High-throughput one-pot micro-volume chemical derivatization combined with targeted lipidomics for the analysis of oxylipins in cells

An efficient method for the analysis of oxylipins in cells was developed. Eight oxylipins were identified in single macrophage cells. Pre-treatment of 54 cell samples could be completed within 10 minutes. Fourteen oxylipins were identified in macrophages samples containing 1000 cells. Polarized and normal macrophages could be distinguished via oxylipins profiling. Oxylipins, generated from polyunsaturated fatty acids (PUFAs), are essential for regulating fundamental physiological processes, such as tissue repair, immunity, and inflammation. However, their intracellular concentrations are inherently low, and many are rapidly transformed or degraded through enzymatic or non-enzymatic reactions, resulting in extremely short half-lives. The complex and time-consuming pretreatment, typically involving sequential cell lysis, solvent extraction, solid-phase clean-up, evaporation, and off-line chemical derivatization, undermines the accuracy of oxylipin analysis in cells. A high-throughput one-pot micro-volume chemical derivatization combined with targeted lipidomics analysis method was developed. Initially, cells were sorted into a 54-well micro sample tube array using flow cytometry. Then, a one-pot method was employed to simultaneously perform cell disruption, oxylipin extraction, and N, N-diethyl-1,3-diaminopropane (DEPA) labeling. The derivatization of 54 cell samples was completed within 1 minute at room temperature, enabling the entire pretreatment to be completed in 10 minutes. Finally, an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was utilized for profiling oxylipins. A total of 14 oxylipins were identified in 1000 RAW 264.7 cell samples, and 8 distinct oxylipins were successfully detected in single-cell analysis including 12-HEPE, 15-HEPE, 12-HETE, PGD2, and PGE2. The proposed method exhibited high sensitivity, with concentration ranges of 0.03–2.98 nmol/L, excellent linearity (R² = 0.992–0.999), and a limit of detection as low as 0.2 pmol/L, demonstrating good accuracy and precision. This protocol eliminates solvent transfers, evaporation, and reconstitution through a one-step micro-volume reaction, drastically minimizing sample loss and oxidation risk. The entire workflow is completed in 10 min for 54 samples with -level sensitivity. Fully compatible with flow-sorted cells, automated liquid handling, and on-line MS, it delivers precise, reproducible oxylipin profiles from rare primary cells, clinical biopsies, or single cells, thereby opening new frontiers in single-cell lipidomics and precision medicine research.