| High performance liquid chromatography (HPLC) as an important modern measurement of separation and analysis is developed on the basis of classical Liquid Chromatography. Due to its convenience, high-speed, sensitivity and accuracy, it has been widely used in the areas of biology engineering, pharmacy, foodstuff, environment and petroleum so far.This thesis establishes several detection methods by Reversed-phase High-performance Liquid Chromatography (RP-HPLC). It includes two sections-review and research report. In the review section, information about high performance liquid chromatography such as characteristic, the development of chromatography technology and application in the medicine analysis was summarized. In the next research report section, analysis methods of several herbal medicine and antibiotic-netilmicin were reported. That is:Trace analysis of netilmicin; Etimicin; amikacin; Isepamicin in rat plasma by pre-column derivatization and high-performance liquid chromatography with fluorimetic detection. And netilmicin; amikacin; Isepamicin in pharmacokinetics the method has been successfully applied to the netilmicin; Etimicin; amikacin; Isepamicin determination in parenteral pharmaceutical formulations.This dissertation consists of five chapters.Chapter 1:In this chapter, the current status of HPLC technique is given by complete description at characteristic and development of HPLC and the application of HPLC in study of the kinds of aminoglycoside antibiotics pharmacokinetics in rat plasma.Chapter 2:A new, simple and sensitive method based on pre-column derivatization by reversed-phase high performance liquid chromatographic (HPLC) is described for the separation and quantification of netilmicin in plasma, using 9-fluorenylmethyl chloroformate (FMOC-Cl) as the derivatization reagent. Its pharmacokinetics is also presented. The derivatization modes and conditions for this method were optimized. The separation was performed on an Agilent XDB-Cg column (150 mm×4.6 mm,5μm) with a mixture of water-acetonitile (15:85, v/v) as mobile phase and the flow rate was 1.0 mL·min-1. The analytes were detected at excitation 265 nm and emission 315 nm. Analytical linearity was obtained for the method over the concentration range of 0.045-8.88μg·mL-1 and the correlation coefficient (r)= 0.9993. The limit of detection (LOD) (S/N= 3) was about 0.01μg-mL-1, and the limits of quantification was 0.03μg-mL-1 (3 LOD) for netilmicin. The relative standard deviation was less than 3% for intra-day assay (n=5) and 3.5% for inter-day assay (n=5) and the relative recovery was in the range of 96.62-100.84%(n=3). The plasma volume of 30μL was sufficient for the determination of netilmicin. The method provides a reliable bioanalytical methodology to carry out netilmicin pharmacokinetics in rat plasma.Chpter 3:A simple and sensitive reversed-phase liquid chromatographic method has been developed for the determination of amikacin by derivatization. The method is based on the pre-column derivatization of amikacin with 9-fluorenylmethyl chloroformate (FMOC-Cl). Isepamicin was used as the internal standard. The derivatization reaction proceeds in aqueous solution at room temperature with a borate buffer of pH 7.3. The formation of the corresponding derivative of amikacin is instantaneous and it is stable for more than 48 h. Detection was performed by fluorescence. Several factors influencing the derivatization reaction yields were studied and optimized. The system offered the following analytical parameters:limit of detection (LOD) of 90 ng ml-1 (3σ), linear correlation coefficient of 0.9998 and linear range response from 0.45 to 21.60μg-mL-1. The precision of the method was< 6%. As a preliminary application, the method has been successfully applied to the amikacin determination in parenteral pharmaceutical formulations.Chapter 4:A simple and novel LC method has been developed for determination of isepamicin (ISP) in rat plasma, an aminoglycoside antibiotic agent. After protein precipitation and clean-up procedure to remove lipophilic contaminants, ISP is derivatized by pre-column with 9-fluorenylmethyl chloroformate for fluorescence detection. Chromatographic separations are achieved using C18 column and mobile phase consisting of water and acetonitrile (68/32, v/v). Amikacin was used as an internal standard. The calibration curve was linear over a concentration range of 0.625-15μg/ml. The limit of quantification was 0.45μg/ml. The intra-and inter-day variabilities of ISP were both less than 5%. Both derivatives were stable for at least a week at ambient condition. This assay procedure should have useful application in therapeutic drug monitoring of ISP. The limit of detection was 0.10μg/ml. The specificity, assay linearity, low level assay linearity and assay repeatability were also investigated. The established method provides a reliable bioanalytical method to carry out isepamicin pharmacokinetics in rat plasma.
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