Unambiguous regiochemical assignment of sulfoquinovosyl mono- and diacylglycerols in parsley and spinach leaves by liquid chromatography/electrospray ionization sequential mass spectrometry assisted by regioselective enzymatic hydrolysis

Rationale Sulfoquinovosylmonoglycerides (SQMG) and sulfoquinovosyldiglycerides (SQDG) in the lipid extracts of parsley (Petroselinum crispum) and spinach (Spinacia oleracea) leaves were investigated. The aim of this work was to assess and establish the chemical characterization of fatty acyl chains in sulfolipids (SQMG and SQDG) and their regiochemistry. Methods A key component of this approach is a combination of hydrolysis reactions catalyzed by Lecitase® Ultra, which is a sn1-regioselective hydrolase enzyme, and reversed-phase liquid chromatography with electrospray ionization and sequential mass spectrometry (RPLC-ESI-MS) by collision induced dissociation (CID)-MSn (n=2,3). Results The occurrence of SQMG bearing 16:0 or 18:3 acyl chain was established for the first time. A regiochemistry-dependent fragmentation pattern of SQMG was attained whereby the sulfoquinovosyl anion ([C6H11O8S]¯ at m/z 243.0) provides a diagnostic product ion. Regioselective enzymatic treatment also provided a posteriori confirmation of a widely-accepted fragmentation rule for SQDG. The sulfoquinovosyl anion was found to play a role also in the fragmentation pattern of SQDG, whose regiochemical assignment could be ultimately confirmed by MS3 experiments. Conclusions The predominant sulfolipid in leaf extracts of raw parsley (Petroselinum crispum) and spinach (Spinacia oleracea) was identified as SQDG 18:3/16:0, along with of SQMG 18:3/0:0 and SQMG 16:0/0:0. The present CID MS-based method can be considered a successful approach to validate the regiochemical characterization of sulfolipids paving the way for their unambiguous characterization.