A post-column 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 post-column reaction was based on tetrodotoxin conversion to a quinazoline fluorescent compound in strong alkaline conditions. All post-column 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 bases 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 1,500 μL polyetheretherketone knitted coil with 0.010″ internal diameter was optimum exhibiting higher pressure tolerances than Teflon coils. The HPLC post-column reaction method was evaluated in Downloaded by [University of Arizona] at 09:01 08 June 20163 terms of linearity, sensitivity, accuracy, precision, and ruggedness. The linear dynamic ranges for tetrodotoxin and 4, 9-anhydrotetrodotoxin were 40 to 3,000 ppb and 80 to 3,000 ppb, respectively. The limits of detection and quantification were 12 and 41 ppb for tetrodotoxin and 26 and 85 ppb 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 allowed confirmation of by highperformance liquid chromatography -tandem mass spectrometry using identical mobile phase conditions.