Determination Of Tetrodotoxin In Difference Tissue Of Pufferfish By Mouse Bioassay And HPLC

Investigate the toxicity of pufferfish which were collected from dandong,qingdao and nantong, they are Fugu pseudommus,Fugu flavidus,Fugu rubripes and Fugu xanthopterus.In this paper determine TTX in difference tissue of pufferfish by mouse bioassay and HPLC.Mouse bioassay is the earlist detection standard of TTX, which established by American scholar at 1940's. The principle bases on the derect proportion relationship between the reverse death time of mouse and received TTX of unit weight, established the curve of reverse death time of mouse-TTX.Now the method is the authority detection method of TTX in Japan. The toxicity is showed by MU, 1MU means the content of TTX which can kill a male mouse in 30 minutes.(1MU=0.22μg TTX).The result of The Mouse bioassay is ,the content of TTX in differ tissue is diverse. The ovary is the most toxin, the next is liver, the last is muscle and skin, but confined by the reason., breed and quantity ,some statistic still need farther consummated, the quality of TTX and the rate of toxicity is diverse , though they are in same area and breed. The recoveries rare is 86.35%-88.03%, liver is 1-171.20MU/g, ovary is 1-739.68MU/g, muscle and skin is 1-10 MU/g.The sample preparation is pivotal to determine TTX by HPLC, included the sample extraction and purification involving the recovery and impurity removing. Many sample preparations were parallel with different researchers'report . First , the tissues were extracted with acetic acid , then applied to columns of activated charcoal to remove impurities. The adsorption is very strong , so the TTX is hard to be eluted , resulting in low recovery. Then the extraction was ultrafiltrated though membrane( cut-off limit 1000MW) or applied to a Bio– Gel P-2 column to remove macromolecules. At last , the liquid was applied to cation exchange resin two or three times to get rid of impurity . The chromatogram is good , but the recovery is low . It is hard to determine the content of TTX. This experiment used activated SPE HLB cartridges which can replace numerous and low effect purification process.It can be activated by CH3OH and H2O, By HPLC method, the chromatogram show the peak of TTX aparted from the matrix, and the recovery is high.At this experiment, a RP-HPLC method which associates with phosphate buffer and Photo-diode Array detector to determinate of TTX.method:Waters 600E;PDA detector;column Hypersil BDS C18 (5μm, 250mm*4.6mm i.d), mobile phase of phosphate(0.05mol/LNaH2PO4:0.05mol/LNa2HPO4=1:1, with Heptane-1-sulfonic acid sodium salt(5mmol/L) as an ion-pairing reagent, flow rate:0.6ml/min,column temperature:25℃,detection wave:205nm, injection volume :20μl. The linear relationship of calibration culibration curve was good in the range of 250-100mg/L for TTX(R=0.9998), The detection limit was 40ng, and the recovery was 91.42%-94.39%, the relative standard deviation was 2.16-3.14%. The method is simple, accurate and sensitive. It can be adopted to quantification of Tetrodotoxin in pufferfish.