Analysis Of Toxic Components Of Horseshoe Crab And Rearchon Emergency Poisoning Detection Methods And Its Application

In recent years,food poisoning accidents witch caused by horseshoe crabs occurred frequently in our country,and already becomed a serious threat to people’s health and life safety.But the toxicity in horseshoe crabs which led to food poisoning accidents is not clear,and the toxicity and composition of each tissue is unknown.And,when the poisoning accident occure we are short of relevant emergency detection methods to handle it.So,detection methods for TTX and its analogues,lipophilic shellfish toxin,hydrophilic shellfish toxin in horseshoe crabs had been developed by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS),combining with pre-column derivation high performance liquid chromatography(HPLC)and mouse biological experiment to test the horseshoe crabs and analyse the composition and content differences between different tissues of horseshoe crabs in this study.The results showed that muscle had the greatest toxicity,followed by egg and connective tissue.TTX and its ramifications were detected in every tissue of horseshoe crabs,and the highest content was TTX.The samples were free from lipophilic shellfish toxin.Only low levels of dcGTX2 and dcSTX of hydrophilic shellfish toxin were checked out in egg and muscle.1 Confirmed the composition and content of TTX and its analogues in horseshoe crabsConfirmed the Chromatography mass spectrometry analysis condition of TTX and its analogues,developed a method for the determination of TTX and its analogues by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS).The horseshoe crabs samples were extracted by 3% acetic acid/methanol solution,Extract solution was purifies by MCX solid-phase extraction cartridges.The TTX and its analogues were separated on Atlantis HILIC Silica column,using acetonitrile and10mmoL/L formic acid-5mmoL/L ammonium formate solution as mobile phase and then detected by ultra performance liquid chromatography-mass/mass spectrometry under multiple reaction monitoring(MRM)mode.The results showed that TTX and its ramifications were detected in every tissue of horseshoe crabs by UPLC-MS/MS,and the highest content was TTX,followed by deoxyTTX.Muscle had the greatest toxicity,followed by egg and connective tissue in female horseshoe crabs.Muscle had the greatest toxicity,followed by connective tissue in male horseshoe crabs.In the muscle,egg and connective tissue of female horseshoe crabs maximum content of TTX were3.593μg/g,1.747μg/g and 1.406μg/g,and the maximum content of total content of TTX and its analogues were 3.977μg/g,1.978μg/g and 1.511μg/g.In the muscle and connective tissue of male horseshoe crabs maximum content of TTX were2.701μg/g and 1.324μg/g,and the maximum content of total content of TTX and its analogues were 3.344μg/g and1.598μg/g.2 Confirmed the composition and content of hydrophilic shellfish toxin in horseshoe crabsConfirmed the Chromatography mass spectrometry analysis condition of hydrophilic shellfish toxin,developed a method for the determination of hydrophilic shellfish toxin by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS).The horseshoe crabs samples were extracted by 80% acetonitrile(0.1% formic acid).Extract solution was purifies by Sep-Pak C18 solid-phase extraction cartridges.The hydrophilic shellfish toxin were separated on Atlantis HILIC Silica column,using 0.5%formic acid/acetonitrile and 0.5% formic acid-5mmoL/L ammonium formate solution as mobile phase and then detected by ultra performance liquid chromatography-mass/mass spectrometry under multiple reaction monitoring(MRM)mode.A detection method of STX,dcSTX and GTX2&3 was developed on the basis of literature review by pre-column derivation high performance liquid chromatography.The results showed that dcGTX2 and dcSTX were detected in horseshoe crabs by UPLC-MS/MS with a low level.DcGTX2 and dcSTX were mainly detected in the muscle and egg of horseshoe crabs.In the egg and muscle of female horseshoe crabs the content of dcGTX2 were 0.056~0.725μg/g and 0.045~0.366μg/g,and the content of dcSTX were 0.030~0.069μg/g and 0.012~0.062μg/g.Egg had the greatest toxicity,followed by muscle.In the muscle of male horseshoe crabs the content of dcGTX2 were0.435μg/g and 0.074μg/g,and the content of dcSTX were 0.010μg/g and 0.039μg/g.DcSTX were detected in muscle and egg of horseshoe crabs by pre-column derivation high performance liquid chromatography.In the egg and muscle of female horseshoe crabs the content of dcSTX were 0.022~0.063μg/g and 0.018~0.057μg/g.Egg had the greatest toxicity,followed by muscle.In the muscle of male horseshoe crabs the content of dcSTX were 0.029μg/g.Its content was slightly lower than UPLC-MS/MS method.The results of UPLC-MS/MS and pre-column derivation high performance liquid chromatography were essentially identical.3 Confirmed the composition and content of lipophilic shellfish toxin in horseshoe crabsConfirmed the Chromatography mass spectrometry analysis condition of lipophilic shellfish toxin,developed a method for the determination of lipophilic shellfish toxin by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS).The horseshoe crabs samples were extracted by methanol.The lipophilic shellfish toxin were separated on ACQUITY UPLC BEH C18 column,using 95% acetonitrile(20mmoL/L formic acid-2mmoL/L ammonium formate)and 20mmoL/L formic acid-2mmoL/L ammonium formate solution as mobile phase and then detected by ultra performance liquid chromatography-mass/mass spectrometry under multiple reaction monitoring(MRM)mode.The results showed that the lipophilic shellfish toxin were not detected in all 10 horseshoe crabs.4 Validated the toxicity of horseshoe crabs by mouse biological experimentThe horseshoe crabs samples were extracted by 0.1%(V/V)acitic acid.The clan grade ICR mice(18~22g)by intraperitoneal injection of extract solution.The survival time of mice under hyperthermia were observed and used to calculate the content of toxins in samples.The results showed that muscle had the greatest toxicity,followed by egg and connective tissue in the female horseshoe crabs,and the maximum content in muscle,egg and connective tissue were 16.96 MU/g(3.731μg/g),8.77MU/g(1.929μg/g),6.70 MU/g(1.474μg/g).The muscle had the greatest toxicity,followed by connective tissue in the male horseshoe crabs,and the maximum content in muscle and connective tissue were 14.18 MU/g(3.120μg/g)and 7.00MU/g(1.540μg/g).