Quality Analysis Of Tetrodotoxin And Its Pharmacokinetics Study

Tetrodotoxin, TTX, its molecular formula is C11H17N3O8, relative molecular weight is 319.1016, which is a non-protein, low molecular weight, high active neurotoxin.The toxicity of TTX is 1000 times stronger than cyanide. Tetrodotoxin mainly exists in the puffer fish testis, ovary, liver, spleen, roe, eye and blood. In 1909, Japanese scholar Tahara Ryojun extracted a kind of crude toxin from the dolphin species eggs, named tetrodotoxinOn the other hand, TTX has many pharmacological effects such as analgesic, regional anesthesia and spasmolysis. It is especially capable of easing many dull pains and sharp pains obviously. Clinical, TTX injection can be used instead of morphine, dolantin, atropine and South America tubocurarine. Given these conditions, we should search scientific and accurate methods for TTX quality control and determination of TTX in plasma as soon as possible.1、The quality analysis of tetrodotoxin.First, an LC-MS/MS method for the determination of Tetrodotoxin (TTX) in Fugu roe and liver was established. The analyte was vortex-mixed, extracted with acetonitrile by ultrasound, and followed by centrifugation. The separation was performed on an Innovation HP Amide column (100×3.0 mm,5 μm) using acetonitrile-0.3% formic acid (v/v,70:30) as mobile phase. Quantification was through tandem-mass spectrometry with positive electro-spray ionization (ESI) and multiple reaction monitoring (MRM) at m/z 320.1�'162.1 for TTX. Calibration curve was linear over the TTX concentration range of 0.0313-2.00 μg·mL-1, (r= 0.9999). The lower limit of quantification of TTX was 0.30 ng·mL-1. The average recovery of TTX was 95.9-103.9%. The validated method was shown to be simple, sensitive, rapid, reproducible and suitable for the determination of TTX in Fugu roe and liver.Then, the purity of tetrodotoxin was determined and the related substances were researched, using a quick liquid chromatography-high resolution mass spectrometer and Peakview software. The purity of tetrodotoxin was more than 99%.It was also founded three kinds of related substances which was, m/z 482,304,272, and their structures were proposed.Last, a determination method of tetrodotoxin in the content of tetrodotoxin soft capsule was established. The separation was performed on a ZORBAX RX-SIL column (4.6x 150 mm,5μm) using methano 1-0.1% formic acid (v/v,60:40) as mobile phase. The linear range was 0.0125-0.800 μg·mL-1, the recoveries were between 95.00%-100.32%, the method was simple, rapid, high sensitivity, suitable for the quantitative determination of tetrodotoxin.2, Study on pharmacokinetics of tetrodotoxin in rat. An LC-MS/MS method for determing tetrodotoxin (TTX) concentration in rat plasma was developed and its pharmacokinetics in rats was studied. The analytes were extracted from plasma samples by liquid-liquid extraction and matrine was selected to be the internal standard. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The ion combination of m/z 320.1�'162.1 and m/z 249-1�'148.1 was used to qualify TTX and the internal standard, respectively. After a single oral dose of 8 μg·kg-1 TTX, the concentrations of TTX were detected and the pharmacokinetic parameters were calculated by DAS 2.0. The calibration curve was in good linearity over the range of 0.625-80 ng·mL-1 (r=0.9992) for TTX. The extraction recovery was higher than 85%. The main pharmacokinetic parameters of Cmax,Tmax and T1/2 were (1.874±0.803)μg·L-1,(1.305± 0.573) h, (9.761±4.687) h for TTX, respectively. The method is shown to be simple, accurate, sensitive and specific for the simultaneous determination of TTX in rat plasma and can be used for the pharmacokinetic study.