Lipids play a crucial role in cells and organisms by acting as the key components of cell membranes,the sources for chemical-energy storage and the messengers for cellular signaling,etc.It is generally believed that the physiological processes of cells highly rely on the chemical complexity and structural diversity of lipid compounds.In addition,the progression of many diseases such as diabetes,atherosclerosis,neurodegenerative diseases and tumorigenesis might be also related to the dysregulated lipid metabolism.Therefore,the precise structural characterization of lipids plays a vital role in exploring their biological functions.Mass spectrometry(MS) is the first choice for lipid analysis and lipidomics investigation due to its merits of high sensitivity,good specificity and informative,etc.,while the advent of ambient ionization MS provides an important tool for in situ,real-time,direct and rapid analysis for lipids and lipidomics investigation under ambient and open-air conditions,facilitating the in-depth investigation on the roles and mechanisms that lipids play in organisms.With the continual development of ambient ionization MS techniques in lipid analysis and lipidomics,it is of great importance to comprehensively characterize the structures.The recent progresses in high-resolution MS(e-g-,orbitrap MS and fourier transform ion cyclotron resonance MS) and tandem MS techniques have substantially promoted the development of lipid analysis and lipidomics investigation by ambient ionization MS.High-resolution MS provides the ability to obtain molecular formulas by accurate mass measurements,while tandem MS enables the detailed structural composition of the fatty acyl chain and head group of a mass-selected lipid ion via collision-induced dissociation(CID) or high-energy collision dissociation(HCD).In spite of this,the full structural identification of lipid species remains challenging.Unsaturated lipids are a series of lipid species containing one or more carbon carbon double bond(C‖C) in their fatty acyl chains.The locations of C‖C might have remarkable effects on their biological functions,and even could serve as the biomarkers of disease pathology.However,conventional tandem mass spectrometry techniques such as CID and HCD are unable to provide the molecular information for precise location of C‖C bonds.To address these problems,scientists have spent great effort on incorporated derivatization and ion activation/dissociation methods in ambient ionization MS to achieve the precise structural elucidation on lipid C‖C locations in an isomer.The typical derivatization and ion activation/dissociation methods that have been successfully applied in ambient MS include Paternò-Büchi(PB) reaction,epoxidation,ozoneinduced dissociation(OzID) and ultraviolet photodissociation(UVPD).In this article,the ambient MS techniques based on PB reaction,epoxidation,OzID,UVPD are described in detail,and the applications of these techniques in accurate identification of lipid C‖C location and isomer differentiation are also discussed.The development trends of ambient MS in precise structural analysis of lipids are also prospected.