ANALYSIS OF BREVETOXINS BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY AND LASER-INDUCED FLUORESCENCE DETECTION

Micellar electrokinetic capillary chromatography (MEKC) with laser-ind uced fluorescence (LIF) detection was used to measure four red tide br evetoxins at sub-attomole levels. The separation of four brevetoxins b y 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 produc ts. Brevetoxins with a terminal aldehyde group were reduced to the alc ohol with sodium borohydride prior to derivatization with the coumarin . Three derivatized brevetoxins (PbTx-3, PbTx-5, and PbTx-9) were sepa rated by MEKC and detected using He/Cd laser excitation at 354 nm and fluorescence emission at 410 nm. A fourth brevetoxin (PbTx-2) was conv erted to PbTx-3 prior to derivatization and was then determined by sub traction. Instrumental detection limits for all four toxins were appro ximately 0.10 fg or about 10(6)-fold more sensitive than existing liqu id chromatographic methods. Brevetoxins were isolated from cell cultur es and fish tissue using an alumina column/gel-permeation chromatograp hy procedure. Method detection limits fbr the brevetoxins in fish tiss ue were approximately 4 pg/g. These method detection limits are at lea st 100-fold better than previous chromatographic and/or electrophoreti c methods. The MEKC-LIF method reported here allows measurement of bre vetoxins at the trace levels considered critical for understanding tox in metabolism and mode of action.