Arsenosugar Phospholipids (As-PL) in Edible Marine Algae: An Interplay between Liquid Chromatography with Electrospray Ionization Multistage Mass Spectrometry and Phospholipases A₁ and A₂ for Regiochemical Assignment

The chemical identity of arsenosugar phospholipids (As-PL) as mono- (i.e., lyso, L-As-PL) and diacyl-arsenosugar PL in four edible and common marine alga samples, such as nori (<i>Porphyra spp</i>.), wakame (<i>Undaria pinnatifida</i>), dulse (<i>Palmaria palmata</i>), and kombu (<i>Saccharina japonica</i>), was successfully investigated. Adopting negative polarity electrospray ionization (ESI), not common for As-PL, conjugated with hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry (MS), performed either at low resolution using a linear ion trap (LIT) with sequential MS<i>ⁿ</i> (<i>n</i> = 2, 3) or at high resolution using a high-resolution/high-accuracy Fourier-transform MS (FTMS), based on an orbital trap instrument, more than 20 As-PL and 2 L-As-PL species were identified. The absence of As-PL standard compounds encouraged us to generate an in-house-built database of As-PL/L-As-PL for a rapid and simple classification. Despite their compositional diversity, tandem MS of deprotonated As-PL and L-As-PL ([M – H]⁻) demonstrated the occurrence of a highly diagnostic product ion at <i>m</i>/<i>z</i> 389.0 ([AsC₁₀H₁₉O₉P]⁻). The fatty acid composition and distribution of As-PL were easily assigned on the basis of the ratio intensity between <i>sn</i>-1 and <i>sn</i>-2 product ions. Indeed, the preferential formation of [R₁C₃H₅O₄P]⁻ ions over [R₂C₃H₅O₄P]⁻ ions, both containing the glycerol backbone, enabled the regiochemical assignment of As-PL. These outcomes were confirmed by MS<i>ⁿ</i> (<i>n</i> = 2, 3) analyses and using <i>sn</i>-1- and <i>sn</i>-2-regioselective hydrolase enzymes (i.e., phospholipases A₁ and A₂). The predominant As-PL’s in samples of nori (red alga), wakame, and kombu (both brown algae) were identified as containing palmitic acyl chains (i.e., As-PL958 (As-PL 16:0/16:0) with ca. 66 ± 3, 82 ± 4, and 58 ± 3% as relative abundances, respectively), while the main species in dulse (red alga) samples was As-PL982 (As-PL 18:1/16:1) at ca. 38 ± 3%.