High-pressure liquid chromatography and capillary electrophoresis for the analysis of gliclazide, corticosteroids and methotrexate

• Main Authors: Chien-Yuan Kuo, 郭建源
• Other Authors: 吳秀梅
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/43961061131163017254

博士 === 高雄醫學大學 === 藥學研究所博士班 === 94 === High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have been broadly applied to the analysis and separation of drugs. This thesis includes a HPLC method for establishing the concentration-time profile of gliclazide (GL) in plasma and CE methods for the assay of six corticosteroids in commercial pharmaceuticals and the simultaneous determination of methotrexate (MTX) and its metabolites in whole blood and plasma. One HPLC method was established to determine GL in plasma using an electrochemical detector (ED). The analysis was performed on a C18 column using a mobile phase of borate buffer (70 mM, pH 7.5) and acetonitrile (73.5:26.5, v/v) and detected at 800 mV. During method validation, the regression equations of GL possessed good linearity (r?d0.9990) over the calibrated ranges of 50.0 nM ~ 4.0 ?嵱. The relative standard deviation (RSD) and relative error (RE) values in intra- and inter-day assays were below 5.30 %, which showed good precision and accuracy. The limit of detection (LOD) of GL was 10.0 nM (S/N=3, injection 10 ?尳). This proposed method was applied to monitor the GL concentration of a male volunteer (27 yr, 70 kg) dosed one 80-mg Diamicron?? tablet. Another part of this thesis was to develop a micellar electrokinetic chromatography (MEKC) for the assay of six corticosteroids (betamethasone, BMS; cortisone, CTS; prednisolone, PNL; methylpredisolone, MPL; triamcinolone, TCL; prednisone, PNS) in commercial pharmaceuticals. An uncoated capillary was used (with an effective length of 30 cm) for the analysis. The optimized conditions were using borate buffer (80 mM, pH 9.0) containing 60 mM sodium cholate and 60 mM sodium deoxycholate at 20 kV, and detected at 254 nm. During method validation, the regression equations of corticosteroids showed good linearities (r?d0.9969) over the calibrated ranges of 10.0 ~ 100.0 ?嵱. The RSD and RE values in intra- and inter-day assays were below 4.91 %, which showed good precision and accuracy. The LOD was 5.0 ?嵱 for each analyte. This method was feasible for the application of content assays of commercial formulations (Prednisolone??, Methylprednisolone??, Rinderon??). All of the analytical values fell within labeled amounts required by the USP XXV and ChP V. We also established a simple, rapid and selective capillary zone electrophoresis (CZE) method to simultaneously determine MTX and its eight metabolites in whole blood. The separation was performed in an uncoated capillary (effective length 30 cm) using 1.2 M glycine buffer (pH 9.3) under 20 kV by UV detector (300 nm). The baseline separations could be done within 10 minutes. During method validation, the regression equations (10.0 ~ 50.0 ?嵱) possessed good linearities (r?d0.9965). The RSD and RE values in intra- and inter-day assays were below 7.06 %, which showed good precision and accuracy. The optimized method was successfully applied to monitor the blood concentration of MTX and its eight metabolites from one 8-yr-old boy dosed 5.0 g MTX / m2/ 24h IV infusion, and the data fitted that of the pharmacokinetics in literatures. Large-volume sample stacking (LVSS) is a sensitivity-enhancing technique without additional instrument modification in CE. To improve the sensitivity of MTX, 7-OHMTX and DAMPA in plasma, large volume sample solution (3.0 psi, 70 s) was introduced into the capillary, which was filled with electroosmotic flow modifier, poly(ethylene oxide). The sample matrices would be pumped out from the capillary by electroosmotic flow in the sample zone. Analytes could be stacked at the interface between the buffer zone and the sample zone, and then separated. The optimized conditions were using phosphate buffer (70 mM, pH 6.0) containing 0.01 % (w/v) poly(ethylene oxide) at 25 kV (detection at anode side) and detected at 300 nm. During method validation, the regression equations of MTX, 7-OHMTX and DAMPA had good linearities (r?d0.9958) over the calibrated ranges of 0.1 ~ 10.0 ?嵱 for MTX and 7-OHMTX and 0.3 ~ 10.0 ?嵱 for DAMPA. The RSD and RE values in intra- and inter-day assays were below 9.69 %, which showed good precision and accuracy. The LODs were found to be 0.05 ?嵱 for MTX and 7-OHMTX, and 0.1 ?嵱 for DAMPA. The sensitivities of MTX and its two main metabolites in plasma could be increased about one-hundred fold compared with CZE method under the optimized method. This method was successfully applied to determine the plasma concentration after treating with MTX in clinical practice.