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

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 PbTx-9) were separated by MEKC and detected using He/Cd laser excitation at 354 nm and fluorescence emission at 410 nm. A fourth brevetoxin (PbTx-2) was converted to PbTx-3 prior to derivatization and was then determined by subtraction. Instrumental detection limits for all four toxins were approximately 0.10 fg or about 10(6)-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.