Determination of domoic acid in shellfish by liquid chromatography with electrospray ionization and multiple tandem mass spectrometry

Amnesic shellfish poisoning is a potentially lethal human toxic syndrome which is caused by domoic acid (DA) that originates in marine phytoplankton belonging to the Pseudonitzschia genus. A new sensitive liquid chromatographic–mass spectrometry (LC–MS) method has been developed for the determination of DA in various marine biological samples. The characteristic fragmentation pathways for DA were established using multiple stage MS on selected daughter ions, which were sequentially trapped and fragmented. Chromatography was performed using a gradient of acetonitrile–water (5:95 to 40:60), containing trifluoroacetic acid (0.05%), over 25 min at 0.2 ml/min with a C18 column (Luna-2, 150×2.0 mm, 5 μm). Using electrospray ionisation, multiple tandem MS experiments were performed with an ion-trap mass spectrometer (Finnigan MAT LCQ). The protonated DA molecule was the precursor ion, m/z 312, and the relative collision energies were optimised for multiple MS (MSn, n=2–4) studies. LC–MS3 using the ions, m/z 266 and 220, from the loss of two HCOOH molecules, produced the best sensitivity data. Calibration data for various MS modes were: MS (0.05–10 μg DA/ml, r2=0.9973); MS2 (0.025–10 μg DA/ml, r2=0.9997); MS3 (0.025–10 μg DA/ml, 0.9994). The detection limits (3:1 signal:noise) were better than 0.02 μg DA/ml for LC–MS, 0.014 μg DA/ml for LC–MS2 and 0.008 μg DA/ml for LC–MS3. This method was applied to determine DA in scallop (Pecten maximus) tissues, which subsequently led to the closure of several shellfish harvesting sites on the west coast of Ireland.