Evaluation of Helically Coiled and Knitted Open Tubular Reactors for the Efficient Post-Column Determination of Tetrodotoxin by High-Performance Liquid Chromatography

Abstract

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.

Tetrodotoxin and Its Analogues in Food: Recent Updates on Sample Preparation and Analytical Methods Since 2012

“…Sometimes, TTX standards contain its analogues, for example, a TTX standard solution from CIFGA (Lugo, Spain) contains TTX, 4,9-anhydroTTX, 11-deoxyTTX, 4-epiTTX, and trace levels of 5,6,11-trideoxyTTX and 11-norTTX-6-ol . However, almost all the analogues of TTX are not commercially available, and they have to be isolated from available sources (with further chemical modification in some cases), except 4,9-anhydro TTX, which is available from NRC (Halifax, Nova Scotia, Canada), Cifga (Lugo, Spain), or Biomol GmbH . Future availability of certified reference materials for various TTX analogues may provide new opportunities to extend the validation and application of different methods for TXT and its analogues …”

“…For one thing, the use of reversed-phase columns for the direct separation of TTX and its analogous is limited, because they are highly polar and hard to sufficiently retain in these columns. For another, ion-pair reagents, such as heptanesulfonic and heptafluorobutyric acid, are usually required to improve analytes retention when using a C 18 column, but these reagents can negatively affect MS measurement. Hence, to solve the problem, a hydrophilic interaction chromatography (HILIC) column becomes a preferable choice.…”

“…Because TTX does not exhibit a natural fluorescence intensity, postcolumn derivatization using NaOH is usually needed, and the excitation and emission wavelengths are set at about 365 and 510 nm to detect the fluorescent products. , The detection of TTX and its analogues (4-epiTTX and 4, 9-anhydroTTX) in pufferfish using HPLC-FLD were first developed by Yasumoto and Tooru . Lately, Tanis et al established an efficient HPLC-FLD postcolumn derivatization method for the selective determination of TTX and 4,9-anhydroTTX. In this work, helically coiled and knitted open tubular reactors were evaluated and compared, and results revealed that knitted reactors are more efficient in terms of sensitivity and band broadening.…”