A derivatization strategy for comprehensive identification of 2- and 3-hydroxyl fatty acids by LC-MS.

2/3-Hydroxy fatty acids (2/3-OHFAs) are a class of important biological molecules closely related to many diseases, of which the unsaturated ones (2/3-OHUFAs) obtain special recognition for their bioactivities. However, the comprehensive identification of 2/3-OHFAs has been a daunting task due to the similarity of isomeric structures and lack of authentic standards. Herein, we report a strategy for the 2/3-OHUFA identification by using an in-house synthesized derivatization reagent, 4-amino-1,1-dimethylpiperidin-1-ium iodide hydrochloride (ADMI). Through ADMI derivatization, the diagnostic ion m/z 155.1 or 171.1 were produced by liquid chromatography-mass spectrometry (LC-MS) analysis, which could distinguish the 2-OH or 3-OH group, respectively. Then the meta-chloroperoxybenzoic acid (mcpba) was used to resolve the locations of double bonds in 2/3-OHUFAs by the characteristic cleavage of the newly formed epoxides. Thus, the identification of 2/3-OHUFAs was enabled by all these characteristics. Moreover, in order to simplify the whole analysis, an isotope-labeled ADMI was designed to quickly pick out the low-abundant 2/3-OHFAs from complex biological matrices. Finally, a combined derivatization strategy was established to analyze mouse lung tissues with melanoma metastasis. Different long-chain 2-OHFAs and 3-OHFAs were identified, including FA 12:0-3OH, FA 12:1(Δ9)-3OH, FA 14:0-3OH, FA 14:1(Δ5)-3OH, FA 16:0-2/3OH, and FA 18:0-2/3OH. The results showed that the contents of most identified 2/3-OHFAs were lower in the cancer group than in the control group, suggesting the plausible role of 2/3-OHFAs in cancer development. In summary, the new derivatization method provides a powerful analysis strategy for 2/3-OHUFAs, which sheds light on a better understanding of the biological functions of 2/3-OHUFAs.