unambiguous identification of saxitoxin using lc-ms/ms & hplc-fld

Saxitoxin (stx) is a member of the paralytic shellfish neurotoxins that are produced by certain marine dinoflagellates and freshwater cyanobacteria [1]. these organisms are responsible for harmful algal blooms (hab) or red tides. stx is transferred from toxin-producing algae to human through seafood such as shellfish, lobster, fish; or contaminated drinking water (from cyanobacteria) and cause intoxication. stx binds to the sodium channels in nerve cells, thereby blocking the influx of sodium ions into cells, preventing impulse-generation in the peripheral nerves and skeletal muscles, leading to paralysis, respiratory failure and death [2]. the lethal dose, ld50 (mouse, oral) is 263 µg/kg. it is also being included in the both chemical and biological weapon conventions (cwc & bwc) [3], so unambiguous identification of this neurotoxin is very critical. the hplc-fld data that usually is used for identification is not unequivocal, so for unambiguous identification, a data rich spectrometric technique (lc-ms/ms) is needed. the aim of this work was isolation of stx from some simple and complicated environmental samples and unambiguous identification using hplc-fld and lc-ms/ms (mrm) as confirmatory approach. sample preparation for simple water samples was developed using different solid phase extractions. finally, envi-carb cartridge was selected. for complicated matrix (such as orange juice, milk, sea water, algae and mussel), after protein precipitation using acetonitrile, at first lipophilic interfering materials were isolated using dsc-18 cartridge. then, after solvent exchange to water, an envi-carb was used for extraction of stx. other interfering matrix components were washed with water. stx was eluted with 2% formic acid, 20% acetonitrile solution, concentrated and analyzed using lc-ms/ms (mrm) and hplc-fld (after hydrogen peroxide pre-column oxidation, fluorescence detection (ex=350 nm, em=395 mn)).after optimization of methods and instrument parameters, the on-column limit of detection (lod) was determined 3 picogram. the results showed that stx (100 ppb, 1 gram of oyster homogenized material) was extracted and analyzed successfully. this method was used in the 6th opcw (organisation for the prohibition of chemical weapons) international biotoxin sample analysis exercise and results showed that stx was identified successfully in the samples. the method can be used for analysis of various environmental samples and seafood.