returning to the starting conditions for re-equilibration giving a total run time of 12 min. The column effluent was monitored using a SYNAPT-XS mass spectrometer operated in negative (ESI) mode

Results: LC/MS analysis of phosphorylated and acidic lipids from biological samples is a critical step, especially for low abundant signaling lipid classes such LPA, PA, LPS and PS. The phosphate group of the phosphatidic acids (PA and LPA) and the carboxylate group of the phosphatidylserines (PS and LPS) bind to metal ions to form chelation complexes that causes peak tailing and smearing during chromatographic analysis. The HST system/column reduced peak tailing by 65-80% for LPA, PA, LPS and PS( compared to the conventional configuration. The peak width at FWHM was reduced by 70-86% for LPA and PA; and by 25-50% for LPS and PS using the HST system/column combination compared to the conventional mode. These improvements in chromatographic performance for these acidic phospholipids are most probably due to the elimination of the interactions of phosphate and carboxylate groups with the metal surfaces is the conventional system by the HST. These improvements in chromatographic performance can be attributed to the fact that the phosphate groups in LPA and the carboxylate group in LPS are, due to their chemical structure, more exposed to metal surfaces compared to PA and PS respectively. The increase in sensitivity obtained with the HST system/column was illustrated for the analysis of LPA at the concentrations of 50 and 5 pg/µL respectively. The LPA was undetectable at 5 pg/µL with the conventional system/column due to analyte loss caused by interaction with metal surfaces where as it was clearly visible with the HST system.

This new HST technology was also applied to the analysis of egg (chicken) PA and brain (porcine) PS extracts and the results compared to those obtained with the conventional system/column. The results of this comparison clearly show that the PA lipid species at eluted with very broad tailing peaks using the conventional system and column whereas with the HST system the peaks show significant improvements in shape and intensity. The HST system/column provided four additional baseline separated peaks at retention times of 3.1, 3.3, 4.7 and 5.8 min respectively which were not observed with the conventional system. Based on exact mass and high energy fragment ion information these peaks were identified as PA(38:6) , PA(36:4) , PA(36.2) and PA lipids. In contrast, it was difficult to make confident molecular species identification with the data obtained using the conventional system/column as there was a large amount overlap of the fragment ions because of peak tailing as well as very low MS signal intensity.

Conclusion: The HST system/column combination clearly provided a significant improvement in sensitivity, reduced peak tailing and increased the recovery of phosphorylated and carboxylate lipids compared to a conventional system and CSH C18 column. The HST system/column combination reduced peak tailing by 65-80% and peak width by 70-86% for LPA and PA. Moreover, increased signal intensities of up to 12.7 times were observed for LPA with the HST approach compared to the untreated system. This methodology can be applied to other phosphorylated and carboxylated lipid classes, such as phosphatidylinositol phosphates with mono, di and tri phosphates, ceramide-1-phosphate and sphingoid base 1-phosphates. The HST allowed for the simultaneous analysis of these acidic phospholipid classes at the same time as other lipid classes without adverse effects on the coverage of these biologically important lipids.


Overlayed negative ESI base peak extracted ion chromatogram of LPA (16:0/0:0) at a concentration of 250 pg/µL using the conventional system/CSH C18 column, the conventional system/HST CSH C18 column or the HST system/CSH C18 column.


Avanti polar lipids egg chicken PA extract (10 ng/µL) measured using (A) the conventional system/CSH C18 column or (B) the HST system/CSH C18 column