Differentiating ether phosphatidylcholines with a collision energy-optimized MRM method by RPLC-MS/MS and its application to studying ischemia-neuronal injury

Highlights: • We developed the collision energy (60 eV)-optimized method to facilitate differentiation between PC(O-) and PC(P-). • The novel method uses specific alkenyl ion-to-ring fragment ratio with conventional triple quadrupole mass spectrometry. • Oxygen-glucose deprivation-treated primary neuronal cultures show distinct regulation between diacyl-PC, PC(O-), and PC(P-). The identification of ether-phosphatidylcholine (ether-PC) isomers, including alkyl-PC (PC(O-)) and plasmalogen-PC (PC(P-)), is technically challenging in MS/MS analysis, which hinders scientists from gaining a deeper understanding of such important lipids. In this study, we developed a sensitive and specific LC-MS/MS-MRM method to accurately identify PC(O-) and PC(P-). We first deciphered the specific fragmentation rules from LPC(O-) and LPC(P-) isomers, in which the product ion of LPC(P-) would be dominated by alkenyl ions (A). In contrast, LPC(O-) only provided a ring-structure fragment (R) without further fragmentation to the alkyl ion, showing completely different characteristics between LPC(O-) and LPC(P-) in negative ion mode. Next, to overcome the sensitivity issue, the MRM approach based on fragmentation rules was used to differentiate PC(O-) and PC(P-). The CE-optimized MRM method increased the alkenyl-to-ring ratio (A/R) between PC(O-) and PC(P-), in which A/R was almost equal to zero for PC(O-) but A/R ≥ 3 for PC(P-). This highly selective property of the CE-optimized MRM method provides accurate identification of PC(O-) and PC(P-) in whole blood samples. The proposed method was applied in primary neuronal cultures with oxygen-glucose deprivation (OGD) treatment to investigate the regulation of PCs under hypoxic stress. The results showed that the regulation of ether-PCs was mainly related to the sn-1 chain length, and the concentration changes of diacyl-PCs were highly dependent on the degree of unsaturation. In summary, the CE-optimized MRM method enables users to distinguish between PC(O-) and PC(P-) in a simple way.