| Liver is the main organ for the detoxification of foreign bodies, most of the drugs in the body will be broken down in the liver, and then be excreted through the kidney or the bile. Drug metabolism can be divided into two general categories, i.e., in vivo and in vitro. The former one is analyze the blood, urine, bile, tissues and other biological samples and separate of drugs sample and their metabolites after a period of of time of the body on medication. The concentration of the drug and its metabolites in the body is low because of many drugs are widely distributed. The organs and the enzymes which the metabolic conversion required is another reason. So that the detection of metabolites is difficult. In contrast, in vitro metabolism method could get a large number of metabolites in a short time, and can easily control some certain metabolic conditions, make the metabolites separated and extracted easily, and have a highlighted advantages of the determination of the structure of metabolite. However, in vitro metabolism have some real differences with in vivo metabolism, it can not fully reflect the body's metabolism.HPLC-MS method is commonly used in drug metabolism research. In this study, a strong chemiluminescence was observed during the reaction of repaglinide with tris(2,2'-bipyridyl) ruthenium(Ⅱ) [Ru(bpy)32+] in resin and potassium permanganate (KMnO4) under acidic conditions combined with microdialysis sampling. Repaglinide have a short half-life and can be easily be metabolised completely in the liver. This experiment achieved real-time monitoring of the drug in vitro metabolism in the liver cells process. This paper also established other methods for several drugs of high performance liquid chromatography-immobilized ruthenium(Ⅱ) bipyridine chemiluminescence for the future metabolism study.This thesis includes two sections-review and report. The review section informations about the status of high performance liquid chromatography-immobilized ruthenium(Ⅱ) bipyridin-chemiluminescence detection(HPLC-CL), the principle and methods of microdialysis sampling techniques, and applications in life science. Commonly used methods of the drugs in vitro metabolism in pig liver research are also reviewed.The report consists of three parts: 1 High performance liquid chromatography with immobilized Ru(bpy)32+-KMnO4 chemiluminescence detection and it's application in metabolism of repaglinide in pig liver microsomesA novel high performance liquid chromatography-chemiluminescence(HPLC-CL) method for investigation of in vitro metabolism of repaglinide was developed. The detection was based on the chemiluminescence reaction of repaglinide with acidic potassium permanganate(KMnO4) and tris(2,2'-bipyridyl) ruthenium(III) [Ru(bpy)33+], which was immobilized on the cationic ion-exchange resin for obtaining high sensitivity and reducing consumption of expensive reagent. Under optimum conditions, the linear range for repaglinide was 4.0×10-8-1.0x10-5 g/mL with the detection limit of 2.0×10-8 g/mL (3σ). The relative standard deviation (RSD) was 2.1%(n=11) for 2.0×10-6 g/mL repaglinide. The proposed method had been successfully applied to study the in vitro metabolism of repaglinide in pig liver microsomes with microdialysis sampling technique.2 Determination of ranitidine by high performance liquid chromatography with immobilized tris(2,2'-bipyridyl)ruthenium(II)-potassium permanganate chemiluminescence detectionA sensitive method was developed for the determination of ranitidine by HPLC-CL detection. The detection was based on the chemiluminescence reaction of ranitidine with acidic potassium permanganate (KMnO4) and tris(2,2'-bipyridyl) ruthenium(III), which was immobilized on the cationic ion-exchange resin. Under optimum conditions, the linear range for ranitidine was 2.0x10-7-4.0×10-5 g/mL with the detection limit of 6.7x10-8 g/mL(3σ). The relative standard deviation (RSD) was 2.2%(n=11) for 2.0×10-6 g/mL ranitidine. Ranitidine in the serum samples were analyzed by the proposed method with satisfactory results.3 Determination of rufloxacin by high performance liquid chromatography with immobilized tris(2,2'-bipyridyl)ruthenium(II)-Ce(IV) chemiluminescence detectionA sensitive, selective and simple method was developed for the determination of rufloxacin by HPLC-CL detection. The detection was based on the chemiluminescence reaction of rufloxacin with Ce(IV)-HNO3 and tris(2,2'-bipyridyl)ruthenium(III), which was immobilized on the cationic ion-exchange resin. Under optimum conditions, the linear range for rufloxacin was 6.0×10-8-3.0x10-5 g/mL with the detection limit of 3.0×10-8 g/mL. The relative standard deviation (RSD) was 1.6%(n=11) for 5.0×10-7 g/mL rufloxacin. Rufloxacin in the serum samples were analyzed by the proposed method with satisfactory results. |
