Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids containing two or more double bonds and a carbon chain length of 18 to 22 carbon atoms, which are divided into n-3 and n-6 PUFAs. It has been reported that dietary n-3 PUFAs exert significant benefits against multiple diseases, including neurodegenerative diseases cardiovascular diseases and metabolic disorders, especially docosahexaenoic acid DHA) and eicosapentaenoic acid (EPA). The concentration of EPA in the brain is over 250-fold lower than that of DHA, compared to 4- and 5-fold lower levels of EPA vs. DHA in plasma and erythrocytes, respectively. In contrast, EPA and DHA enter the brain at similar rates. Thus, it is essential to clarify EPA metabolism in the brain to explain the considerable differences in concentration and efficacy, In addition, PUFAS can be metabolized into diverse classes of oxylipins under enzymatic or nonenzymatic catalysis like lipoxygenase (LOX), cyclooxygenase (COX), cytochrome P450(CYP) and reactive oxygen species (ROS). These oxylipins derived from n-3 PUFAs exert predominantly anti-inflammatory, vasodilatory effects and anti-platelet aggregation, which are functionally adverse with n-6 PUFA-derived oxylipins. Furthermore, it can also be integrated into the phospholipids of cell membranes, which will affect the membrane's fluidity and mediate various signal pathways. Due to the wide variety of metabolites produced by complex metabolic processes, there are many challenges to the research of PUFAs: i) the structural diversity and high similarity; ii) trace amounts in biological samples and the complicated matrix; ii) the high hydrophilicity and low ionization efficiency; iv) accurate quantification with high sensitivity; v) identification of metabolites with highly selective to uncover the underlying metabolic networks of PUFAs. In this thesis, to overcome these problems, chemical derivatization and click chemistry-based strategies have been applied to analyze the derivatives of n-3 PUFAS and track their metabolic processes. In Chapter 2, a sensitive method was developed to quantify n-3 PUFAs/oxylipins by chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Standards labeled with cholamine-d9 were used as one-to-one internal standards to achieve accurate quantification. After cholamine derivatization, the MS sensitivity and chromatographic performance of n-3PUFAs/oxylipins were substantially improved. In addition, the relationship between retention time and substituent position of regioisomers was investigated, which can facilitate the identification of unknown regioisomers, Furthermore, the developed method was applied to quantify the target n-3 PUFAs/oxylipins in serum and brain tissue from fish oil-supplemented mice, which exhibited its great potential and practicability. In Chapter 3, a click chemistry-based enrichment (CCBE) strategy for tracing the cellular metabolism of eicosapentaenoic acid (EPA) in neural cells was established. Terminal alkyne-labeled EPA (EPAA) used as a surrogate was incubated with N2a cells. mouse neuroblastoma cells, and alkyne-labeled metabolites (ALMs) were selectively captured by an azide-modified resin via a Cu(l)-catalyzed azide-alkyne cycloaddition(CuAAC)reaction for enrichment. After removing unlabeled metabolites, ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatograph mass spectrometry (LC-MS) analysis. The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from complicated matrices, In addition, the method can overcome current detection limits by improving analytical performance, including MS sensitivity of targets, chromatographic separation of sn-position glycerophospholipid regioisomers, facilitating structural characterization of ALMs by a specific MS/MS fragmentationsignature, and versatile fluorescence detection of ALMs for cellular distribution. The developed methods in the thesis could facilitate better elucidation of the roles of PUFAs in physiological and pathological events.