大白鼠血漿中褪黑激素之高效液相層析分析   全文下載

Determination of Melatonin in Rat Plasma by High Performance Liquid Chromatography with Electrochemical Detection

本研究利用高效能液相層析儀配合電化學偵測器以定量分析大白鼠血漿中褪黑激素之濃度，結果顯示：此分析方法具有良好之線性關係（R^2=1），偵測極限為1.3 ng／ml；利用乙氰直接將血漿樣品液去蛋白並同時萃取褪黑激素，以簡化縮短前處理之步驟，其添加於血漿樣品液之回收率為92～99%；同日內之穩定性試驗其偏差係數為1.5～2.2%，異日間之偏差係數為3.5～5.0%，顯示此分析方法具有簡單、快速、高準確性且再現性好之優點。利用此分析方法以探討Sprague Dawley品系大白鼠血漿中褪黑激素之清除率，經由動物體股靜脈給予褪黑激素（0.67 mg／Kg）， 結果顯示褪黑激素在注射0.5～1.0小時後於血液中濃度達到最高峰，於0.5、1.0、1.5、2.0、2.5與3.0小時後血漿中褪黑激素之濃度分別為102.8 ± 24.3、51. 5 ± 13.0、30.2 ± 7.1、22.2 ± 7.2、20.4± 9.8與17.9 ± 8.1 nM （平均值± SEM,n=8），其清除率相當快速，約3小時後可完全清除血漿中褪黑激素；而此SD品系大白鼠之實驗動物模式將可廣泛應用在探討大白鼠對褪黑激素抗氧化相關主題之研究上。 Melatonin（5-methoxy-N-acetyltryptamine） plays a role in a number of distinct physiological functions. We have developed a convenient high performance liquid chromatography assay with electrochemical detection（HPLC / ECD） for the accurate determination of melatonin in biological samples. This assay involves direct addition of rat plasma to acetonitrile for simultaneous protein precipitation and melatonin extraction. The assay was validated by addition of melatonin to rat plasma with a recovery rate between 92 and 99 %. The correlation coefficient of calibration curve was 1.000. The relative standard deviations for intraday and interday analyses were 1.5 ~ 2.2 % and 3.5 ~ 5.0 %, respectively. Melatonin clearance rate of biological samples were determined by this ‧method. Melatonin（0.67 mg / kg) were given to Sprague Dawley rats by femoral vein injection. The melatonin concentrations were determined by HPLC / ECD at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hrs. The concentrations were reached the peak levels at 0.5 - 1.0 hrs and cleared after 3.0 hrs. The concentrations of melatonin were 102.8 ± 24.3, 51.5 ± 13.0, 30.2 ± 7.1, 22.2 ± 7.2, 20.4 ± 9.8 and 17.9 ± 8.1 nM (mean± S.E.M., n = 8） at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hrs, respectively. This HPLC / ECD method can be useful in its application to biomedical studies of melatonin."

Melatonin（5-methoxy-N-acetyltryptamine） plays a role in a number of distinct physiological functions. We have developed a convenient high performance liquid chromatography assay with electrochemical detection（HPLC / ECD） for the accurate determination of melatonin in biological samples. This assay involves direct addition of rat plasma to acetonitrile for simultaneous protein precipitation and melatonin extraction. The assay was validated by addition of melatonin to rat plasma with a recovery rate between 92 and 99 %. The correlation coefficient of calibration curve was 1.000. The relative standard deviations for intraday and interday analyses were 1.5 ~ 2.2 % and 3.5 ~ 5.0 %, respectively. Melatonin clearance rate of biological samples were determined by this •method. Melatonin（0.67 mg / kg) were given to Sprague Dawley rats by femoral vein injection. The melatonin concentrations were determined by HPLC / ECD at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hrs. The concentrations were reached the peak levels at 0.5 - 1.0 hrs and cleared after 3.0 hrs. The concentrations of melatonin were 102.8 ± 24.3, 51.5 ± 13.0, 30.2 ± 7.1, 22.2 ± 7.2, 20.4 ± 9.8 and 17.9 ± 8.1 nM (mean± S.E.M., n = 8） at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hrs, respectively. This HPLC / ECD method can be useful in its application to biomedical studies of melatonin.