Quantitation Of Sirolimus In Whole Blood And Tissues Using Liquid Chromatography-tandem Mass Spectrometry (HPLC-MSMS)

The objective of this study is to develop and validate sensitive, specific and rapid methods to quantitatively determine Sirolimus in whole blood and tissues using liquid chromatography-tandem mass spectrometry (HPLC-MSMS) for preclinical pharmacokinetic (PK) studies, and served as a reference to further support clinical PK studies.The First Chapter of this thesis describes the background and significance of this study. Goronory Heart Disease (CHD), a serious threat to the physical and mental health of modern people, causes a heavy social burden to public health. The threatments of CHD include drug therapy, coronary artery bypass grafting (CABD), and percutaneous coronary artery interventional therapy (PCI). PCI was applied to clinical for the first time in 1970s, and it has become one of the most important approaches for the treatment of CHD after decades of development. Intervention stent restenosis has been found to be a major factor affecting the long-term effects of PCI for CHD treatment, however, after drug-eluting stents, especially when sirolimus drug-eluting stents has been applied to clinical since 2002, the incidence of restenosis is significantly reduced, which has greatly improved the long-term treatment effect of PCI. As a result, drug-eluting stents has shown good effect and prospects in the treatment of CHD. In addition, many researchers are evaluating new formulations and routes of administration to improve the bioavailability and treatment of sirolimus. Therefore, in order to achieve therapeutic effect and to minimize the incidence of adverse reactions at the same time, it is important to understand the PK characteristics of sirolimus in vivo; and thus, to develop a more sensitive, specific, and rapid method to determine the concentrations of sirolimus in biological samples becomes essential.The main subject in Chapter Two is to develop methods to quantitatively determine Sirolimus in whole blood and tissues using LC-MSMS. The LC conditions, MSMS conditions, and sample preparation procedures have been optimized; and simple sensitive and specific methods with great reproducibility have been established which can be simultaneously applied to analysis Sirolimus in different biological matrices. A linear calibration curve is obtained in the concentration range of 50-10,000 ng/mL for whole blood, and 50-25,000 pg/mL for tissue homogenate, respectively. The intra-and inter-run precisions, expressed as the relative standard deviation (RSD), are less than 12.2%,11.9%,5.7%,6.1%,4.5%,5.1% and 7.9% for Sirolimus in pig whole blood, artery, myocardium, liver, kidney, lung and spleen homogenate, respectively; and the corresponding accuracies, expressed as the relative error (RE,) are within-11.0%-4.7%,-7.2%-0.6%,0.9%-6.4%,-8.9%-0.0%,-4.7%-10.0%,-2.6%-10.0% and-3.6%-5.8%, respectively. In rat whole blood, the intra-and inter-day precisions are less than 12.1%, and the accuracies) are within-1.6%～13.6%. In human whole blood, the intra-and inter-day precisions are less than 6.6%, and the accuracies are within -0.8%～14.3%.It can be concluded that in "the current study, sensitive, specific, and rapid methods have been successfully developed and validated for quantitative determination of Sirolimus in whole blood and tissue homogenate using LC-MSMS. The methods have also been successfully applied for the determination and evaluation of preclinical PK studies of elution of Sirolimus in the swine coronary artery model.