Study On The Determination Of Some Pharmaceuticals In Rat Plasma By Using High Performance Liquid Chromatography With Other Devices

High performance liquid chromatography (HPLC) is one of high performance and fastly speed analysis and separation technology emerging in the late1960’s. Since it was invented, it has been widely used in food industry, chemical industry and biochemistry, medical research and other areas due to its convenience, high-speed, sensitivity and accuracy. As an important analysis method, it not only possesses extending good advantages such as high sensitivity, selectivity, its convenience but also it can analyze the structure of unknown compounds. It is widely applied in analysis of drugs and metabolites, natural products, residues, biomacromolecules and clinical assay in recent years.This thesis established several detection methods by high performance liquid chromatography (HPLC) with mass spectrometry (MS). It included two sections:preface and research report. In the preface section, it included preface and research report. Information about HPLC and MS such as the history, their characteristics, the development of the hyphenated techniques and their pharmacokinetic application in the medicine analysis by HPLC-MS was stated in the preface section. In the other research report section, analysis methods of the determination of bezafibrate in in plasma were reported, Determination of phillyrin in plasma and its application in the study of pharmacokinetics in rats by high performance liquid chromatography coupled with mass spectrometry was also studied; HPLC method for the quantitatively determination of diprophylline in rat plasma and urine with UV detection and Its application to a pharmacokinetic study.This dissertation consists of four chapters.Chapter1:PrefaceThe preface mainly described the history and current status of HPLC by complete description at characteristic and history of HPLC and HPLC with mass spectrometry. Meanwhile it simply introduces the pharmacokinetics, application and development of HPLC-MS in ananlysis of the drugs in plasma.Chapter2:quantitative determination of bezafibrate in rat plasma using HPLC-MSA simple, rapid, sensitive HPLC-MS method was developed and validated for determining bezafibrate in rat plasma. Chromatographic separation was achieved on a reverse phase Gemini C18(150×4.6mm,5μm Phenomenex, CA, USA) at35℃using acetonitrile, methanol and0.03%aqueous solution of formic acid (60:15:25, v/v/v) as mobile phase at flow rate of0.3mL/min. The wavelength was set at235nm and the injection volume was5μL. Under this conditions, The detection was performed by negative electrospray ionization, monitoring the transitions m/z359.65→273.70for bezafibrate and m/z212.95→127.08for the IS (internal standard). A good linearity was achieved within the concentration range of0.073μg/mL-7.884μg/mL (r=0.9995) for bezafibrate and the mean recovery of samples ranged from94.3%to103.1%at three concentrations tested. The intra-day and inter-day precisions (as relative standard deviation) were below6%. The limit of quantification (LOQ) was30ng/mL and the limit of detection (LOD) was9ng/mL. This HPLC-MS method was reproducible and sensitive enough for routine analysis of bezafibrate in human plasma.Chapter3:determination of phillyrin in rat plasma by high performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic studyA simple, fast and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method was developed and validated to quantitatively determinate phillyrin in rat plasma using reserpine as internal standard (IS), The sample preparation method was simple only using0.6mL acetonitrile as protein precipitant, The supernatant was dried at50℃in a Vacuum Drying Oven and then dissolved in l.OmL organic phase consisted with methanol and acetonitrile (1:9, v/v). The analytical samples were separated on a reversed-phase C18column (150×4.6mm,5μm Phenomenex, CA, USA) at30℃, using0.05%acetic acid aqueous solution and acetonitrile (56:44, v/v) as mobile phase, at a flow rate of0.3mL/min. The detection were performed by electrospray ionization (ESI) mass spectrometry using extracted ion chromatogram (EIC) detection in positive ion mode while monitoring target molecular ions at [M+Na]+m/z557.57for Phillyrin and [M+H]+m/z609.45for the I.S. Good linearity was generated over the concentration range of0.069-8.97μg/mL (r=0.9995) with the limit of detection and quantification of7.04and21.12ng/ml, respectively. Intra-day and inter-day precisions were less than6%and the mean recoveries at three tested concentrations ranged from96.27%to100.63%. Those results indicated that the proposed method was fast, sensitive and feasible enough to be applied to the pharmacokinetic study in rats after intravenous injection of phillyrin. Chapter4:HPLC method for the quantitatively determination of diprophylline in rat plasma and urine with UV detection and its application to a pharmacokinetic studyA simple, fast and sensitive HPLC method has been developed for determination of diprophylline in rat plasma and urine and was applied to a pharmacokinetic study in the present study. The plasma and urine samples were separated and analyzed on a NH2column(250x4.6mm,5μm, Phenomenex, CA, USA), using acetonitrile and0.01%acetic acid aqueous solution (80:20, v/v) as a mobile phase at a flow-rate of0.3mL/min.The column temperature was kept at35℃and UV detection was performed at273nm. The injection volume was20μL. The method was validated over the concentration range of0.088μg/mL-46.63μg/mL with a lower limit of detection of7.88ng/mL and8.3ng/mL in rat plasma and urine, respectively. The RSDs for the intra-day and inter-day determinations were lower than1.96%, the mean extraction recovery of diprophylline in rat plasma ranged from98.61%to99.18%and the average recoveries for diprophylline from urine were between99.12%and101.15%. This validated method was successfully applied to the determianation of diprophylline in rat plasma and urine and investigated the pharmacokinetic properties of diprophylline in rats after intravenous administration (6mg/kg). The results also showed that the method provided us a reliable bioanalytical method to study the pharmaceutical formulation in rat plasma.