Assessment of acylation routes and structural characterisation by liquid chromatography/tandem mass spectrometry of semi-synthetic acyl ester analogues of lipophilic marine toxins.

Esterification is one of the most important metabolic routes of lipophilic marine toxins in shellfish. In this work we assessed several chemical acylation reactions aimed at obtaining acyl ester analogues via partial synthesis from the free toxins. The procedures developed including sensitive and selective methods based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be applied to obtain reference materials that may be used as analytical standards (internal/external) for method development and calibration, as well as to perform toxicological in vitro and in vivo studies. Acylation systems involved both anhydrous and non-anhydrous fatty acid or acid halides as a source of the acyl radical, and several catalysers of the reaction. A series of mass spectrometric experiments involving product ion scans and multiple reaction monitoring (MRM) were used to confirm the identity and to elucidate the fragmentation pathways of the synthesised products. Reaction yields regarding reaction time and temperature were examined at sub-nmol scale for the acylation system consisting of palmitic anhydride and 4-(dimethylamino)pyridine (DMAP) in anhydrous pyridine, showing the best conditions at 75 °C for 60 min, 75 °C for 120 min and 100 °C for 270 min for cyclic imines, azaspiracid-1 and pectenotoxin-2, respectively. The esterification approach was verified at a larger scale for the esterification of gymnodimine-A (GYM-A), which kept a good yield >90% for the synthesis of 10-O-palmitoyl-GYM-A. Acyl ester analogues of lipophilic marine toxins have been synthesised and their structure elucidated by LC/MS/MS. For acyl ester analogues identical to natural metabolites, the procedures developed have potential to be applied for the semi-synthesis of metabolites in a sustainable, scalable and controlled way, avoiding extensive and tedious isolation and purification procedures from naturally contaminated shellfish. For the semi-synthetic esters structurally different than those found in shellfish, they may have applicability as internal standards for accurate quantifications of natural metabolites present in complex matrices.