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.

醚-磷脂酰胆碱 (ether-PC) 异构体，包括烷基-PC (PC(O-)) 和缩醛磷脂-PC (PC(P-)) 的鉴定在 MS/MS 分析中具有技术挑战性，这阻碍了科学家获得更深入地了解这些重要的脂质。在本研究中，我们开发了一种灵敏且特异的 LC-MS/MS-MRM 方法来准确识别 PC(O-) 和 PC(P-)。我们首先从 LPC(O-) 和 LPC(P-) 异构体中破译了特定的碎裂规则，其中 LPC(P-) 的产物离子将由烯基离子 (A) 主导。相比之下，LPC(O-)只提供一个环结构碎片(R)，没有进一步裂解成烷基离子，在负离子模式下LPC(O-)和LPC(P-)表现出完全不同的特性。接下来，为了克服敏感性问题，基于碎片规则的 MRM 方法用于区分 PC(O-) 和 PC(P-)。CE优化的MRM方法增加了PC(O-)和PC(P-)之间的烯基环比(A/R)，其中PC(O-)的A/R几乎等于0，但A对于 PC(P-)，/R ≥ 3。CE 优化 MRM 方法的这种高选择性特性可准确识别全血样品中的 PC(O-) 和 PC(P-)。将所提出的方法应用于氧-葡萄糖剥夺 (OGD) 治疗的原代神经元培养，以研究缺氧应激下 PC 的调节。结果表明，ether-PCs的调控主要与sn-1链长有关，二酰基-PCs的浓度变化高度依赖于不饱和度。总之，