| The paper discussed the synthesis of four kind of restricted access material(RAM) chromatograph supports. The experiments developed an online sample preparation HPLC column switching system by self-made RAM C18 backing column, and applied this system to analyze the concentration of carbamazepine and donepezil in human serum. The experiments also developed a method for determining fudosteine in human serum and its pharmacokinetics. There are five chapter in this paper.Chapter one introduced the development of pharmaceutical analysis and online biosample preparation as well as the research of RAM chromatograph supports. It also introduced the technology of HPLC/MS/MS.Chapter two introduced the synthesis of four kind of RAM chromatograph supports and evaluated its hydrophobic character as well as anion retention character. The protein eluting ability and durability of the supports had also been evaluated. The Elemental analysis results showed the carbon percent of the RAM C18 5μm silica was 12.58% and hydrogen percent was 2.30%. The carbon percent of the RAM C18 10μm silica was 15.89% and hydrogen percent was 2.82%. The carbon percent of the Anion exchange RAM-1 10μm silica was 7.46%, hydrogen percent was 1.51%, and nitrogen percent was 0.75%. The carbon percent of the Anion exchange RAM-2 10μm silica was 8.39%, hydrogen percent was 1.73%, and nitrogen percent was 1.61%.Carbamazepine, pentobarbital sodium, barbital, phenobarbitone had been used as the evaluation drug of RAM C18 column, the mobile phase was water-methol(60:40, v/v), the flowrate was 1.0 mL/min. The eluting sequence was pentobarbital sodium(0.65 min), barbital(0.73 min), phenobarbitone(1.82 min) and carbamazepine(4.28 min). The results showed that the RAM C18 supports displayed good hydrophobic ability. pentobarbital sodium, aminobenzoic acid, sodium para-aminosalicylate had been used as the evaluation drug of Anion exchange RAM-1 and RAM-2. The mobile phase was water-methol(60:40, v/v), the flowrate was 1.0mL/min, injection volume was 20μL. The retention time of four evaluation drugs were more than 15 min. The results showed that the anion exchange RAM-1 and RAM-2 supports displayed good anion exchange ability. LiChrospher ADS C18 had been used as reference while the relative protein flushing rate of four kind of RAM chromatograph supports had been evaluated. The evaluation results indicated that the RAM supports definitely had restricted access class character. The durability results showed persistent column pressure which indicated that the RAM packing columns had good durability.Chapter three introduced the development of a simple, sensitive and online sample preparation HPLC column switching system method with UV absorbance detection for the quantification of carbamazepine and donepezil.A self-made RAM C18 pre-column (4.6 mm×10 mm,10μm) excluded the macromolecules and focused the carbamazepine by a mobile phase consisting in a mixture of water and methanol (95:5, v/v) at flow rate of 2 mL/min; afterwards, a second mobile phase consisting in a mixture of water and methanol (40:60, v/v) elutes the analyses to a C18 analytical column in back flush mode at flow rate of 0.8 mL/min. The UV detection was performed at 285 nm. The column switching time was 3.0 min and the retention time of carbamazepine was 9.2 min, the time of an analysis cycle was 12.0 min. Results indicated that the calibration curve showed a linearity range from 2.0 to 40.0μg/mL (r = 0.9997). The LOQ was 2μg/mL. The RSDs of intra-day and inter-day were less than 5%, the method recoveries were more than 95%.The sample preparation of donepezil had been processed online. A self-made RAM C18 pre-column (4.6 mm×10 mm,10μm) excluded the macromolecules and focused the donepezil by a mobile phase consisting in a mixture of water and acetonitrile (99:1, v/v) at flow rate of 2 mL/min; afterwards, a second mobile phase consisting in a mixture of 20 mM phosphate buffer, acetonitrile and acetonitrile (65:35:0.1, v/v) elutes the analyses to a C18 analytical column in back flush mode at flow rate of 1.0 mL/min. The UV detection was performed at 315 nm. The column switching time was 5.5 min and the retention time of donepezil was 11.5 min, the time of an analysis cycle was 16.0 min. Results indicated that the calibration curve showed a linearity range from 0.02 to 1.0μg/mL (r = 0.9997). The LOQ was 0.02μg/mL. The RSDs of intra-day and inter-day were less than 7%, the method recoveries were between 85% to 90%.The online sample clean-up extraction process was consisted of a column-switching system coupling a restricted access material (RAM) pre-column tandem an C18 analytical column. The process had short analysis time and decreasing sample preparation time as well as good recovery compare to off line procedue. The developed system showed to be adequate and attractive, demonstrating a large potential for sample preparation.Chapter four introduced the establishment of a sensitive method with HPLC/MS/MS technology for the quantification of fudosteine in human serum and the pharmacokinetic parameters of fudosteine. The mass spectrometer had used MRM mode and ESI source. Tandem mass spectrometer's main working parameters were as follows: source temperature 500 ?C, dwell time per transition 200 ms, ion source gas 60～70 psi, curtain gas 20 psi, collision gas 5 psi, ion spray voltage 5500 V, declustering potential 16 V, entrance potential 10 V, collision energy 22 V, collision cell exit potential 14.7 V, mode of analysis positive, ion transition for fudosteine 180.2/91.0. The chromatography parameters were as follows: Microsorb-MV C18 analysis column(4.6 mm×150 mm),separation temperature was 30℃, mobile phase was 20 mM formic acid- acetonitrile (75:25, v/v),flowrate was 0.40 mL/min. The results indicated that the serum endogenous did not disturb the detection of fudosteine sample. The retention time of fudosteine was 3.6 min, the time of an analysis cycle was 4.0 min. Results indicated that the calibration curve showed a linearity range from 100 to 20000 ng/mL (r = 0.9998). The RSDs of intra-day and inter-day were less than 5%, the method recoveries were more than 80%. There was no need of derivatization pre- or post-column as detection of fudosteine. The procedure was simple and decrease sample preparation time as well as variation. The research provided a new strategy for the direct high throughput detection of amino acids in biological specimen. Chapter five introduced the establishment of a RP-HPLC method to Simultaneous detect three kinds of tanshinones(cryptotanshinone, tanshinone I and) in traditional Chinese medicinal. Separation was achieved with the mobile phase consisting of methanol-tetrahydrofuran-water (25:30:45, v:v).Mobile phase was 20 mM formic acid- acetonitrile (75:25, v/v),flowrate was 0.40 mL/min. Results indicated that the calibration curve showed a linearity range from 0.1~30μg/mL(cryptotanshinone)0.5~30μg/mL (tanshinone I and tanshinone IIA). The UV detection was performed at 254 nm and temperature was at ambient temperature. The RSDs of intra-day and inter-day were less than 5%, the method recoveries were more than 90%. The proposed method has the advantages of recoveries, precision, and reliability, allowing it to apply in quality control of manufacturing process of traditional Chinese medicinal preparations containing Radix salvia miltiorrhiza.
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