Sensitive determination of domoic acid in mussel tissue using dansyl chloride derivatization and liquid chromatography-mass spectrometry

Abstract

This paper describes a new method for sensitive determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP), in shellfish. The method involves extraction of tissue homogenates with 50% methanol followed by a highly selective strong anion exchange solid phase extraction and a derivatization with dansyl chloride (DNS-Cl) to form the dansyl derivative of domoic acid (DNS-DA). Reaction times were very rapid and proceeded under ambient conditions to yield stable derivatives. A study of the collision-induced dissociation of ESI-produced protonated DNS-DA was carried out to identify the most sensitive transitions to use in development of a selected reaction monitoring detection method. Compared with un-derivatized DA, DNS-DA showed a 5-fold increase in sensitivity of MS/MS detection and improved retention on a reversed phase LC stationary phase. Resolution of DNS-DA and its isomers was achieved using isocratic elution in 15 min. A quantitative verification of the new method was carried out by analyzing a mussel tissue certified reference material (CRM) containing 49 mg kg⁻¹ DA, as well as a toxin-free mussel tissue CRM spiked at levels ranging from 0.003 to 10 mg kg⁻¹. Results showed good recovery (83–107%) with a between-sample variability of ≤5% RSD. The LC-MS/MS method presented is suitable for DA analysis over a broad range of concentrations spanning from above the regulatory limit of 20 mg DA per kg tissue down to near the method detection limit of 1.1 μg DA per kg mussel tissue. The resulting method serves as a confirmatory method with alternative selectivity to existing methods. It is also suitable for quantification of low levels of DA in shellfish as an early warning sign for toxic events or in forensic applications after intoxication has occurred.