■Bto SOO bL nde am fiasks aader vaaniiB atpiraiMCL Arrfopc tfttuiiotu arc truuicrrco u> mojviau^ loag 

 ■ed SOO mL ranad boooB fiaikt and eadi  fiasb ewiponted oo a rotary evaporator operating at nduccd 

 flrtsuxre. Reclaimed ioKeal  placed is five pBoe Bed drmas for appropriate rtnpnaal. 



(4) Hooogenate fiber caken, togetber with the fiber papert, are placed back iato their mdivuluil SOO mL 

 beakerv muI ^OO mL chtorofarm it added. Hooogeaizaticxi and fibratioc it rcpeaiti 



(5) The cfalorofonn fihrates are added to the appropriate acetooe residues from ctep (3) and the chlorofons 

  remowed by rotary flasb-evaporation. [Note 1]. 



(6) Ib the SOO mL flasks «^n«ifm^£ the fiver residues, add 20 g dry silica gel (ICH chromatograpby grade 

 or better) and follow with 100 mL acetone. Swirl each flask nitQ the silica gel scours the residue from the flask 

 liie. and is uniformly tan to brown in color. Flash evaporate acetooe and apply the dried brown silica gel/liver 

 enraa to the top of a 120 mL silica gel flash chromatography column (Baker Chemic a l ). Add 2S mL acetone 

 to the original round bottom flask to rinse any remaining material from the sides and pipette this toUuion oo 

 top of the dried sibca gel in the column. R^ieat once. 



(7) Assemble the flash chromatography column resevoir and add Z7D oiL acetooe. Apply 4 psi nitrogen 

 (99.999%) pressure to the column qiparatus and ooDect aD ehoed niateiial into dean SOO mL loqg neck 

 round bonom flasks. Flash evaporate loivenl in each case. [Note 1]. 



(8) Repeat step (6). Substitute 20 mLehlorofonnimethanoLacetic add (100:10:1) flask rinse for the acetone 

 rinse in step (6). 



(9) AssemUe the flash chromatogra|^ column resevoir and add 310 mL cfaloroformaaethanokacetic acid 

 (100:10:1). Apply 4 pu nitrogen pressure to the column qiparatns and collect aD ehaed material imo a dean 

 SOO mL loog neck round bottom flasL Flash evaporate. [Nou 1, Note 2]. 



(10) The residue is redissolved m L0-2J) mL solvent (either acetooe or cUoroform is fine) and is appfied to 

 a preparative fluorescent silica gel presorbant thin-layer chrooiatqgraphy plate (20 x 20 cm., 1000 » **«i<*»»^». 

 Whatman PK6F or equivalent) using an Applied ScicBcrs TLC breaking system, nates are chromatogn^ihed 

 using 100 mL of mobile phase ***«i«*"£ ctf acetooedight prtrolrmn (30:70). [Note 1, Note 3). 



(11) Toxic fractions frt» the TLC plate are scrqied m a safety hood, groimd to a fine powder using a 

 porcelis mortar and pestle [Note 4), and ehtted from the siEca gd in 30 mL sintered ^ass fibers using 2S mL 

 afnot>f or methanol Eluted tooon is placed in individual SO mL round bottom flasks and flash evaporated. 



(12) Toadc frsefioBS tre redisstrfved m methanol, ud applied to semi-preparative fhioreseeat nlica gd thin- 

 layer chromatography plates C^ x 20 bO., SOO a thirknr.w, Ar^hr^i Uoiplate or equivalenl) and 

 chromatographed using 100 mL ethyl acetaieJight petrofeum (SChSO) as molnle phase. [Note 1, Note 3). 



(U) Repeal txep (11) for toobc fractions. 



(14) TooDC fractions are redissolved in methanol, and applied to semi-preparative fluorescent silica gel thin- 

 layer chromatograiAy plates (20 x 20 cm., SOO a riiifknrti, Anahedi Uniplate at equivalettt) and 

 chromatographed using 100 mL ethyl acetateJighi petroleinn (70-30) as mobile phue. [Note 1. Note 3J. 



(15) Repeat st^ (11) for tooac fraaioos. 



(16) Toobc fraetioes are £ssoh«d from SO mL round bottom flasks nsiag '"i"^*"*' (03-10 mL ^lic grade 

 methanoL High perfo r m ance liquid chromatography is performed in ana^ticd mode using a Ranun C-18 

 analytical reverse phase column (4j6 mm diameter x 2S ^ long) using isocratic 8S% methanol in water, 1.4 

 mL/minute flow rate. Effliimt is moohored at 215 am [Note 1). f^itmin are caEbrated with known 

 cooceatrationt of analytical grade purified brevetoxins FbTx-L PbTx-2, and FbTx-3, the &ree predominant toxins 

 m natural or cultured systems. Excepting note below and ""'**>^">£ steps, we «^ coofirm the identity of the 

 |^)lc peaks by mixing equal proportioos of anthentv brevetooon and suspect br e v tt oo un from samples. Mixed 

 samples would then be sAjeaed to oo-migratioo in hplc analysis. Quantification of the amount of bt e v ct oa an 

 m the origina] unknown samples can be calculated from hplc data. 



119 



