單克隆免疫金標試紙應用于血液中藥物篩選的可靠性研究
發(fā)布時間：2019-05-20 07:58
【摘要】：目的:1.通過空白添加、動物實驗,探索單克隆試紙對血液中苯二氮?類;巴比妥類;毒品類等藥(毒)藥物篩選的檢測閾值及LC-MS/MS確認方法的研究;2.通過實際案例驗證單克隆試紙用于血液中苯二氮?類;巴比妥類;毒品類等藥(毒)物篩選的可靠性。方法:1.血液用單克隆免疫金標試紙檢測前的處理方法的研究通過血液空白添加實驗,分析了不同p H值緩沖液條件、不同離心轉速以及不同溫度下對血液前處理的影響,得到血液中不同藥物的檢測閾值。2.動物實驗,建立血液篩選的檢測閾值及檢驗方法的研究稱重,按大鼠的LD50給動物灌胃,在動物血液藥物濃度達到最大值時解剖取血液,按照第二部分的血液前處理,使用單克隆免疫金標試紙檢測血液藥物濃度,結果呈陽性,則減少1/2灌胃劑量,直至單克隆免疫金標試紙呈陰性。這時,陰性結果的前一次陽性灌胃劑量就是現(xiàn)有單克隆免疫金標試紙檢測所能達到的最低血液藥物濃度。取1m L的動物血液,加9m L乙酸乙酯,振蕩,超聲5min,離心,取上清液于蒸發(fā)皿中,60℃水浴揮干,加1m L的乙腈復溶,有機膜過濾,LC-MS/MS檢測分析,得到血液藥物濃度。3.通過實際案例驗證單克隆免疫金標試紙應用于血液中藥物篩選的可靠性研究實際案例的血液經過上述的前處理,使用單克隆免疫金標試紙檢測和LC-MS/MS檢測驗證,得出結果。結果:1.使用單克隆免疫金標試紙對血液進行藥(毒)物篩選時,有必要向血液中加入磷酸鹽緩沖液,這樣能得到較好的檢測結果。另外,V(血液):V(磷酸鹽緩沖液)=1:1進行等量添加,效果最好。2.使用Sigma 3-30K高速臺式冷凍離心機在12000轉、4℃條件下離心處理得到的檢測限最低,檢測效果最好,在條件允許時最好在篩選時選用高速冷凍離心機離心,能夠獲得最佳結果。但是這種離心機成本高、普及率低,因此,一般使用低速離心機也是可以的。3.磷酸鹽緩沖液的p H值對各類藥物檢測閾值的影響不一致,其中對氯硝安定和氯胺酮的影響較大。4.實際案例。2012-2013年的38個實際案例的LC-MS/MS檢驗結果與用單克隆免疫金標試紙檢測結果相比較,其中的89.47%結果一致。結論:1、用于尿液篩選的單克隆免疫金標試紙在應用于血液檢材的篩選時差別不大。2、使用磷酸鹽緩沖液稀釋血液,用高速冷凍離心機離心,取上清液用于單克隆免疫金標試紙篩選血液能夠獲得較好的結果。3、根據空白實驗結果,在使用單克隆免疫金標試紙篩選血液中苯二氮?類藥物時,應注意分別使用p H6-8及p H11兩種緩沖液分別稀釋血液后進行篩選,以免漏掉氯硝安定這種需要在堿性條件下才能用單克隆免疫金標試紙較好篩選出的藥物。4、使用單克隆免疫金標試紙篩選血液中巴比妥類藥物時,使用p H6-8的緩沖液稀釋血液可以滿足檢測需要。5、在使用單克隆免疫金標試紙篩選血液中的毒品時,嗎啡類、苯丙胺類使用緩沖液p H6-8,均可滿足檢測要求,但氯胺酮篩選存在較大問題,在p H6-8時,閾值為4000ng/m L,在p H11時,閾值為2000ng/m L,均與試紙廠家說明書上標注的1000ng/m L相差較大。6、根據本課題研究,目前單克隆免疫金標試紙檢測藥(毒)物的閾值如表5-1,建議廠家對氯胺酮單克隆免疫金標試紙加以改進,以滿足實際案件需要。
[Abstract]:Objective:1. Through the blank addition, animal experiment, explore the monoclonal test paper to the blood of the benzodiazepines? Class; barbiturates; drug-like drug (toxic) drug screening detection threshold and LC-MS/ MS validation method;2. Use a practical case to verify that the monoclonal test paper is used in the blood of benzodiazepines? Class; barbiturates; drug-like drugs (toxic) screening reliability. Method:1. The study of the treatment method before the detection of the monoclonal immunogold-labeled test paper for blood was carried out by a blank addition experiment, and the effects of different pH buffer conditions, different centrifugal rotation speeds and different temperatures on the pretreatment of the blood were analyzed, and the detection threshold of different drugs in the blood was obtained. an animal experiment is carried out to establish a test threshold for blood screening and a study weighing of the test method; the LD50 of the rat is given to the animal for the stomach, and the blood is dissected and taken according to the blood pre-treatment of the second part when the blood drug concentration of the animal reaches the maximum value; The concentration of blood drug was detected by using the monoclonal immunogold standard test paper, and the result was positive, and the dose of 1/2 was reduced until the monoclonal antibody test paper was negative. At this time, the first positive intragastric dose of the negative result is the minimum blood drug concentration that can be achieved by the prior monoclonal antibody test paper. 1 m L of animal blood,9 m L of ethyl acetate, shaking, ultrasonic for 5 min, centrifuging, collecting the supernatant in the evaporating dish, volatilizing at 60 & deg; C water bath, adding 1 ml of acetonitrile for reconstitution, filtering the organic membrane, and detecting the LC-MS/ MS to obtain the blood drug concentration. The actual case was used to verify the reliability of the application of the monoclonal immunogold standard test paper to the drug screening in the blood. The blood of the actual case was subjected to the above-mentioned pretreatment, and the test and the LC-MS/ MS detection and verification were performed using the monoclonal immune gold standard test paper and the results. Results:1. When using the monoclonal immunogold standard test paper to screen the blood, it is necessary to add a phosphate buffer to the blood, so that a better detection result can be obtained. In addition, V (blood): V (phosphate buffer) = 1:1 was added in the same amount, with the best effect. The lowest detection limit is obtained by using a Sigma 3-30K high-speed bench freezing centrifuge at the temperature of 12000 rpm and 4 鈩，
本文編號：2481485
[Abstract]:Objective:1. Through the blank addition, animal experiment, explore the monoclonal test paper to the blood of the benzodiazepines? Class; barbiturates; drug-like drug (toxic) drug screening detection threshold and LC-MS/ MS validation method;2. Use a practical case to verify that the monoclonal test paper is used in the blood of benzodiazepines? Class; barbiturates; drug-like drugs (toxic) screening reliability. Method:1. The study of the treatment method before the detection of the monoclonal immunogold-labeled test paper for blood was carried out by a blank addition experiment, and the effects of different pH buffer conditions, different centrifugal rotation speeds and different temperatures on the pretreatment of the blood were analyzed, and the detection threshold of different drugs in the blood was obtained. an animal experiment is carried out to establish a test threshold for blood screening and a study weighing of the test method; the LD50 of the rat is given to the animal for the stomach, and the blood is dissected and taken according to the blood pre-treatment of the second part when the blood drug concentration of the animal reaches the maximum value; The concentration of blood drug was detected by using the monoclonal immunogold standard test paper, and the result was positive, and the dose of 1/2 was reduced until the monoclonal antibody test paper was negative. At this time, the first positive intragastric dose of the negative result is the minimum blood drug concentration that can be achieved by the prior monoclonal antibody test paper. 1 m L of animal blood,9 m L of ethyl acetate, shaking, ultrasonic for 5 min, centrifuging, collecting the supernatant in the evaporating dish, volatilizing at 60 & deg; C water bath, adding 1 ml of acetonitrile for reconstitution, filtering the organic membrane, and detecting the LC-MS/ MS to obtain the blood drug concentration. The actual case was used to verify the reliability of the application of the monoclonal immunogold standard test paper to the drug screening in the blood. The blood of the actual case was subjected to the above-mentioned pretreatment, and the test and the LC-MS/ MS detection and verification were performed using the monoclonal immune gold standard test paper and the results. Results:1. When using the monoclonal immunogold standard test paper to screen the blood, it is necessary to add a phosphate buffer to the blood, so that a better detection result can be obtained. In addition, V (blood): V (phosphate buffer) = 1:1 was added in the same amount, with the best effect. The lowest detection limit is obtained by using a Sigma 3-30K high-speed bench freezing centrifuge at the temperature of 12000 rpm and 4 鈩，
本文編號：2481485
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