本文關(guān)鍵詞： 乙醇 頂空氣相色譜 穩(wěn)定性 死后再分布 自生醇類　出處：《山西醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 1建立生物樣品中乙醇和自生醇類的直接進(jìn)樣和頂空進(jìn)樣氣相色譜檢測方法; 2建立乙醇的死后再分布、保存檢材中穩(wěn)定性和自生醇類的研究模型和方法; 3研究犬體內(nèi)乙醇的死后再分布規(guī)律、保存檢材中乙醇的穩(wěn)定性和自生醇產(chǎn)生規(guī)律,為涉及酒精案件檢材的采集、保存、檢測和結(jié)果分析提供科學(xué)依據(jù)。 方法: 1乙醇及自生醇的氣相色譜檢測方法 1.1直接進(jìn)樣氣相色譜法:體液、組織(勻漿)離心后取上清,加入內(nèi)標(biāo)液,柱頭直接進(jìn)樣,TRACE2000氣相色譜儀(FID檢測器,PEG融硅石英毛細(xì)管柱)檢測乙醇及自生醇,保留時(shí)間定性,內(nèi)標(biāo)法定量。 1.2頂空氣相色譜法:取血樣于頂空瓶中,加入內(nèi)標(biāo)液及蒸餾水,平衡后自動頂空進(jìn)樣器進(jìn)樣,Agilent6820氣相色譜儀(FID檢測器,DB-5毛細(xì)管柱)檢測乙醇及自生醇,保留時(shí)間定性,內(nèi)標(biāo)法定量。 2死后再分布研究 2.1動物模型 犬6只,分別于10min內(nèi)經(jīng)胃管灌入3.5ml/kg劑量的乙醇,1.5h后CO_2處死。 2.2樣品采集與處理 實(shí)驗(yàn)犬的心電、血壓和呼吸全部消失后,置于室溫下,分別于死后0、2、4、8、12、24、48、72、96、120h取左心室血、左心房血、右心室血、右心房血、下腔靜脈血、玻璃體液、膽汁、肝臟、脾臟、肺臟、腎臟、大腦和肌肉,直接進(jìn)樣氣相色譜法檢測乙醇及正丙醇含量,同時(shí)觀察是否有自生醇產(chǎn)生。 3保存檢材中乙醇的穩(wěn)定性 3.1模型 死亡12h內(nèi)人尸體血液,經(jīng)檢測無自生醇后,用乙醇溶液添加成初始濃度分別為20.12、51.73、77.9、142.29、314.05mg/100ml的樣本。 3.2樣品保存與處理 樣本置于不同器皿(塑料袋,試管,注射器)中,分別于室溫、4℃和—20℃中保存,在2、4、7、15、30天經(jīng)頂空氣相色譜法檢測其中乙醇濃度。 4保存檢材中自生醇的研究 4.1保存人血液中自生醇的研究 離體模型:空白人尸體樣本分別置于不同器皿(塑料袋,試管,注射器)中,室溫、4℃和—20℃條件中保存,在2、4、7、15、30天經(jīng)頂空氣相色譜法檢測乙醇和正丙醇含量,并觀察其它自生醇的產(chǎn)生情況。 4.2犬體內(nèi)自生醇的研究 在體模型:犬3只,經(jīng)CO_2處死,心電、血壓和呼吸全部消失后,置于室溫下,分別于死后0、2、4、8、12、24、48、72、96、120h取左心室血、左心房血、右心室血、右心房血、下腔靜脈血、玻璃體液、膽汁、肝臟、脾臟、肺臟、腎臟、大腦和肌肉,每次取材后將肌肉,皮膚逐層縫合,盡量保持機(jī)體的完整性。上述檢材經(jīng)直接進(jìn)樣氣相色譜法檢測乙醇及正丙醇含量,并觀察其它自生醇的產(chǎn)生情況。 5統(tǒng)計(jì)學(xué)方法 采用SPSS11.5統(tǒng)計(jì)軟件處理數(shù)據(jù),結(jié)果以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示 結(jié)果: 1氣相色譜法 本研究建立的直接進(jìn)樣和頂空氣相色譜檢測生物樣本中乙醇及自生醇(甲醇、丙酮、正丙醇、異丙醇、正丁醇和異丁醇)方法,使乙醇和自生醇分離良好,線性范圍為2～400mg/100ml,最低檢出濃度是0.15mg/100ml,回收率均在95%以上。 2死后再分布研究 2.1分布 犬經(jīng)灌胃后1.5h,體內(nèi)乙醇含量順序?yàn)椴Ａw液(373.04±51.64)＞左心室血(266.52±39.47)、左心房血(275.67±52.29)、右心室血(255.06±35.28)、右心房血(277.65±28.91)、下腔靜脈血(264.01±25.06)、尿(234.82±162.18)＞肝臟(162.01±5.45)、脾臟(177.07±43.35)、肺臟(199.70±29.28)、腎臟(203.27±34.20)、大腦(214.47±22.47)＞膽汁(169.27±52.74)＞肌肉(152.54±27.93)。玻璃體液中乙醇含量為心血和下腔靜脈中乙醇含量的1.41倍;膽汁與心血和下腔靜脈中乙醇含量之比為0.67。 2.2死后再分布 與0h相比,2、4、8、12、24、48、72、96h左心室、左心房、右心室、右心房血中乙醇濃度變化無統(tǒng)計(jì)學(xué)意義,120h時(shí)左心室、左心房血乙醇濃度顯著升高,且均高于右心室和右心房血中乙醇濃度(P＜0.05)。玻璃體液中乙醇濃度在0～2h明顯下降(P＜0.05),2～72h變化無統(tǒng)計(jì)學(xué)意義。0～120h肌肉中乙醇濃度變化無統(tǒng)計(jì)學(xué)意義。 3保存檢材中乙醇的穩(wěn)定性研究 保存溫度、器皿類型和初始濃度均對血樣中乙醇濃度有不同程度的影響。不同初始濃度的變化趨勢大致相同,15天后,初始濃度為314.05mg/100ml的樣本變化趨勢趨于降低,其他組變化微小。隨著保存溫度的升高,樣本中乙醇濃度的下降速度增加。4℃保存時(shí)樣品中乙醇濃度在15天內(nèi)無顯著變化(P＞0.05),而-20℃保存的樣品中乙醇濃度的變化均無顯著性差異。塑料袋保存血中的乙醇濃度在第7天時(shí)就出現(xiàn)了明顯的下降,而其他容器中乙醇濃度的變化趨勢基本一致,在前15天變化平穩(wěn),之后有小幅度的下降,但均無顯著性差異。 4保存檢材中自塵醇的研究 4.1保存人血液中自生醇的研究 室溫保存大部分樣本第7天時(shí)有少量乙醇、異丙醇、異丁醇和丙酮生成,30天也只有個(gè)別樣本中檢測出正丙醇;4℃保存樣本在第30天時(shí)可檢測到少量的乙醇;-20℃保存樣本在30天時(shí)仍未檢測到任何自生醇, 4.2犬體內(nèi)自生醇的研究 大部分組織如心血、下腔靜脈血、尿、膽汁、肝臟、脾臟、肺臟和腎臟在死后12h就有不同程度乙醇和正丙醇產(chǎn)生,其中,120h尿中乙醇濃度可以達(dá)104.92mg/100ml。肌肉,玻璃體液,大腦分別在死后48、72、96h檢出乙醇。犬體內(nèi)乙醇和正丙醇的生成存在正相關(guān)。 結(jié)論: 1本研究建立了生物樣品中乙醇和自生醇類直接進(jìn)樣和頂空氣相色譜檢測方法,可使乙醇和自生醇達(dá)到良好分離,靈敏度高,可應(yīng)用于酒精中毒的法醫(yī)學(xué)研究和法醫(yī)學(xué)鑒定。 2醉酒劑量灌胃后1.5h,犬體內(nèi)乙醇含量順序?yàn)椴Ａw液＞左心室血、左心房血、右心室血、右心房血、下腔靜脈血、尿＞肝臟、脾臟、肺臟、腎臟、大腦＞膽汁＞肌肉。玻璃體液中乙醇含量與心血和下腔靜脈乙醇含量之比為1.41;膽汁與心血和下腔靜脈中乙醇含量之比為0.63。 3乙醇在灌胃犬體內(nèi)可發(fā)生死后再分布現(xiàn)象,左心血受到很大影響,比較而言,玻璃體液、肌肉和腦組織受到死后再分布影響較少。且死亡犬體內(nèi)會產(chǎn)生乙醇、正丙醇、丙酮和異丙醇。在酒精中毒案件的法醫(yī)學(xué)鑒定中,應(yīng)綜合考慮死后再分布對體內(nèi)酒精含量的影響。 4保存溫度,器皿類型和初始濃度均對血樣中乙醇濃度有不同程度的影響,其中保存溫度和器皿類型的影響較為顯著。而且除了保存溫度與保存時(shí)間不存在交互作用以外,其它因素兩兩之間都存在交互作用。鑒定人在保存和檢測樣本過程中只有加強(qiáng)各個(gè)環(huán)節(jié)的質(zhì)量控制,才能提供客觀準(zhǔn)確的檢測數(shù)據(jù)及保證復(fù)查結(jié)果的有效性。 5在體模型和離體模型均會產(chǎn)生自生醇,溫度對其影響較大。大部分組織如心血、下腔靜脈血、尿、膽汁、肝臟、脾臟、肺臟和腎臟在死后12h就有不同程度的乙醇和正丙醇產(chǎn)生,其中,120h尿中乙醇濃度可以達(dá)104.92mg/100ml。肌肉,玻璃體液,大腦分別在死后48,72,96h檢出乙醇,犬體內(nèi)乙醇和正丙醇的生成存在正相關(guān);人空白血樣在-20℃放置30天未檢出任何自生醇。在涉及乙醇的法醫(yī)學(xué)檢案中,特別是腐敗樣品,應(yīng)注意自生醇的檢測,尿中檢出乙醇不能判定生前飲酒,應(yīng)采集不易腐敗的組織如玻璃體液、肌肉和大腦,檢測其乙醇含量,并觀察與正丙醇比例關(guān)系來進(jìn)行綜合判定。
[Abstract]:Objective:
1 the determination of ethanol and autogenic alcohols in biological samples by direct injection and headspace injection gas chromatography (GC) was established.
2 the post - death redistribution of ethanol was established, and the research models and methods of the stability and autogenic alcohols in the materials were preserved.
3, we studied the rule of postmortem redistribution of ethanol in dogs, preserved the stability of ethanol and the rule of spontaneous alcohol production, and provided a scientific basis for the collection, preservation, detection and result analysis of samples collected from alcohol cases.
Method:
Determination of 1 ethanol and autogenic alcohol by gas chromatography
1.1, direct injection gas chromatography: body fluid, tissue (homogenate) centrifugation, take the supernatant, add the internal standard solution, column head directly into the sample, TRACE2000 gas chromatograph (FID detector, PEG fused silica capillary column) to detect ethanol and autogenic alcohol, the retention time is qualitative, internal standard method is quantitative.
1.2 headspace gas chromatography: blood samples were taken from headspace bottles, added the internal standard solution and distilled water. After balanced, the automatic headspace sampler was injected. The Agilent6820 gas chromatograph (FID detector and DB-5 capillary column) were used to detect ethanol and autogenic alcohol, the retention time was qualitative, and the internal standard method was used for quantitative analysis.
Postmortem redistribution study of 2
2.1 animal model
In 6 dogs, 3.5ml/kg was injected into the gastric tube in 10min, and CO_2 was executed after 1.5h.
2.2 sample collection and treatment
Dogs ECG, blood pressure and respiration all disappeared after being placed at room temperature, respectively, after the death of 0,2,4,8,12,24,48,72,96120h left ventricular blood, left atrial blood, right ventricular blood, right atrial blood, inferior vena cava blood, vitreous fluid, bile, liver, spleen, lung, kidney, brain and muscle, chromatography determination of ethanol and n-propanol content directly into the sample gas, and observe whether there is self generated alcohol.
3 the stability of ethanol in the stored material
3.1 model
After the autogenic alcohol was detected in the dead human body of 12h, a sample of 20.12,51.73,77.9142.29314.05mg/100ml was added to the initial concentration by the ethanol solution.
3.2 preservation and treatment of samples
Samples were placed in different containers (plastic bags, tubes, syringes), respectively at room temperature, save 4 DEG C and - 20 DEG C, in 2,4,7,15,30 days the top air chromatography of the ethanol concentration.
Study on the preservation of autogenic alcohol in 4 samples
4.1 study of autogenic alcohol in human blood
In vitro human cadaveric model: blank samples were placed in different containers (plastic bags, tubes, syringes), room temperature preservation, 4 C and 20 C and conditions, in 2,4,7,15,30: Headspace Gas Chromatography Determination of ethanol and n-propanol content, and observe the other authigenic alcohols produced conditions.
Study on autogenic alcohol in 4.2 dogs
In the model: the 3 dogs were sacrificed by CO_2, ECG, blood pressure and respiration all disappeared after being placed at room temperature, respectively, after the death of 0,2,4,8,12,24,48,72,96120h left ventricular blood, left atrial blood, right ventricular blood, right atrial blood, inferior vena cava blood, vitreous fluid, bile, liver, spleen, lung and kidney. The brain and muscles, each biopsy after muscle, skin sutured, try to keep the integrity of the body. The samples by direct injection gas chromatographic method for the determination of ethanol and n-propanol content, and observe the other self generated alcohol production.
5 statistical method
SPSS11.5 statistical software is used to deal with data, and the results are expressed in mean + + standard deviation ((?) + s)
Result:
1 gas chromatography
The detection of alcohol and self generated alcohol in biological sample directly into the sample and headspace gas chromatography was established in this study (methanol, acetone, isopropanol, n-propanol, n-butanol and isobutanol) method, ethanol and self generated alcohol were good. The linear range is 2 ~ 400mg/100ml, the minimum detectable concentration is 0.15mg/100ml, the recovery rate were more than 95%.
Postmortem redistribution study of 2
2.1 distribution
The dog after intragastric administration of 1.5h, in order for the ethanol content of vitreous humor (373.04 + 51.64), the left ventricular blood (266.52 + 39.47), left atrial blood (275.67 + 52.29), right ventricular blood (255.06 + 35.28), right atrial blood (277.65 + 28.91), inferior vena cava blood (264.01 + 25.06) urine, (234.82 + 162.18), liver (162.01 + 5.45), spleen (177.07 + 43.35), lung (199.70 + 29.28), kidney (203.27 + 34.20), brain (214.47 + 22.47), bile (169.27 + 52.74), muscle (152.54 + 27.93). The content of ethanol in the vitreous of 1.41 double heart and inferior vena cava blood alcohol content in blood and bile; ethanol content and ratio of 0.67. in inferior vena cava
2.2 postmortem redistribution
Compared with 0h, 2,4,8,12,24,48,72,96h, left ventricle, left atrium, right ventricle, right atrium blood alcohol concentration change was not statistically significant, left ventricular 120h, left atrial blood ethanol concentration was significantly increased, and the ethanol concentration was higher than that of the right ventricle and right atrium blood (P < 0.05). The concentration of ethanol in the vitreous fluid decreased significantly in 0 ~ 2H (P < 0.05), no significant changes in 2 ~ 72h ~.0 no significant change of ethanol concentration 120h muscle.
Study on the stability of ethanol in 3 stored samples
All types of vessels preservation temperature, and initial concentration have different effects on blood ethanol concentration. The change trend of different initial concentrations are roughly the same, 15 days after the initial concentration of sample 314.05mg/100ml change trend tends to decrease, the other group of small changes. With increasing storage temperature, ethanol concentration in the sample rate of decline increased.4 when the samples stored at the ethanol concentration in 15 days no significant change (P > 0.05), but there was no difference in ethanol concentration in the samples stored at -20. The concentration of ethanol in blood preservation bags appeared significantly decreased at day seventh, and the change trend of ethanol concentration in other container consistent changes smoothly in the first 15 days, after a small drop, but there was no significant difference.
Study on self dust alcohol in 4 preservation materials
4.1 study of autogenic alcohol in human blood
Most of the samples stored at room temperature for seventh days when there is a small amount of ethanol, isopropanol, isobutanol and acetone, 30 days only individual samples detected propanol; 4 C preservation sample can detect small amounts of ethanol on the thirtieth day; -20 samples stored at 30 days has not detected any self generated alcohol,
Study on autogenic alcohol in 4.2 dogs
Most organizations such as blood, urine, inferior vena cava blood, bile, liver, spleen, lung and kidney after the death of 12h have different degrees of ethanol and n-propanol, among them, 120h urine concentration of ethanol can reach 104.92mg/100ml. muscle, vitreous humor, brain 48,72,96h respectively after death. There was a positive correlation detection of ethanol formation in dogs ethanol and n-propanol.
Conclusion:
1, a direct injection and headspace gas chromatographic method for the determination of ethanol and autogenic alcohols in biological samples is established. The ethanol and self generated alcohols can be well separated, and the sensitivity is high. It can be applied to the forensic research and forensic identification of alcoholism.
2 drunken dose after intragastric administration of 1.5h dogs ethanol content order of vitreous, left ventricular blood, left atrial blood, right ventricular blood, right atrial blood, inferior vena cava blood, urine, liver, spleen, lung, kidney, brain, bile, muscle. In the vitreous fluid and blood alcohol content of ethanol and inferior vena cava the content ratio is 1.41; ethanol content of bile and blood and inferior vena cava in the ratio of 0.63.
3 ethanol in the postmortem redistribution phenomenon can be poured in the stomach of dog, left heart has been greatly affected, in comparison, vitreous fluid, muscle and brain tissue from postmortem redistribution is less affected. And death dogs will produce ethanol, propanol, acetone and isopropanol. In the case of alcoholism in forensic identification that should consider the postmortem redistribution effect on alcohol content.
4 types of vessels were temperature and initial concentration have different effects on blood alcohol concentration, the effect of temperature and the type of vessel is significant. In addition to storage temperature and storage time of interaction does not exist outside, there are other factors. The interaction between the 22 experts in the preservation and detection process only samples to strengthen the quality control of each link, in order to provide objective and accurate detection data and guarantee the validity of the review results.
5 in vivo and in vitro model model will produce self generated alcohol, temperature has more effect on it. Most tissues such as heart, inferior vena cava blood, urine, bile, liver, spleen, lung and kidney after the death of 12h have different levels of ethanol and n-propanol, the ethanol concentration can reach 120h in urine 104.92mg/100ml. muscle, vitreous humor, brain 48,72,96h detected ethanol after death respectively, are related to the generation of dogs ethanol and n-propanol; blank blood samples at -20 DEG C for 30 days were not detected in any spontaneous alcohol. Histologic examination involved in ethanol forensic case, especially corruption samples should be tested in situ alcohol could not determine his drinking, alcohol detection in urine should be collected is not easy to corrupt organizations such as vitreous fluid, muscle and brain, to detect the content of ethanol, and to observe the relationship with n-propanol in proportion to the comprehensive judgment.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：D919

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