| OBJECTIVE:1. To establish the postmortem distribution model of methamidophos in dogs; To establish the decomposition kinetics model of methamidophos in preserved specimens and buried cadaver of dogs2. To improve a GC equipped with an FPD and a GC-MS analysis for methamidophos determination.3. To investigate the postmortem distribution of methamidophos in dogs and the decomposition kinetics of methamidophos in preserved specimens and buried cadaver of dogsMETHODS:1. Study on postmortem distribution:Six dogs were given an intragastric administration of methamidophos with a dose of 8LD50. As soon as the blood pressure, respiration and ECG disappeared, the dogs were dissected, and the specimens such as heart, liver, spleen, lung, kidney, brain, gastric wall, heart-blood, bile, breast muscle, right hindlimb muscle, the right forelimb muscle were collected and preserved at-20℃. After the acidification the specimens were extrcted with dichloromethane. Analysis was performed with a GC equipped with an FPD and a GC-MS. The analysis identification was based on retention time in the chromatographic system coupled with the ion fragmentation spectrum in the mass spectrometer. The quantitative analysis was on an internal standard method.2. Study on the decomposition kinetics of methamidophos in preserving specimen:After being given an intragastric administration of methamidophos with a dose of 8LD50,6 dogs were executed, and the blood and liver of every dog were sampled and divided into four parts. Three of them were preserved at 20℃,4℃,-20℃respectively. Another blood containing l%NaF or liver fixed with 4% formaldehyde solutionwere preserved at 20℃. Methamidophos in them was determined by a GC equipped with an FPD and a GC-MS at 0h,7d,16d,60d and 180d. The mean C-T data of methamidophos in preserved blood and liver was manipulated by WinNorLin's pharmacokinetics software. The kinetic model was decided by AIC information criterion. The decomposition kinetic equation and parameter were estimated and calculated.3. Study on the decomposition kinetics of methamidophos in buried cadavers.(1) Decomposition kinetics of methamidophos in buried cadavers:30 dogs were given an intragastric administration of methamidophos with a dose of 8LD50. As soon as the blood pressure, respiration and ECG disappeared, the dogs were put into plastic unsealed bags, and buried in the field in East Mountain, Taiyuan City. Three of them were dugged out, dissected and the specimen were collected for analysis of methamidophos at 0d,29d,58d,89d,139d,211d and 225d after the burying by a GC-FPD and GC/MS.(2) The effection of burial way on the decomposition kinetics of methamidophos in buried cadavers. Nine dogs were given an intragastric administration of methamidophos with a dose of 8LD50. As soon as the blood pressure, respiration and ECG disappeared, the dogs were put into plastic unsealed bags, woven bags and wooden cases (coffins) respectively and buried in the field in East Mountain, Taiyuan City. They were dugged out, dissected and the specimen were collected for analysis of methamidophos at 75d after the burying by a GC-FPD and GC/MS.(3) The effection of burial temperature (season) to the decomposition kinetics of methamidophos in buried cadavers:Six dogs dogs were given an intragastric administration of methamidophos with a dose of 8LD50. As soon as the blood pressure, respiration and ECG disappeared, the dogs were put into plastic unsealed bags respectively. Three of them was buried in 09/3/28 in the field in East Mountain, Taiyuan City. The other three of them was buried in 09/8/24. They were dugged out, dissected, and the specimen were collected for analysis of methamidophos at 75th day after the burying by a GC-FPD and GC/MS.4. Statistics Data was expressed as mean±SD model and analysed statistically with SPSS 11.5 software by One-Way ANNOVA's t-test.RESULTS:1. Postmortem distribution:The order of methamidophos concentration detected in poisoning death dogs was as follows:stomach (99.84±60.87μg/g)> spleen (46.87±28.67μg/g), liver (43.82±22.74μg/g), kidney (43.79±29.04μg/g), heart-blood (35.36±13.98μg/ml), lung (35.25±18.59μg/g), urine (34.81μg/g)> breast muscle (19.23±17.18μg/g), right forelimb(16.92±8.98μg/g)> heart (15.09±6.11μg/gμg/g), right hindlimb (12.83±7.63μg/g)> brain (10.91±4.13μg/g), bile (6.75±1.45μg/ml), vitreous humor (6.22±4.97μg/ml) (t-test, p<0.05).2. The decomposition kinetics of methamidophos in preserving specimen Methamidophos content in preserved blood and liver of dogs showed a sharp descend followed by a slow one. Methamidophos concentration detected in preserved blood at 20℃,4℃,-20℃,20℃(1%NaF)descended significantly to 55.1±18.2%,73.7±33.9%,61.5±11.0%, 4.2±3.5%(p< 0.05) and 0.2±0.2%,1.4±1.2%,4.5±2.1%,0.6±0.2%(p< 0.05)of initial concentration on 7th day,16th day,7th day,4th day and on 180th day,180th day,180th day and 12nd day; Methamidophos content detected in liver preserved at 20℃,4℃,-20℃and 20(fixed with 4% formaldehyde) descended significantly to 25.8±12.7%,36.7±16.8%,51.2±14.1%, 6.9±2.2%(p<0.05) and 2.6±1.5%,2.9±1.1%,5.2±2.8%,2.1±0.3%) on 7th day,7th day,16th day,7th day and on 180th day. Except for in 1%NaF blood group, the decomposition kinetics of methamidophos in the other preserved blood and liver met the tow compartment open model with a first order kinetics, and could be expressed as C,=Co-αt+Cle-βt. In 1%NaF blood group that met a one compartment open model with a first order kinetics and and could be expressed as Ct=Co-αt.The fast and slow decomposition half-lifes (t1/2αand t1/2β) of methamidophos in livers of poisoned dogs, which were stored at 20℃,4℃,-20℃and 20℃(in 4% formaldehyde) were 3.37 days,9.46days,16.28 days,1.38 days and 0 day(none),354.72 days,195281.2 days,113.93 days. The fast and slow decomposition half-lifes (t1/2αand t1/2β) of methamidophos in preserved blood of dogs, which were stored at 20℃,4℃,-20℃and 20℃(1%NaF), were 2.38 days,14.42 days,10.04 days,0.68 days and 15.61 days,47696.27 days,206.78 days. The observed value of mean methamidophos concentration was close to the predicted value calculated with Ct=Co-αt+Cle-βt or Ct=Co-αt.3. Decomposition kinetics of in methamidophos in buried cadavers of dogs Methamidophos content detected in buried cadavers of dogs, which died from poisoning after an intragastric administration of 8LD50, showed a rise followed by a descend in 225 days. Methamidophos contents in heart, liver, spleen, lung, kidney, stomach and heart-blood ascended from 100% on 0th day to 427.5%±154.7%-1270.9%±1136.2% on 29th days, then descended to 10.5%±2.4%-86.7%±32.8% on 88th day and 1.1±0.6-6.4±2.6% on 225th day. Methamidophos content in right forelimb, right hindlimb, breast muscle and brain ascended from 100% on 0th day to 4254.6%±246.2%-304.2%±43.7% on 58th day, then decended to 9.8%±3.8%-27.2±8.1 on 139th day and 0-6.0%±2.3% on 225th day.The research on the burial way's affection showed that methamidophos content detected in the heart, spleen, lung, kidney, brain and breast muscle of dog cadavers buried in plastic bags on 75th day was significantly higher than those in woven bags. Methamidophos contents detected in the spleen, kidney and brain buried in the plastic bags were significantly higher than those buried in the wooden boxes (coffins).The research on the burial seasons's affection showed that methamidophos content detected in the heart, stomach, right hindlimp of dogs buried from March to June was significantly higher than those buried from August to November. There was not a statistics difference in methamidophos contents detected in liver, lung, kidney, right forlimp and breast between dogs buried from March to June and from August to November. Methamidophos contents detected in dog cadavers buried from March to June was 3.74±0.84μg/ml, while no heart-blood was sampled from those buried in August-November.CONCLUSION:1. The postmortem distribution, decomposition kinetics model of methamidophos (8LD50,ig) in dogs have been developed, which can be applied to forensic identification and study on forensic toxicokinetics of decomposition of methamidophos poisoning death case.2. The developed GC-FPD and GC/MS analysis, of which recovery, linear range and sensitivity meets the demand for analysis of poison in biological specimen, can be used in the forensic identification and forensic toxicokinetics study of methamidophos poisoning death case.3. The order of the methamidophos concentration detected in poisoned death dogs after an 8LD50 dose is stomach>spleen>liver> kidney> heart-blood> lung>urine> breast muscle>right forelimb> heart> brain>bile>vitreous humor. Besides blood, stomach, spleen, liver and kidney should be taken for analysis in the forensic identification of methamidophos poisoning case. The postmortem distribution relation can be applied to estimating the methamidophos concentration range in blood in extreme case (example for dismembered body).4. Methamidophos in preserved blood and liver can be decomposed. Specimen's being stored at low temperature can make the decomposition slows down. It suggests that the specimen for analysis should be submitted within 7-16 d in the forensic identification of methamidophos poisoning death case, otherwise, they should be frozen, submitted and analysed as soon as possible. The bacteriostat (NaF) or aseptic (formaldehyde) are forbidden to be added in preserved specimen.5. The decomposition kinetics of methamidophos in stored blood and the liver meets the one or tow compartment open model with a first order kinetics. In the forensic identification of methamidophos poisoning death, the equation as Ct=Co-αt+Cle-βt or Ct=Co-αt and decomposition kinetics parameter can be used to estimated the methamidophos concentration range in the specimens at sampled time, and provide a scientific evidence for the forensic identification of methamidophos poisoning death case.6. Methamidophos content detected in buried cadavers of dogs with 8LD50 showed a rise followed by a descend in 225 days, in which the highest one was on 29th or 58th day. By 225th day after the burial, methamidophos ccould had been detected in the heart, liver, spleen, lung, kidney, brain, stomach, right forelimb, right hindlimp (Cmax:1.13±0.61μg/g). The buried way coule affected the decomposition of methamidophos, of which dogs buried in the woven bags and in the plastic bags were the fastest and slowest. The buried season also affected the decomposition of methamidophos, of which dogs buried in March-June was slower than in August-November. In the forensic identification of buried cadavers of methamidophos poisoning death, combined with the administration way of poison and antemortem treatment, the value and possibility of cadaver-dugging should be estimated according to the effection of buried time, buried way and buried season on decomposition of methamidophos. Cadaver-dugging and toxic analysis should be carried out as soon as possible. It can approximately be estimated the methamidophos concentration range in buried cadavers according to the poision analysis and decomposition rule.
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