D. Shea, ANALYSIS OF BREVETOXINS BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY AND LASER-INDUCED FLUORESCENCE DETECTION, Electrophoresis, 18(2), 1997, pp. 277-283
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.