DETERMINATION OF DIARRHEIC SHELLFISH TOXINS IN MUSSELS BY MICROLIQUIDCHROMATOGRAPHY TANDEM MASS-SPECTROMETRY

A fast, sensitive, and specific procedure for determining toxins that cause diarrheic shellfish poisoning (DSP) using microliquid chromatogr aphy coupled with tandem mass spectrometry (micro-LC-MS-MS) is reporte d. The lipophylic polyether acidic toxins okadaic acid (OA), its isome r dinophysistoxin-2 (DTX-2), the 35-methylokadaic acid dinophysistoxin -1 (DTX-1), and the novel toxin dinophysistoxin-2B (DTX-2B; recently i solated from Irish mussels) were extracted from shellfish tissues with acetone and chromatographed by isocratic elution at 10 mu L/min with CH3CN-H2O, 80 + 20 (v/v), containing 0.1% trifluoroacetic acid, throug h a C-18 reversed-phase column (1.0 mm id). The chromatograph is coupl ed via an ion spray interface to an atmospheric pressure ionization so urce. Collision-induced-dissociation (CID) ion mass spectra of the pro tonated molecule, [M + H](+), at m/z 805 for OA, DTX-2, and DTX-2B and at m/z 819 for DTX-1, were obtained in MS-MS experiments to identify 2 diagnostic fragment ions for each analyte that could be used for sel ected-reaction-monitoring (SRM) micro-LC-MS-MS analysis. The CID spect rum of DTX-2B confirmed it to be a new OA isomer, like DTX-2. Standard curves obtained by SRM micro-LC-MS-MS were linear (r(2) greater than or equal to 0.9992) over the range 0.05-1.00 mu g/mL (i.e., 0.10-2.00 mu g toxin/g hepatopancreas), and a detection limit of 15 pg/injection was obtained for each DSP toxin. Average recoveries ranged from 95 to 101%, and coefficients of variation ranged from 1.8 to 3.4%. This nov el SRM micro-LC-MS-MS method was used to confirm acidic DSP toxins in Irish and Italian toxic mussels. It offers a high degree of specificit y because analyte confirmation is based on retention time, molecular w eight, structural information obtained from the presence of 2 diagnost ic fragments for each analyte, and ion ratios. OA was found in both Ir ish (less than or equal to 0.7 mu g/g hepatopancreas) and Italian (les s than or equal to 1.5 mu g/g hepatopancreas) mussels. DTX-1 was found only in Italian mussels (less than or equal to 0.3 mu g/g hepatopancr eas). DTX-2 (less than or equal to 6.1 mu g/g hepatopancreas) and DTX- 2B (less than or equal to 0.08 mu g/g hepatopancreas) were unique to I rish shellfish.