| Pharmacokinetics is the study of the time course of a drug within the body and incorporates the processes of absorption, distribution, metabolism and excretion. To better interpret the effect and toxicity of the drug, an analytical method should be established to determine the drug and its metabolites quantitatively, which help us to know about their mechanisms of action and their pharmacokinetic properties during the initial state of drug development and in clinical therapy. Danshen, the dried roots of Salvia miltiorrhiza, is a widely used traditional Chinese medicine. Danshensu (DSS) is one of the phenolic acids in Danshen, and it has been reported that these monomers, especially DSS, could protect the cardiac muscle by reducing oxygen free radical generation, inhibiting perxidative damage, preventing platelet aggregation and calcium antagonizing. The purpose of the present work was to develop and validate a sensitive, reliable liquid chromatography tandem mass spectrometric method for the determination of DSS in rat plasma, urine and bile. Moreover, a preliminary study on the metabolites in vitro and in vivo was carried out in this paper. The results provided a solid basis for the clarification of the pharmacokinetics and metabolism of DSS.1. The establishment of analytical method of DSS in biological matrixThe method validation plays an important role in pharmacokinetics study. For the determination of DSS in biological samples, the analytes were separated on a Dikma Diamonsil C18 column (5μm, 200mm×4.6mm i.d) eluted at 0.8mL/min with a mobile phase of methanol-water (80:20, v/v) containing 0.001% formic acid. The split ratio was 3:2 and the rate that flew in the MS equipment was 0.32mL/min. The mass spectrometer was operated in the negative MRM mode. The column and the autosampler temperature were kept at 25℃and the injection volume was 20μL. The validation results demonstrated that this method was significantly specific, accurate, precise, which was suitable for the preclinical pharmacokinetic studies on DSS. 2. The pharmacokinetics study of DSS in SD ratsThe validated LC-MS/MS method was used to investigate the plasma profiles of DSS after a single intravenous dose of 15, 30, 60mg/Kg respectively. The concentration-time data were analyzed by non-compartmental method. The t1/2 values were 2.73h, 2.37, 1.95h and the AUC0-∞ values of DSS were 15.29μg·h/mL, 34.58μg·h/mL and 58.49μg·h/mL for 15, 30 and 60mg dose, respectively, which increased with the dose, showing apparent dose-dependent relationship (r=0.9836). Urine was collected after a single intravenous dose of 30mg/Kg to rats and the accumulative excretion amount of DSS in urine was 46.99 % within 96h. The accumulative excretion amount of DSS in bile was 0.82% within 24h. The results showed that DSS was excreted mainly in urine.3. Preliminary study on the metabolites in vitro and in vivo of DSSAn LC-MS method was applied in analyzing rat's urine and bile samples. Blank urine was collected and compared with that after administration. Under the established HPLC-MS conditions, totally 7 compounds were detected including methylation DSS [M-H+14]-, sulfated DSS [M-H+80]-, sulfated DSS after methylation [M-H+94]- and acetylated DSS [M-H+42]-. We only found methylation DSS in bile. In the experiment of in situ rat single-pass intestinal perfusion model, the in situ rat loop model and rat liver perfusion model, methylation DSS could be found. In rat's hepatic microsome incubation system, the main metabolite was methylation DSS. The results of the enzyme kinetics showed that the Vmax was 185.19ng/(mL·min·mg)protein,Km was 98940.74ng/mL, that is 5.0×10-4mol/L, Clint was 1.87×10-3mL/(min·mg)protein. The results of the enzyme kinetics of COMT showed that the Vmax was 243.90ng/(mL·min·mg)protein , Km was 102156.10ng/mL, that is 5.16×10-4mol/L, Clint was 2.34×10-3mL/(min·mg)protein.
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