The Study On Reliability Of Golden-labeled Immunochromatograp Hic Assay To Screen Drugs In Blood Samples

Objectives:1. To research the detection threshold for screening the benzodiazepines, barbiturates and Drug classes in the blood samples with monoclonal test paper, which is formally for the urine samples screening, and study the established method of LC/MS through the animal experiments and addition of the blank.2. To validate the reliability of the blood samples screening for the benzodiazepines,barbiturates and Drug classes with monoclonal test paper through practical cases.Methods:1. Study on the pretreatment method of the blood samples detecting with monoclonal test paper.After the influence of different PH buffer solution conditions、rotational speed and temperature were analysed on the blood samples pretreatment through blank tests,detection thresholds of different drugs were obtained in the blood samples.2. Animal experiments, to establish the detection threshold and method of the blood samples screening.Being weighed, the rats were given intragastric administration with LD50, and dissected when the blood concentration rose to the maximum level. After the pretreatment of the blood samples in the same way of the second part, the blood concentration showed positive with golden-labeled monoclonal test paper detection. Then the intragastric administrations were reduced continually until the results changed negative.9ml ethyl acetate was added to 1ml rat blood, supernate were collected in the evaporating dish after shocking and ultrasound for 5 minutes, which was redissolved with1 ml acetonitrile after being dried on the water bath pot at 60℃. After the solution was filtered with organic membrane, the blood concentration was obtained by LC-MS/MS.3. The study of the reliability for the screen of blood samples with monoclonal test paper after they were validated with practical cases.The blood samples of the practical cases were pretreated in the above way, the results were obtained through detection of the golden-labled monoclonal test paper and the verification of LC-MS/MS.Results:1. It is necessary to add phosphate buffer to the blood samples to get the results more easily when monoclonal test papers were used to conduct poisonous substance or illicit drugs in the blood samples screen. Further more, it came to the best effect when the equate adding was made in the way as V(blood samples): V(phosphate buffer) =1:1.2. The lowest limit and the best effect of detection appeared when the Sigma 3-30 K high speed tabletop refrigerated centrifuge was used at 12000 rpm and 4℃. If permitted, it is best to choose high speed tabletop refrigerated centrifuge to acquire the results of high quality. While the low speed centrifuge is a good choice for the high price and popularizing rate of the high speed centrifuge.3. The PH value of phosphate buffer showed variant influence on the detection threshold of drugs in different categories, where clonazepam and ketamine endured the higher impact.4. Practical cases. Compared the results of 38 practical cases from 2012 to 2013 done by LC-MS-MS with those done though golden-labeled immunochromato, we could find that 89.47 perscent of them was consistent.Conclusions:1. The difference was not obvious in the screen of blood samples and other samples with golden-labled test paper for urine samples.2. The blood dilution with phosphate buffer, centrifugation with high speed tabletop refrigerated centrifuge provided excellent results as well as collected supernate for the screen of the blood samples with golden-labled test paper.3. The blank test caused noticed that the blood dilution should be conducted with buffer solution of p H6-8 and p H11 respectively before the screen, in avoid of the lack of the situation that clonazepam could be detected better with the test paper in the alkaline condition.4. The buffer solution of p H 6-8 for the dilution of blood samples met the need whenthe barbital drugs were screened with golden-labled test paper.5. The requests of morphine and benzedrine on the p H value of buffer solution were not high when the screen of the blood samples were conducted with golden-labled test paper, and the p H6-8 can satisfy the need, while the screen of ketamine encountered problems, in which the minimum detectable amount was 4000ng/m L at p H6-8, and the amount reached 2000ng/m L at p H11. All of this presents the large difference from the1000ng/m L which is signed on the test paper.6. According our research, the threshold value of poisonous substance and illicit drugs detected by golden-labled monoclonal test paper were showed in the table 30-32.Manufacturers should be advised to modify the test paper of ketamine and decrease the threshold value to meet the requests of practical cases.