Analysis of brevetoxins by micellar electrokinetic capillary chromatography and laser‐induced fluorescence detection

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Abstract
Micellar electrokinetic capillary chromatography (MEKC) with laser‐induced fluorescence (LIF) detection was used to measure four red tide brevetoxins at sub‐attomole levels. The separation of four brevetoxins by MEKC was achieved with a sodium borate/sodium dodecyl sulfate buffer at pH 9.3. Brevetoxins with a terminal alcohol group were derivatized with an acyl azide coumarin to form stable, highly fluorescent products. Brevetoxins with a terminal aldehyde group were reduced to the alcohol with sodium borohydride prior to derivatization with the coumarin. Three derivatized brevetoxins (PbTx‐3, PbTx‐5, and Pb Tx‐9) were separated by MEKC and detected using He/Cd laser excitation at 354 nm and fluorescence emission at 410 nm. A fourth brevetoxin (Pb Tx‐2) was converted to Pb Tx‐3 prior to derivatization and was then determined by subtraction. Instrumental detection limits for all four toxins were approximately 0.10 fg or about 106‐fold more sensitive than existing liquid chromatographic methods. Brevetoxins were isolated from cell cultures and fish tissue using an alumina column/gel‐permeation chromatography procedure. Method detection limits for the brevetoxins in fish tissue were approximately 4 pg/g. These method detection limits are at least 100‐fold better than previous chromatographic and/or electrophoretic methods. The MEKC‐LIF method reported here allows measurement of brevetoxins at the trace levels considered critical for understanding toxin metabolism and mode of action.