Glycerophospholipids play important roles in iron-induced lipid peroxidation during cerebral ischemia-reperfusion, making it essential to investigate changes in their varieties and concentrations under these conditions. However, the wide range of glycerophospholipid contents, particularly the low-abundance species in actual biological samples, posed a challenge for comprehensive analysis. In this study, an iterative quadrupole time-of-flight mass spectrometry (Q-ToF-MS/MS) method was established with the aim of comprehensively detecting glycerophospholipids. This method was a data acquisition strategy implemented through iterative analyses. In each iteration, ions detected in previous runs were excluded, allowing low-abundance glycerophospholipids that were missed by the usual analysis to be extensively detected by a simplified operational process. Using this strategy, 254 glycerophospholipids including 157 PCs, 67 PEs, 19 PGs, 9 PIs, 7 PSs and 5 PAs in rat brain samples were identified after four iterations, and the number of glycerophospholipid species increased by 93.9% compared to a single assay, significantly enhancing the coverage of glycerophospholipid detection. Furthermore, the characteristic fragmentation patterns of six glycerophospholipid subclasses were systematically summarized to improve the accuracy of qualitative identification. In addition, these patterns were also used to construct an ion pair database containing 254 glycerophospholipids, enabling targeted multiple reaction monitoring (MRM) analysis under the optimized high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) conditions. By comparing the changed glycerophospholipids of rat brains from the normal and cerebral ischemia-reperfusion injury groups, 29 glycerophospholipids were recognized as the potential biomarkers for cerebral ischemia-reperfusion injury, among which nine glycerophospholipids were particularly detected by four iterations. Overall, this iterative MS/MS approach extensively expanded the coverage of low-abundance components, and has been proven to be an effective approach in biomarker screening of cerebral ischemia-reperfusion injury.

Keywords: Cerebral ischemia–reperfusion injury; Glycerophospholipid identification; Iterative MS/MS; MRM ion pair database; Relative quantification.

© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.

甘油磷脂在脑缺血再灌注过程中铁诱导的脂质过氧化中起重要作用，因此研究其种类和浓度的变化非常重要。然而，由于实际生物样本中的甘油磷脂含量范围广泛，特别是低丰度物种的存在，对其进行全面分析存在挑战。本研究建立了一种迭代四极杆飞行时间质谱（Q-ToF-MS/MS）方法，旨在全面检测甘油磷脂。该方法是一种通过迭代分析实施的数据采集策略，在每次迭代中排除之前运行中检测到的离子，使低丰度甘油磷脂能够通过简化操作流程被广泛检出。采用此策略，经过四次迭代后，在大鼠脑样本中鉴定出了254种甘油磷脂，包括157个PC、67个PE、19个PG、9个PI、7个PS和5个PA，与单一检测相比，甘油磷脂种类增加了93.9%，显著提高了对甘油磷脂的覆盖范围。此外，还系统总结了六种甘油磷脂亚类的特征裂解模式，以提高定性鉴定的准确性。这些模式还被用于构建一个包含254种甘油磷脂的离子对数据库，在优化后的高效液相色谱-三重四级杆质谱（HPLC-QQQ-MS/MS）条件下进行靶向多重反应监测（MRM）分析。通过比较正常组和脑缺血再灌注损伤组大鼠大脑中变化的甘油磷脂，确定了29种潜在的脑缺血再灌注损伤生物标志物，其中九种甘油磷脂特别在四次迭代中被检测到。总体而言，这种迭代MS/MS方法广泛扩展了低丰度成分的覆盖范围，并已被证明是筛查脑缺血再灌注损伤生物标志物的有效方法。

关键词：脑缺血-再灌注损伤；甘油磷脂鉴定；迭代MS/MS；MRM离子对数据库；相对定量。