950 (2002) 139–147 Journal of Chromatography A, www.elsevier.com / locate / chroma Liquid chromatography with electrospray ion trap mass spectrometry for the determination of five azaspiracids in shellfish * ˜ M. Lehane, A. Brana-Magdalena, C. Moroney, A. Furey, K.J. James Ecotoxicology Research Unit, Chemistry Department, Cork Institute of Technology, Cork, Ireland Received 24 October 2001; received in revised form 18 December 2001; accepted 20 December 2001 Abstract Azaspiracid poisoning (AZP) is a new human toxic syndrome that is caused by the consumption of shellfish that have been feeding on harmful marine microalgae. A liquid chromatography–mass spectrometry (LC–MS) method has been developed for the determination of the three most prevalent toxins, azaspiracid (AZA1), 8-methylazaspiracid (AZA2) and 22- demethylazaspiracid (AZA3) as well as the isomeric hydroxylated analogues, AZA4 and AZA5. Separation of five azaspiracids was achieved on a C column (Luna-2, 15032 mm, 5 mm) with isocratic elution using acetonitrile–water 18 containing trifluoroacetic acid and ammonium acetate as eluent modifiers. Using an electrospray ionisation (ESI) source with 1 an ion-trap mass spectrometer, the spectra showed the protonated molecules, [M1H] , with most major product ions due to the sequential loss of two water molecules. A characteristic fragmentation pathway that was observed in each azaspiracid was due to the cleavage of the A-ring at C –C for each toxin. It was possible to select unique ion combinations to 9 10 3 distinguish between the isomeric azaspiracids, AZA4 and AZA5. Highly sensitive LC–MS analytical methods were compared and the detection limits were 5–40 pg on-column. Linear calibrations were obtained for AZA1 in shellfish in the 2 range 0.05–1.00 mg/ml (r 5 0.9974) and good reproducibility was observed with a relative standard deviation (%RSD) of 3 1.8 for 0.9 mg AZA1/ml (n 55). The %RSD values for the minor toxins, AZA4 and AZA5, using LC–MS (A-ring fragmentation) were 12.3 and 8.1 (0.02 mg/ml; n 57), respectively. The selectivity of toxin determination was enhanced using LC–MS–MS with high energy WideBand activation.  2002 Elsevier Science B.V. All rights reserved. Keywords: Azaspiracid poisoning; Toxins; Azaspiracids 1. Introduction cramps. This severe gastrointestinal disturbance was not due to the presence of known diarrhetic shellfish Human poisonings in The Netherlands in 1995 poisoning (DSP) toxins, okadaic acid and were caused by the consumption of contaminated dinophysistoxin-2, which were present only at low mussels (Mytilus edulis) from Killary Harbour, Ire- concentrations. Azaspiracid was isolated from mus- land. The associated human symptoms consisted of sels and structurally elucidated [1,2]. Azaspiracid nausea, vomiting, severe diarrhoea and stomach contains fused polyether rings similar to other classes of marine toxins [3] but there are also rare structural features including a trispiro ring and an azaspiro ring *Corresponding author. Tel.: 1353-21-4326317; fax: 1353- assembly, together with a terminal carboxylic acid 21-4345191. E-mail address: kjames@cit.ie (K.J. James). group (Fig. 1). A second intoxication incident in 0021-9673 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0021-9673(02)00003-1