The analysis of domoic acid using alkaline UPLC-MS/MS A. O’Neill, C. Baker, Cefas (Weymouth), UK Aims and objectives Domoic acid (DA) is the principal toxin responsible for incidents of amnesic shellfish poisoning (ASP). A high performance liquid chromatography with ultra-violet detection (HPLC-UV) method is currently applied at Cefas and is the reference method for the analysis of DA. However, with liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) becoming the reference method for the analysis of lipophilic toxins (LT), the simultaneous analysis of DA and LT by LC-MS would offer time and cost reduction benefits to Cefas and its customers. The work presented includes the investigation of different extraction procedures as well as the initial LC-MS/MS analysis of DA in calibration solutions, ASP laboratory reference material (LRM) and LT freeze-dried material (FDMT) also containing DA, to assess linearity, recovery, precision and reproducibility. Method Three different extraction methods were used to extract three replicate LRMs (mean value: 23.07 µg/g DA) currently included for quality control in DA analysis by HPLC-UV: - 50:50 methanol:water currently used for ASP extraction 100% methanol currently used for LT extraction 90:10 methanol:water following LT extraction procedure The extracts were then analysed using the current ASP HPLC-UV method and the current LT LC-MS method modified, following detector optimisation, to include DA on three separate days. One FDMT was extracted with 100% methanol and analysed on two of the three days. The HPLC-UV analyses were carried out on an Agilent 1100 instrument and the UPLC-MS/MS analysis conducted on a Waters I-class UPLC coupled to a Xevo TQ-S mass spectrometer. Five transitions of Domoic acid FDMT toxin retention times using UPLC-MS/MS Results Mean recoveries of DA as determined by HPLC-UV and UPLC-MS/MS. Discussion and further work Recovery of DA determined by HPLC-UV, was satisfactory (>95%) using 50:50 and 90:10 MeOH:H2O. Following UPLC-MS, correlation coefficients of the standards were all >0.98, inter batch precision was good along with reproducibility. DA was seen to elute at 0.18 mins following UPLC-MS/MS and poor recoveries (<50%) were determined, potentially attributable to DA signal suppression. Analysis of spiked and naturally contaminated shellfish matrices would be required to further assess method performance. In the event of the method performance characteristics being fit for purpose, UPLC-MS/MS would have to be recognised as a reference method for ASP prior to it being used for official control. 50:50 90:10 100% UPLC-MS/MS performance summary Calibration correlation coefficient (R²) > 0.98 on all runs Inter-batch precision (%RSDs) 0.3 to 1.6% (n=5) Mean reproducibility (%RSDs) 17% (n=15) domoic acid