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ABSTRACT
A postcolumn fluorescence reaction system for the high-performance liquid chromatographic (HPLC) determination of tetrodotoxin in the silver-cheeked toadfish Lagocephalus sceleratus is discussed theoretically and investigated experimentally. Ion-pair chromatography with volatile ammonium perfluoroheptanoate was used for the separation of tetrodotoxin and 4,9-anhydrotetrodotoxin. The postcolumn reaction was based on the tetrodotoxin conversion to a quinazoline fluorescent compound under strong alkaline conditions. All postcolumn parameters were optimized that affected the sensitivity, dispersion, and stability. Helically coiled and knitted open tubular reactors composed of polyetheretherketone were constructed and characterized in detail. The performance of these reactors was evaluated on the basis of sensitivity and dispersion. Their optimal design is reported. The knitted reactors were more efficient than the relevant helically coiled reactors when higher reaction times are required. A 1500-µL polyetheretherketone knitted coil with 0.010″ internal diameter was optimum exhibiting higher pressure tolerances than Teflon coils. The HPLC postcolumn reaction method was evaluated in terms of linearity, sensitivity, accuracy, precision, and ruggedness. The linear dynamic ranges for tetrodotoxin and 4,9-anhydrotetrodotoxin were 40–3000 and 80–3000 ppb, respectively. The limits of detection and quantification were 12 and 41 ppb, respectively, for tetrodotoxin and 26 and 85 ppb, respectively, for 4,9-anhydrotetrodotoxin. The accuracy was evaluated by recovery measurements and the values for tetrodotoxin were between 90.7 and 93.6%. The use of a volatile perfuorocarboxylic acid as an ion-pair reagent allows confirmation of analytes in sample matrix by liquid chromatography tandem mass spectrometry using identical mobile phase conditions.