| Amnesic shellfish poisoning (ASP) is a kind of strong nerve toxin which is caused by a marine diatom-Quasi nitzschia Pseudo-nitzschia sp, and its chemical name is domoic acid. The first time since 1987 the collective shellfish poisoning occured in Canada, people detected domoic acid from the red tide algae, diatoms nitzschia multi-line spikes. Fish and shellfish filter poisonous algae for food so that domoic acid is enriched in the body. Human poisoned by eating domoic acid contaminated fish or shellfish. In order to prevent poisoning incidents and ensure food safety, Canada made the maximum safe limit of domoic acid in shellfish products, it was 20μg/g. The United States, Europe, Australia and other countries and regions have also adopted this standard. According to EU Commission Directive (2002/226/EC), shellfish meat were forbidden to harvest and come into the market if domoic acid content of these products exceeded the limit.Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) is another emerging technique for the separation and analysis after LC-MS and GC-MS. And has proven potential for development compared with GC-MS. GC-MS was used to analyze volatile, stable and low molecular weight compounds. LC-MS/MS can compensate the lack of GC-MS, it also has advantages such as greater separation ability, small amount of sample, short analysis time, high selectivity, high sensitivity. It has been widely used in studies of protein and peptide genomics, drug metabolism mechanism, kinetics, biochemistry, organic chemistry, environmental analysis, public health immunization and food inspection and other fields. This thesis is divided into two parts, the first part established the detecting method of domoic acid in shellfish residues by liquid chromatography-tandem mass spectrometry. It provides a reliable analysis for accurate and rapid determination of domoic acid in shellfish residues. The second part is domoic acid accumulation in mice, mainly study the distribution and metabolism of domoic acid in mice various organs.One of the purposes of this paper is to establish a liquid chromatography-tandem mass spectrometry method to detect of domoic acid in shellfish residues. According to the most of the literature, HPLC-UV method was always used for domoic acid analysis. The detection limit (LOQ) was 1μg/g. The detection limit of HPLC-TOF/MS method was 0.1μg/g. This paper established a liquid chromatography-tandem mass spectrometry method to determine domoic acid in shellfish. This method is more sensitive, more accurate, and has recognition function to meet current international requirements for toxic substances testing. Determined as follows: optimizing sample preparation methods, samples were extracted with 50% methanol, purified by LC-SAX column, eluted with 3mL 0.1mol/L formic acid; in the selection of chromatographic conditions: injection volume: 25μL; column temperature: 30℃; mobile phase: A: methanol, B: 0.02% formic acid +2 mmol/L ammonium formate solution; flow rate: 0.35mL/min. Then optimized MS conditions: electrospray ionization (ESI), in the positive ion, multiple reaction monitoring mode (MRM) for qualitative and quantitative. The qualitative ions were the m/z 311.98/265.91, m/z 311.98/247.9, m/z 311.98/192.91, and m/z 311.98/265.91 was the quantitative ion. The qualitative method was external standard method. Experimental results show that the method detection limit is 0.01μg/g, limit of qualitation was 0.02μg/g. In the range of 0.02~10μg/mL, the linear correlation coefficient is 0.9999. When the concentration of domoic acid was added 20~1000ng/g, the average recoveries of domoic acid in scallop sample ranged from 81.3%~105.4%, RSD ranged from 3.90%~8.90% (n = 6) ; the average recoveries of domoic acid in the buffy clam sample ranged from 91.8%~95.2%, RSD ranged from 5.70%~9.20% (n = 6); the average recoveries of domoic acid in razor clam sample ranged from 78.0%~90.8%,RSD ranged from 2.00%~4.20% (n=6); the average recoveries of domoic acid in oyster sample ranged from 83.5%～106.6%, RSD ranged from 4.60%～6.40%.( n=6 ) .The method is reliable, sensitive and can meet the requirement for determination of domoic acid in shellfish residue. The second purpose of this paper is to study the distribution of domoic acid in various tissues in mice and the metabolism in mice vitro. First, we verified if the liquid chromatography-tandem mass spectrometry method for determination of domoic acid in shellfish residues was applicable to determine domoic acid in various organs in mice. Experimental results showed: when the added concentration of domoic acid ranged from 20~500ng/g, the average recoveries of domoic acid in liver sample ranged from 83.4%~86.3%, RSD ranged from 5.70%~7.20%; the average recoveries of domoic acid in stomach sample ranged from 79.4%~85.4%,RSD ranged from 5.60%~7.40%; the average recoveries of domoic acid in muscle sample ranged from 81.0%~85.9%, RSD ranged from 3.90%~7.70%; the average recoveries of domoic acid in kidney sample ranged from 78.3%~79.4%, RSD ranged from 4.70%~6.30%; in the range of 0.02~10μg/mL, the peak area of domoic acid responses was linear with the amount of domoic acid. The correlation coefficient was 0.9999. The quantification limit of the method was 0.02μg/g. Therefore the method was suitable for detection of domoic acid in mouse tissues.To study the behavior of mice characteristic, the distribution and excretion of domoic acid in mice tissue, intraperitoneal injection was used to be the method of mice expoure. The liquid chromatography-tandem mass spectrometry method was used to determine the concentration of domoic acid in mouse tissues. Mice were injected at the dose of 0.45μg/g, Kunming male mice were injected with the toxin at different time (1h, 4h, 7h, 10h, 24h, 48h, 72h, 120h) using intraperitoneal injection to determine domoic acid in mice distribution. The results showed that after the mice injected with domoic acid for 1-120 hours, domoic acid mainly distributed in the liver, kidney and stomach. The concentration of domoic acid in various tissues of mice was: liver> stomach >kidney. Five other organs including heart, spleen, lung, blood and muscle had only trace amounts of domoic acid in a short time. 10 hours after the toxin injected, the cencentration of domoic acid in five tissues was lower than the detection limit.At the same time, we also studied the domoic acid accumulation rate in vivo and metabolic rate in vitro in mice. By analyzing the mice feces and urine, we found that after the injection of the toxin for 1-120h, domoic acid in mice in vitro discharge rate basically increased gradually, and domoic acid metabolism in vitro in mice reached the maximum 81.8% at 72h after injection; domoic acid accumulation rate in mice gradually decreased, domoic acid in mice accumulation rate decreased to 5.6% after the injection of 120h. Thus domoic acid accumulation was small in the mice, and metabolism rate was high. Experiments proved that domoic acid in mice have a certain volume within a short time. One week later, 80% of domoic acid toxins were excreted by the body. The metabolism rate of domoic acid toxins is fast. If it is accidentally consumed domoic acid contaminated seafood (domoic acid content is less than food safety standards), as soon as possible to discharge domoic acid toxins from the body. |
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