GENERAL OBSERVATIONS ON SAMPLES RECEIVED 



AQ samples ippeared to be of bver, with « fibrous constitution consistent with dolphin bver examined 

 in the past. Samples ranged in size from a low mass of 12.411 g to a high mass of 110.614 g. Twenty-eight 

 of the samples weighed below the ^jcdfied 45-60 g. Twenty-dght samples were judged to be of good condmon 

 when received with no evidence of decomposition, eight were slightly freezer-burned, an additionaJ four were 

 severely freezer-burned, and ten were received in homogenized condition, except as noted for sac. no sample 

 was deemed unsuitable for testing. Samples were numbered in the laboratory as #]-#50 based on leoeipt and 

 were not re-correlated with NMFS identification untS completion of assays. 



BJOASSAY DURING PURIFICATION 



Based on the size of a substantial number of the sanq>les, bioassay was not performed at neps (1) 

 through (9) of the "Protocol For Liver Analyses: Brevetoxins in Dolphin Liver" [labeled ITEM #1J. Further, 

 based on the consistent quality of the supplied samples, note [2] 'Aberrant Saiiq>les" did not tppYf. 



At step (10) in the protocols, the first preparative thin-layer silica gel diromaiogr^hic step, thirty-five 

 (35) of the samples were non-taodc by Gambtaia affitiis fish bioassay and analyses were terminated in these 

 cases. Negative sao^les at this stage were: 



The remaining sao^les tested positive by fish bioassay in at least one fraction of the panected thin- 

 layer dut>matography plate. Nine of the samples were tone in multiple sections of the plate indicating a 

 multiplicity of toxic materials. Of the samples which tested positive at the first TLC plate, twelve tested 

 negative following thin-byer duomatography on the second plate (stq> 12). The samples «iuch tested negative 

 at this step were: 



Following this diromatogn^hic step, five (5) san^les remained for further analysis. Of these, OO094, 

 2505-5, and 2505-6 were judged to be of limited quantity and were not subjected to the third thin-layer step 

 (Rq> 14). Samples 2505-9 and 2505-10 possessed toxic fractions following cfaromatognqihy according to Step 

 #14_ 



HIGH PERFORMANCE UQUID CHROMATOGRAPHY 



Based on migration of standard brevetoxins, san^les CX3)94, 2SQ5-S, and 2505-6 were negative by 

 HPLC That is to say, they contain < 5 Mg toxinAotal original sample. Samples 2505-9 and 2505-10, when 

 liPLCd, indicated the presence of PbTx-2 and this was confirmed by mixing with authentic PbTx-2 and re- 

 injecting. Co-migration of the mixed samples further suggested authenticity. 



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