【摘要】： 目的 1.建立生物檢材中苯巴比妥的氣相色譜和氣相色譜/質(zhì)譜檢測方法; 2.建立苯巴比妥的毒物動力學、動態(tài)分布、死后分布、死后再分布動物模型; 3.研究苯巴比妥在大鼠體內(nèi)的毒物動力學、動態(tài)分布、死后分布、死后再分布規(guī)律,為苯巴比妥中毒(死)案件的法醫(yī)學鑒定提供實驗依據(jù)。 方法 1.樣品的處理和氣相色譜、氣相色譜/質(zhì)譜檢測:血、組織樣品中加入內(nèi)標物丙烯巴比妥,經(jīng)鹽酸酸化后(pH=1～2),乙醚萃取,氣相色譜/質(zhì)譜聯(lián)用法定性,氣相色譜內(nèi)標法定量檢測苯巴比妥。 2.苯巴比妥在急性中毒大鼠體內(nèi)的毒物動力學及動態(tài)分布的研究:雄性wistar大鼠54只,經(jīng)灌胃勻速注入1/2LD50苯巴比妥(330 mg·kg~(-1)),給藥后觀察大鼠的中毒癥狀,分別于給藥后1、5、10、15、20、30、48、72、96h各處死6只,迅速解剖動物,取心血、心、肝、脾、肺、腎、腦等冷凍保存,酸性乙醚提取,氣相色譜質(zhì)譜法定性、氣相色譜定量檢測其中苯巴比妥。 3.苯巴比妥在大鼠體內(nèi)的死后分布研究:雄性wistar大鼠12只,隨機分LD50組和2LD50組,經(jīng)灌胃勻速注入LD50(0.66 mg·kg~(-1))和2LD50(1.32 mg·kg~(-1))苯巴比妥。觀察給藥到死亡時的生命體征的變化以及中毒癥狀,待呼吸和心跳全部消失時,迅速解剖動物,取心血、心、肝、脾、肺、腎、腦、胃等冷凍保存,酸性乙醚提取,氣相色譜質(zhì)譜法定性、氣相色譜定量檢測苯巴比妥。 4.苯巴比妥在大鼠體內(nèi)的死后再分布研究:雄性wistar大鼠54只,以1/2LD50(330 mg·kg~(-1))灌胃,10小時后處死,室溫(20?C)條件下,于死后0、2、4、8、12、24、48、72、96小時取心血、心、肝、脾、肺、腎、大腦、肌肉,酸性乙醚提取,氣相色譜質(zhì)譜法定性、氣相色譜定量檢測其中苯巴比妥。 5.統(tǒng)計學方法:采用SPSS11.5統(tǒng)計軟件處理數(shù)據(jù),結(jié)果以均數(shù)±標準差( )表示,t檢驗。 結(jié)果 1.氣相色譜和氣相色譜/質(zhì)譜檢測:氣相色譜/質(zhì)譜分析苯巴比妥的特征離子m/z為204、232。心血中苯巴比妥氣相色譜檢測的回歸方程、線性檢測范圍、相關(guān)系數(shù)、回收率、最低檢出濃度分別為Y = 43.476X + 0.8168(μg·mL~(-1))、0.5 -280μg·mL~(-1)、0.9955、97.50±2.0%、0.5μg·mL~(-1);肝組織中苯巴比妥氣相色譜檢測的回歸方程、線性檢測范圍、相關(guān)系數(shù)、回收率、最低檢出濃度分別為Y = 46.867X + 0.0884 (μg·g~(-1))、0.5 -280μg·g~(-1)、0.994、98.5±3.5%、0.5μg·g~(-1)。 2.毒物動力學和動態(tài)分布:1/2LD50劑量苯巴比妥在大鼠體內(nèi)的毒物動力學符合口服給藥一級動力學一室開放模型過程,各臟器中苯巴比妥的毒物動力學符合血管外給藥一級動力學一室開放模型。心血中達峰時間和消除半衰期除為7.39h和26.02h;各臟器中達峰時間和消除半衰期為2.41h~4.73h和8.59h~20.91h;經(jīng)口染毒后不同時間大鼠體內(nèi)苯巴比妥含量順序為肝、心血、心和肺、其他組織,各臟器內(nèi)苯巴比妥含量在5h左右達高峰,之后呈下降趨勢。 3.死后分布:大鼠在苯巴比妥灌胃后活動明顯減少,出現(xiàn)嗜睡癥狀,LD50灌胃組大鼠7.24±3.17h死亡,2LD50灌胃組大鼠1.33±0.25h死亡。各臟器組織苯巴比妥含量由高到低分別為:①LD50組:胃(1261.34±591.83)心(332.12±70.87)、腦(289.81±71.43)、肝(284.68±79.44)、肺(284.41±67.47)、脾(272.56±31.53)、心血(269.52±44.62)腎(167.76±43.72);②2LD50組:胃(1649.21±1212.82)肝(357.83±138.67)、腦(323.19±159.92)、心(310.95±130.73)、心血(309.93±135.60)、肺(308.28±134.48)、脾(299.77±104.58)腎(233.24±91.92)。 4.死后再分布:大鼠在處死后96小時內(nèi),在室溫(20?C)條件下,死后2~4小時的心血、心、肺、腎、腦、肌的苯巴比妥含量與死亡當時含量相比明顯升高(p0.05),而后逐漸降低;死后2小時肝、脾的苯巴比妥含量與死亡當時含量相比明顯升高(p0.05),則出現(xiàn)逐漸升高的趨勢(p0.05)。 結(jié)論 1.本文建立的生物檢材中苯巴比妥氣相色譜/質(zhì)譜檢測方法選擇性好,定性準確,氣相色譜檢測簡便,快速,靈敏,定量結(jié)果準確,可用于苯巴比妥中毒的臨床快速檢驗診斷和苯巴比妥中毒(死)案件的法醫(yī)學鑒定。 2.本文建立了苯巴比妥的毒物動力學、動態(tài)分布、死后分布、死后再分布研究動物模型,可應用于苯巴比妥的法醫(yī)毒物動力學研究。 3.苯巴比妥在大鼠血和各臟器中毒物動力學均符合血管外給藥一級動力學一室開放模型。急性染毒大鼠體內(nèi)苯巴比妥含量順序為,肝、心血、心和肺、其他組織,各臟器內(nèi)苯巴比妥含量在5h左右達高峰,之后呈下降趨勢。 4.苯巴比妥死后分布不均勻,含血豐富和脂質(zhì)含量高的器官如肝、肺、心、腦較其它組織和血液含量高。法醫(yī)鑒定苯巴比妥中毒(死)案件時,應全面正確的采取檢材進行毒物分析。 5.苯巴比妥在染毒大鼠體內(nèi)可發(fā)生死后再分布,死后心血、心、肝、脾、肺、腎、腦和肌中苯巴比妥濃度均出現(xiàn)升高。
[Abstract]:objective
1. establish a gas chromatography and gas chromatography-mass spectrometry (GC / MS) method for the detection of phenobarbital in biological samples.
2. establishment of phenobarbital toxicology, dynamic distribution, postmortem distribution, postmortem redistribution animal model.
3. To study the toxicokinetics, dynamic distribution, postmortem distribution and postmortem redistribution of phenobarbital in rats, and to provide experimental basis for forensic identification of phenobarbital poisoning cases.
Method
1. Sample treatment and gas chromatography, gas chromatography/mass spectrometry detection: blood, tissue samples with the addition of internal standard propylene barbiturate, acidified by hydrochloric acid (pH=1-2), ether extraction, gas chromatography/mass spectrometry qualitative, gas chromatography internal standard quantitative detection of phenobarbital.
2. Study on toxicokinetics and dynamic distribution of phenobarbital in rats with acute poisoning: 54 male Wistar rats were injected with 1/2 LD50 phenobarbital (330 mg (-1)) by gastric perfusion at a uniform rate. The toxic symptoms of rats were observed after administration. Six rats were executed at 1,5,10,15,20,30,48,72,96 h after administration, respectively. Spleen, lung, kidney, brain and other cryopreservation, acid ether extraction, gas chromatography-mass spectrometry, gas chromatography-quantitative detection of phenobarbital.
3. Study on postmortem distribution of phenobarbital in rats: 12 male Wistar rats were randomly divided into LD50 group and 2LD50 group. LD50 (0.66 mg (-1)) and 2LD50 (1.32 mg (-1)) phenobarbital were injected into the stomach at a uniform rate. The changes of vital signs and poisoning symptoms at the time of death were observed and the respiratory and heartbeat symptoms disappeared. Animals, blood, heart, liver, spleen, lung, kidney, brain, stomach and other cryopreservation, acid ether extraction, gas chromatography-mass spectrometry, gas chromatography quantitative detection of phenobarbital.
4. Study on postmortem redistribution of phenobarbital in rats: 54 male Wistar rats were orally administered with 1/2 LD50 (330 mg (-1)) for 10 hours and then executed at room temperature (20?C). Cardiac blood, heart, liver, spleen, lung, kidney, brain, muscle, acidic ether were collected at 0, 2, 4, 8, 12, 24, 48, 72, 96 hours after death. Phenobarbital was detected.
5. Statistical methods: SPSS11.5 statistical software was used to process the data. The results were expressed as mean [standard deviation], t test.
Result
1. Gas Chromatography and Gas Chromatography/Mass Spectrometry: Characteristic ion m/z of phenobarbital was 204,232. Regression equation, linear detection range, correlation coefficient, recovery, minimum detection concentration Y=43.476X+0.8168 (0.5-280ug.mL~(-1), 0.9955, 97. The regression equation, linear detection range, correlation coefficient and recovery rate of phenobarbital gas chromatography in liver tissue were Y=46.867X+0.0884(ug.g~(-1)), 0.5-280ug.g~(-1), 0.994,98.5 (-3.5%) and 0.5ug.g~(-1)), respectively.
2. Toxicokinetics and Dynamic Distribution: The toxicokinetics of 1/2 LD50 dose of phenobarbital in rats conformed to the one-compartment open model of oral administration. The toxicokinetics of phenobarbital in organs conformed to the one-compartment open model of extravascular administration. The peak time and elimination half-life were 2.41h~4.73h and 8.59h~20.91h in each organ, and the phenobarbital content in liver, heart blood, heart and lung, other tissues and organs reached the peak at about 5h, then decreased.
3. Distribution of postmortem: The activity of rats decreased significantly after intragastric administration of phenobarbital, and the symptoms of somnolence were observed. The rats in LD50 group died at 7.24 [3.17 hours] and the rats in 2LD50 group died at 1.33 [0.25 hours] respectively. The contents of phenobarbital in various organs and tissues were: (1) LD50 group: gastric (1261.34 [591.83] heart (332.12 [70.87], brain (289.81 [71.43]), liver (284.68 [79.44]] respectively. (2) 2LD50 group: stomach (1649.21 [1212 12.82) liver (1649.21 [(1649.21 [1212.82), (357.83 [138.67], brain (323.19 [159.92], heart (310.95 [130.73]), heart blood (309.93 [(310.95 [130.73.73], heart (309.93 [309.93 [135.60], heart (308.52 [308.28 [134.28.48].48), spleen (299.77.77 [77.77 [58.58.58.58] kidn (299.58.58.58)] kidney (299.77 3.24 [91.92].
4. Post-death redistribution: Within 96 hours after death, at room temperature (20?C), the phenobarbital content in heart blood, heart, lung, kidney, brain and muscle of rats 2-4 hours after death was significantly higher than that at the time of death (p0.05), and then gradually decreased; 2 hours after death, the phenobarbital content in liver and spleen was significantly higher than that at the time of death (p0.05). The trend is increasing gradually (P0.05).
conclusion
1. The method of gas chromatography/mass spectrometry for phenobarbital in biological samples established in this paper is selective, accurate, simple, rapid, sensitive and accurate. It can be used for rapid clinical diagnosis of phenobarbital poisoning and forensic identification of cases of phenobarbital poisoning (death).
2. In this paper, the toxicokinetics, dynamic distribution, postmortem distribution and postmortem redistribution of phenobarbital were established, which can be used in forensic toxicokinetics of phenobarbital.
3. The toxicokinetics of phenobarbital in rat blood and organs conformed to the one-compartment open model of the first-order kinetics of extravascular administration.
4. The distribution of phenobarbital after death is uneven. The organs with rich blood and high lipid content such as liver, lung, heart and brain are higher than other tissues and blood.
5. Phenobarbital can be redistributed after death in poisoned rats. The concentration of phenobarbital in heart blood, heart, liver, spleen, lung, kidney, brain and muscle increased after death.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：D919

【引證文獻】

相關(guān)期刊論文 前1條

1 陳躍;朱軍;于忠山;喻洪江;劉耀;;毒物代謝動力學研究在法庭科學中的應用[J];中國法醫(yī)學雜志;2012年06期

相關(guān)碩士學位論文 前2條

1 韓亮;苯巴比妥的法醫(yī)毒物動力學研究（二）[D];山西醫(yī)科大學;2011年

2 張曉飛;利眠寧的法醫(yī)毒物動力學研究[D];山西醫(yī)科大學;2012年

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本文編號：2230412