The Development of a Specific and Sensitive LC-MS-Based Method for the Detection and Quantification of Hydroperoxy- and Hydroxydocosahexaenoic Acids as a Tool for Lipidomic Analysis

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid that is highly enriched in the brain, and the oxidation products of DHA are present or increased during neurodegenerative disease progression. The characterization of the oxidation products of DHA is critical to understanding the roles that these products play in the development of such diseases. In this study, we developed a sensitive and specific analytical tool for the detection and quantification of twelve major DHA hydroperoxide (HpDoHE) and hydroxide (HDoHE) isomers (isomers at positions 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 and 20) in biological systems. In this study, HpDoHE were synthesized by photooxidation, and the corresponding hydroxides were obtained by reduction with NaBH4. The isolated isomers were characterized by LC-MS/MS, and unique and specific fragment ions were chosen to construct a selected reaction monitoring (SRM) method for the targeted quantitative analysis of each HpDoHE and HDoHE isomer. The detection limits for the LC-MS/MS-SRM assay were 1−670 pg for HpDoHE and 0.5−8.5 pg for HDoHE injected onto a column. Using this method, it was possible to detect the basal levels of HDoHE isomers in both rat plasma and brain samples. Therefore, the developed LC-MS/MS-SRM can be used as an important tool to identify and quantify the hydro(pero)xy derivatives of DHA in biological system and may be helpful for the oxidative lipidomic studies.

二十二碳六烯酸（Docosahexaenoic acid, DHA）是一种高度富集于大脑的n-3多不饱和脂肪酸，其氧化产物在神经退行性疾病进程中会出现或含量升高。对DHA氧化产物的表征，对于阐明此类产物在这类疾病发生发展中的作用至关重要。
本研究开发了一种灵敏且特异性强的分析工具，可用于检测并定量生物体系中12种主要的DHA氢过氧化物（hydroperoxide, HpDoHE）与羟基化物（hydroxide, HDoHE）异构体，这些异构体分别位于4、5、7、8、10、11、13、14、16、17、19和20号位。
本研究中，HpDoHE通过光氧化法合成，对应的羟基化物则经硼氢化钠（NaBH4）还原制得。通过液相色谱-串联质谱（LC-MS/MS）对分离得到的异构体进行表征，并选取专属且特异的碎片离子，构建了针对每种HpDoHE和HDoHE异构体的靶向定量分析选择反应监测（selected reaction monitoring, SRM）方法。
该LC-MS/MS-SRM检测方法的检出限为：进样至色谱柱的HpDoHE为1~670皮克（pg），HDoHE为0.5~8.5皮克（pg）。利用该方法，可在大鼠血浆与脑组织样本中检测到HDoHE异构体的基础水平。
因此，本研究开发的LC-MS/MS-SRM方法可作为在生物体系中鉴定并定量DHA氢（过）氧基衍生物的重要工具，有望为氧化脂质组学研究提供助力。