Enhanced collision-induced decomposition efficiency and unraveling of fragmentation pathways for anionic adducts of brevetoxins in negative ion electrospray mass spectrometry.

Brevetoxins are a group of natural neurotoxins characterized by polyether ring systems that are found in the blooms of red tide algae. In a conventional water/organic solvent system, without any other additives, deprotonated molecules of brevetoxins do not appear in high abundance in negative mode electrospray mass spectrometry (ES-MS) due to lack of acidic functional groups; thus, they are not well-suited for collision-induced decomposition (CID) experiments in negative mode electrospray. In this study, several anions were tested for their abilities to form anionic adducts by mixing ammonium salts of these anions with brevetoxin-2 and brevetoxin-3. Under CID, [M + Cl](-), [M + Br](-), [M + OAc](-), [M + HCOO](-), and [M + NO(3)](-) adducts all produced only the respective anions in CID experiments and thus provided no structural information. In contrast, upon CID, both [M + F](-) and [M + HCO(3)](-) precursor adducts gave structurally informative fragment peaks that exhibited similarities to those of [M - H](-) ions, which indicated that the first step in gas-phase decomposition of these anionic adducts was loss of neutral HF or H(2)CO(3) molecules, respectively, leaving deprotonated brevetoxin ([M - H](-)) to undergo consecutive fragmentations. In comparison to bicarbonate, fluoride formed adducts in higher abundance and provided more fragment peaks, thus more structural information, in MS/MS experiments. It is therefore the anion of choice to study brevetoxins in negative mode electrospray mass spectrometry using the anion attachment approach. The detailed fragmentation mechanisms are discussed, and diagnostic product ions are proposed for the brevetoxin-2 side chain (m/z 95, 133, and 151), the brevetoxin-3 side chain (m/z 97, 135, and 153), and the type-B brevetoxin backbone (m/z 725, 739, 741, and 797). To test the developed methodology, a sample of brevetoxin-2 subjected to in vitro microsomal incubation was analyzed, and a reduction product of the substrate was confirmed to have the structure of brevetoxin-3.