| Objective: 1.To optimize a qualitative and a quantitative method for simultaneous determination of carbofuran and its main metabolite carbofuran phenol in biological specimens by GCMS/MS.2.To develop the decomposition kinetics method for carbofuran and carbofuran phenol in preserved specimens.3.In order to provide a scientific evidence for forensic identification of carbofuran poisoning death.Methods: 1.Sample extract and analysis After adding internal standard to the blood and liver,the samples were acidized and extracted with dichloromethane.Qualitative and quantitative analysis was performed by GC-MS/MS.2.The stability of carbofuran preparation for 0.1μg/m L,0.2μg/m L,8.0μg/m L carbofuran standard stock solution,stored in 4°C,-20°C refrigerator,detected the concentration of carbofuran at 0d,10.0d,20.0d,34.0d,respectively.The carbofuran standard solution was added to the blank blood(1m L)and liver(1g)respectively to prepar the concentration of carbofuran of 0.1μg/m L(μg/g),0.2μg/m L(μg/g),8.0μg/m L(μg/g).The carbofuran standard solution and citrate sodium(0.5mg/m L,2.5mg/m L and 20.0mg/m L)were added to the blank blood(1m L)and blank liver(1g)respectively to prepare 2.0μg/m L carbofuran solution containing anticoagulant.The samples added carbofuran and citrate sodium were extracted with dichloromethane.The organic layer were evaporating to dryness and sealed,then placed in 4°C refrigerator.The concentration of carbofuran were detected by GC-MS/MS at 0d,10.0d,20.0d and 34.0d,respectively.3.Study on the decomposition kinetics Six dogs(male)were given intragastric administration with 4LD50(13.5mg/kg)of carbofuran.The blood and liver of dogs were taken when the breath and heartbeat of dogs completely disappeared.The blood was divided into five equal parts and preserved at 20°C(1% sodium fluoride),20°C(natrium citrate),20°C,4°C,-20°C.The liver was divided into four equal parts and preserved at 4% formaldehyde solution,20°C,4°C,-20°C.The concentration of carbofuran and carbofuran phenol were detected at different time.The equation and parameter of decomposition kinetics were imitated and calculated with 3p97 software program.The decomposition half-life of carbofuran and carbofuran phenol in different specimens were calculated under different storage conditions.4.Statistical analysis SPSS19.0 statistical software was used to evaluate the experiment data.The paired t test was to compare carbofuran and its metabolite carbofuran phenol in specimens of different storage conditions between different time,compared the effects of different storage conditions and time on the decomposition kinetics of carbofuran and carbofuran phenol.Results: 1.GC-MS/MS analysis The chromatography peaks of carbofuran,carbofuran phenol were symmetrical and can be completely separated.The liner rang for carbofuran and carbofuran phenol in blood and liver were in the range of 0.01~10.00μg/m L,the limit of detection was 0.06~0.54ng/m L and the recoveries were from 93.4% to 112.0%,RSD<6.0%.2.The stability of carbofuran(1)The stability of carbofuran standard stock solution: The concentration of carbofuran with 0.1μg/m L~8.0μg/m L were decended to 78.6%~88.8% of initial concentration after stored 20.0d~34.0d,the deviation were 11.2%~21.4%,P < 0.05,differences were statistically significant.(2)The stability of the residue: The concentration of carbofuran with 0.1μg/m L~8.0μg/m L extracted from the blood and liver preserved at 4°C were decended to 59.5%~88.7% of initial concentration after stored 7.0d~30.0d,the deviation were 11.3%~40.4%,P<0.05,differences were statistically significant.(3)The stability of carbofuran in the residue after extracted from blood added in different concentration of citrate sodium: The residue extracted form the blood added citrate sodium 0.5mg/m L~ 20.0mg/m L preserved at 4°C were decended to 50.3%~62.2% of initial concentration after stored 7.0 d,the deviation were 37.7%~49.8%,P<0.05,differences were statistically significant.3.Decomposition kinetics of carbofuran and its metabolite carbofuran phenol in preserved specimens(1)Decomposition kinetics: The decomposition kinetics of carbofuran and carbofuran phenol in preserved specimens were fitted to the first order kinetics model.The half life of decomposition(t1/2k10)of carbofuran and carbofuran phenol in preserved blood at 20°C(1%Na F),20°C(NC),20°C,4°C and-20°C respectively were 2.0d±0.1d,2.4d±0.1d,3.3d±0.1d,5.6d±0.5d,2.0d±1.0d and 14.4d±6.3d,15.7d±4.9d,16.3d±11.0d,15.9d±10.0d and 23.5d±9.0d.The t1/2k10 of carbofuran and carbofuran phenol in preserved liver at 20°C(4%FA),20°C,4°C and-20°C respectively were 18.5d±4.4d,2.9d±0.3d,3.3d±1.0d,8.3d±1.0d and 19.0d±12.0d,12.3d±9.3d,28.3d±16.8d and 24.7d±23.6d.(2)The tendency of carbofuran and carbofuran phenol concentration change at different preserved conditions: In each condition,the concentration of carbofuran in preserved specimens were found to be significant decreased at 7.0d(P<0.05),then the change was steady.The concentration of carbofuran detected in blood at 20°C(1%Na F),20°C(NC),20°C,4°C and-20°C were decended to 10.2%~51.0% and 0%~6.9% of initial concentration after stored 7.0d and 83.0d,the limited detection time was 83.0d~150.0d.The concentration of carbofuran detected in liver at 20°C(4%FA),20°C,4°C and-20°C were decended to 18.7%~58.7% and 0%~7.2% of initial concentration after stored 7.0d and 150.0d,the limited detection time was 83.0d~150.0d.In each condition,the concentration of carbofuran phenol in preserved specimens showed a increasing trend firstly,then a declined tendency.The carbofuran phenol could be detected at 0h.The concentration of carbofuran phenol in preserved blood were found to be a significant increased at the 15.0d(P < 0.05),the concentration of carbofuran phenol detected in blood at 20°C(1%Na F),20°C(NC),20°C,4°C and-20°C were increased to 195.0%~1339.0% of initial concentration after stored 15.0d and decreased to 0%~97.0% of initial concentration after stored 150.0d,respectively.The limited detection time was 83.0d~150.0d.The concentration of carbofuran phenol detected in liver at 20°C(4%FA),20°C,4°C and-20°C were increased to 182.8%~539.8% of initial concentration after stored 7.0d and decreased to 0%~30.9% of initial concentration after stored 150.0d,respectively.The limited detection time was 83.0d~150.0d.Conclusion: 1.The GC-MS/MS method established for determination of carbofuran and carbofuran phenol in blood and liver is simple,sensitive and reproducible which could meet the requirement of our subsequent research and could be applied to forensic identification of carbofuran poisoning cases.2.The standard stock solution of carbofuran can be decomposed at-20°C and 4°C.The decomposition rate at-20°C is lower than that of 4°C.The higher of carbofuran concentration,the lower of decomposition rate and the deviation.It is suggested that the standard stock solution of carbofuran should be stored at-20°C and with high concentration.The deviation between the standard stock solution concentration and the newly prepared stock solution concentration deviation does not exceed 5%,it suggest that the detection period of 0.1μg/m L,0.2μg/m L,8.0μg/m L carbofuran standard stock solution preserved at 4°C and-20°C is within 10.0d,10.0d,20.0d and 10.0d,20.0d,34.0d,respectively.The concentration of carbofuran in the extracted samples(blood and liver)preserved at 4°C,the stability of high concentration of carbofuran,is higher than that of the low concentration of carbofuran.In the stability study,the acceptable standard for the sample and the true concentration of the deviation is not more than 15%.The results showed that the concentration of carbofuran of 0.1μg/m L,0.2μg/m L and 8.0μg/m L in the extracted blood and liver,the detection period is within 7.0d,15.0d,30.0d and 7.0d,7.0d,15.0d,respectively.The higher the concentration of citrate sodium,the faster decompose of carbofuran in the residue after extracted from blood added in different concentration of citrate sodium.The result indicate that citrate sodium can accelerate the decomposition of carbofuran.It is suggested that the samples of adding of citrate sodium extracted stored at 4°C can not represent the concentration of the sample at the initial time after 7.0d of storage,it is not recommended it as a sample to continue to detect.The detection period of carbofuran in different preserved conditions provide a reference for the detection of carbofuran poisoning.3.Carbofuran and carbofuran phenol in preserved specimens are found to be decomposed.The decomposition is quick at 20°C and slow at ﹣20°C.The concentration of carbofuran and carbofuran phenol descending fast in blood at 20°C(NC)and 20°C(1%Na F).Biological specimens used for forensic identification of the carbofuran poisoning should be stored at refriferated or freezed,and be analyzed as soon as possible.Citrate sodium and sodium fluoride are not suit for anticoagulation and antiputrefactiva.4.Decomposition kinetics of carbofuran and carbofuran phenol in preserved specimens fitting the first order kinetics model.According to the kinetic equation,the theoretical value is in good agreement with the measured value,which could be used to conclude the initial concentration of carbofuran and carbofuran phenol.The decomposition kinetics model and parameters of carbofuran and carbofuran phenol can provide evidence for forensic identification of carbofuran poisoning cases. |
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