The Study Of The New Detecting Method Of The Metobliziton And Of Fluoroacetamide And The Distribution In Tissue

Objectives1.To establish a new apply to on-site testing method for determining of F- through the chip detection method of the electrochemical studies.2. Development of portable, easy chips technology for detecting F- and complete portable F- fast detector.3. To establish a method of high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) for determination of fluoroacetate acid and fluoride citrate4. To establish a model of poisoned rabbit by oral sodium fluoroacetate.5. Research the law of metoabolism of fluoroacetamide and other regicides including fluorine and the pretreatment of samples and distribution in routine cases, to provide scientific basis for forensic identification for Samples taken, testing, result analysis, determine the cause of death and provide scientific basis for forensic identification.6. Research the poisoned rabbits in vivo distribution of fluoroacetate acid and fluoride citrateMethods1. Animal ModelOf 12 rabbits were randomly divided into two groups of six. The first group (six rabbit) is blank control group, normal diet fed; the second group (six rabbit) took lethal dose of oral gavage (10.Omg/kg body weight) of fluoroacetamide.2. Samples taken and treatmentThe poisoned group naturally died by oral medications, then immediately autopsy blood, urine, heart, liver, kidney, brain and other commonly seized material, the control group died from air embolism ear vein, take the same review materials, used as control.3. The research of pre-treatment methods of the fluoride ion:an analysis of different protein precipitation, pH values in different conditions and different temperature conditions on the effects of fluoride ion extraction recovery, optimization of the fluoride ion extraction.4.To optimize the methods of LC-MS/MS for determining fluoroacetate acid and fluoride citrate:use of retention time (Rt)and multiple reaction monitoring (MRM) was used for qualitative and quantitative analysis of fluoroacetate acid and fluoride citrate. 5. The extraction method of the metabolites of fluoroacetamide and other regicides including fluorine.Biological Tissue Samples (such as the liver, kidney, etc.) with the homogenizer mixing machine broken. Take lml blood or 1g organization material samples seized by adding 3ml of water/acetone (1:4, v/v),μltrasound 8 minutes, centrifμge 5 minutes at 4000r/min to remove protein and extract the collection, repeat the extraction process 3 times. After the combined extract three times, add 200.Oμl 10%of diethanolamine alkalization then under the conditions of air drying at 70℃, methanol fixed volume, over membrane for inspection.6. LC-MS/MS Detection:use the retention time and choice of 2-3 pairs of ion characterization for qualitative, Curves do by A pair of ions on the response characteristics of the relatively high peak area and their concentration, using external standard method for quantitative.7. Statistics:SPSS 11.5 statistic software was used for data analysis and output was mean±standard deviation (X±S)Resμlts1. The linear range of Fluoride ion-selective chip is 100～10-6mol/L, linear correlation coefficient of 0.9994. The average slope is 58.14, close to the theoretical slope of 59.35. The detection limit of Fluoride ion-selective chip is 5×10-7mol/L, The average recovery was 76.62%(RSD%= 1.21%).2. In the 10-3～10-6mol/L NaF standard solution, the fluoride ion-selective chip response time<2min, at 10-6～5×10 -7mol/LNaF standard solution, the fluoride ion-selective chip response time<5 min.3. Using the same chip at room temperature to continuous determine of 10-3and the 10-5mol /L standard solution five times, RSD%were less than 0.43 and 0.90; using the same chip at room temperature days to determine of 10-3 and 10 -5 mol/L standard solution once for 5 days, RSD%less than the 0.54 and 1.23, respectively.4. Chip pH Scope:The fluoride ion-sensitive membrane is the optimum pH range at pH 5.0-6.0.5. KF-,OH-about is 10, which meansthe response of chip for F- is 10 times than the OH-KF-,Cl-is about 1000, KF-,Br-and KF-,I-about 1300. 6. Rabbit in the treatment group appears mandatory spasm, paroxysmal convulsion symptoms after administration 1～1.5 hours and died within 2 hours. Rabbit blood poisoning fluoride ion concentration higher 5 to 6 times than the control group; liver, kidney and brain tissue fluoride ion content than the control group increased 2 to 4 times, each index increased significantly (P<0.01).7. LC-MS/MS Method:biological samples in the main toxic metabolite of fluorine-containing fluoroacetic acid and fluoride citrate through a multi-ion monitoring (MRM) mode can be better separated, fluoroacetic acid and fluoride citrate respectively 5.0μg/ml～200.0μg/ml,50.0μg/ml～200.0μg/ml range showed a good linear relationship, correlation coefficient R2 were 0.9995,0.9902, the lowest limit of detection (LOD), respectively 5ng,50ng.8. Fluoroacetic acid and fluoride citrate can be detected from blood,cardiac,liver,kidney, and Fluoroacetic acid can be detected from stomach content. The concentration of Fluoroacetic acid and fluoride citrate in heart isin low levels,comparing with other tissue the heart showed significant difference (P<0.01).The sequence of fluoride citrate content in rabbit is as follow: blood> liver> kidney> heart> stomach contents (concentration= 0);.The sequence of fluoroacetic acid content in rabbit is as follow:stomach content> kidney> liver> blood> heart.Conclusions1. The study subjects of the chip for testing fluoroacetamide and other regicides including fluorine with fluoride ion selective electrode for fluoride ion in high selectivity, sensitivity and potential analysis of simple, convenient and other characteristics, this technology will greatly shorten the testing time, reduce testing costs, and portable testing equipment and realize fluoroacetamide toxins such as on-site determination of fluoride, in order to ban the sale of the use of highly toxic rat poison, rat poison prevention groups, provide the basis for the occurrence of poisoning. The sample to the laboratory testing laboratories to move the road into a field service line, will detect toxic substances as soon as possible to save people's lives, in order to save the patient to gain time, save a lot of manpower and material resources to provide simple, practical method. This method is particμlarly suitable for the promotion at the grassroots level, so that a large number of cases of criminal poisoning of evidence inspection work can be completed at the grassroots level detection.2. Fluoride ion detection chip compared with other common measurement methods, such as ion chromatography, spectrophotometry, the test chip is low price, easy to operate, anti-interference ability and can be quickly detect F'content in the samples, so it can be widely used in environmental samples, body fluids, food content of F-, it is the preferred method for screening of F-content in real samples, test is a screening of F-content in the actual samples the preferred method. Fluoride ion chip samples are given directly to the concentration of fluoride ion, fast response, easier to achieve continuous measurement and automatic control is conducive to the development of a routine detection of fluoride content in a way. The chip can directly give the concentration of fluoride ion, fast response, easier to achieve continuous measurement and automatic control. So it is conducive to the development of a routine detection of fluoride content in a way.3. This study developed the HPLC-MS/MS method of detecting the main toxic metabolite of fluorine-containing fluoroacetic acid and fluoride citrate in biological samples.It is high recovery rate, high sensitivity, good reproducibility and the method is accurate, reliable, easy to operate, the sample does not require derivative treatment, column long life, suitable for biological samples such as fluorine-containing Fluoroacetamide rapid detection of toxic metabolites.4. Fluoroacetic acid and fluoride citrate is generally distributed in blood and tissues of rabbit.The content of fluoride citrate are highest in blood; The concentrations of fluoroacetic acid are highest in stomach content.