Both 2-AG (1) and anandamide (2) exhibit cannabimimetic activities and act as endogenous ligands for cannabinoid receptors in various mammalian tissues. 1 2-AG is known to be an intrinsic natural ligand for the cannabinoid receptors, while anandamide was shown to be a weak partial agonist. 2, 3 MS is an important technique for analyzing these endogenous ligands. Gas chromatography-MS (GC-MS) 4,5 and liquid chromatography-MS (LC-MS) 6,7 have been used as methods for detecting these compounds.

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... ionization is applicable in LC-FAB-MS(/MS) systems, and has been widely used to identify biologically active compounds, e.g., dinucleotide, 8 acetylcholine, 9 and dextromethorphan, 10 or natural products, e.g., cytokinin 11 and oligosaccharide derivative. 12 The FAB-MS/MS technique was applied in the characterization of naturally occurring compounds, e.g., terpene or glycoside 13 and phenolic constituents. 14 High-energy collision activation of ions that contain a stable charge-site results in decompositions that occur remote from the site of charge, i.e., CRF, and has been shown to provide a technique for determining the positions of double bonds, branch points, and other functional groups in a large variety of biological and other molecules. 15,16 A stable charge-site is either anionic, as in carboxylate, sulfate, and sulfonate anions, or cationic, as in protonated quaternary amines, sulfonium, and phosphonium cations, as well as in molecules that have been cationized with metal ions. CRF by CID-MS/MS has been used for determining the double-bond positions in long alkenyl chains, e.g., polyhydroxypolyene 17 from marine sources, and alkenylresorcinol, 18 or alkenylsalicylic acid 19 from natural plants. In our previous study, 20 analyses of anandamide (2) and endocannabinoid-like compounds using high-energy CID-MS/MS in FAB-MS were performed. The CID-MS/MS spectra of lithium-adduct [M+Li] + ions were rich in structurally informative CRF patterns, which provided information on the locations of double bonds in the hydrocarbon chain, which is useful for determining the structure. As a part of research work relating to the high-energy CID-MS/MS reactions of endogenous cannabinoids and several endocannabinoid-like compounds, in the present paper we describe the optimal measurement conditions for the analysis of 2-AG (1) using the CID-MS/MS technique in FAB-MS, focusing on adduct metals, i.e., alkali metals (group 1 elements) and alkaline earth metals (group 2 elements), and matrices. The ability of alkali and alkaline earth metal cations to form a stable charge-site to provide structurally informative CRF ions in the CID-MS/MS measurement is examined. Since 2-AG contains a 1,3-dihydroxyl moiety, the formation of cyclic β-chelation between 2-AG and alkaline earth metal could be expected to give the tightly localized charge-site necessary for CRF. It is well known that coexisting metal halides, such as lithium chloride and magnesium bromide, play an important role in the stereochemical outcome of the nucleophilic addition of an organometallic to αand β-oxycarbonyl compounds through cyclic chelation models. 21, 22 The results have been rationalized in terms of the chelation mechanism by lithium, an alkali metal, and magnesium, an alkaline earth metal. We also analyzed anandamide (2) with the addition of alkali (other than lithium) and alkaline earth metal to investigate the 921 Analysis of 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (anandamide) via alkali or alkaline earth metal-adduct high-energy collision-induced dissociation (CID) in fast-atom bombardment (FAB) ionization-mass spectrometry (MS) is described. The CID-MS/MS of the [2-AG+Li] + or [2-AG+Na] + ion undergoes charge-remote fragmentation (CRF), which is useful for the determination of the double-bond positions in the hydrocarbon chain, while the CID-MS/MS of the [2-AG-H+Cat] + (Cat = Mg 2+ , Ca 2+ , Ba 2+ ) ion provides an abundant fragment ion of the cationized arachidonic acid species, which is derived from cleaving the ester bond via a McLafferty-type rearrangement in addition to structurally informative CRF ions in small amounts. On the other hand, the CID-MS/MS spectra of anandamide cationized with both alkali metal (Li + or Na + ) and alkaline earth metal (Mg 2+ , Ca 2+ , or Ba 2+ ) show CRF patterns: the spectra obtained in lithium or sodium adduct are more clearly visible than those in magnesium, calcium, or barium adduct. The McLafferty rearrangement is not observed with metal-adduct anandamide. The characteristics in each mass spectrum are useful for the detection of these endogenous ligands. m-Nitrobenzyl alcohol (m-NBA) is the most suitable matrix. A lithium-adduct [2-AG+Li] + or [anandamide+Li] + ion is observed to be the most abundant in each mass spectrum, since the affinity of lithium for m-NBA is lower than that for other matrices examined.