高效能液相層析法配合螢光偵檢器分析生物檢體中之Colistin

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標題:	高效能液相層析法配合螢光偵檢器分析生物檢體中之Colistin Assay of Colistin in biological materials by HPLC with fluorescence detector
作者:	李崇維
貢獻者:	嘉南藥理科技大學：藥物科技研究所劉國盛
關鍵字:	液相層析螢光偵檢器 Colistin FMOC-Cl 衍生化人類血漿人類痰液 High performance liquid chromatography Fluorescence detector Coistin FMOC-Cl Derivative Human plasma Human sputum
日期:	2011
上傳時間:	2011-10-27 11:23:41 (UTC+8)
摘要:	Colistin，俗名Polymyxin E，屬於鎖環狀多胜肽狹效性抗生素，為1950年由小山康夫從Bacillus polymyxa var. Colistinus所分離而得，可治療多重抗藥性患者，對革蘭氏陰性菌有抑制作用，但對革蘭氏陽性菌無效。目前有許多抗生素對革蘭氏陰性菌具有多重抗藥性 (Multidrug Resistance，MDR)，Colistin可治療多重抗藥性患者，備受矚目。為了探討人類血漿與痰液中Colistin之濃度，建立一簡易且準確的方法，分析人類血漿與痰液中之Colistin，有其必要性。本研究利用HPLC配合螢光偵檢器分析人類血漿與痰液中之Colistin。人類血漿與痰液經由與螢光衍生化試劑FMOC-Cl (9-Fluorenylmethyl Chloroformate) 在pH=9.5環境下進行衍生化反應1分鐘，隨後加入30% Trichloroacetic Acid去除蛋白質，在4℃下以10,000g離心10分鐘，抽取上清液進行分析。HPLC移動相使用乙腈、四氫呋喃、水之體積比為90：4：6，分析管柱使用C18 Column，流速設定1.0 mL/min，分析時間為25分鐘。本研究在濃度範圍0.5~20.0 mg/L間，可得到一良好的線性回歸 (R2>0.999)，Colistin B、Colistin A滯留時間分別為15和18分鐘，兩成份之解析能力 (Rs) 大於1.5。回收率可達98%以上，同日間與異日間之RSD皆小於10%。反應後形成之衍生物於8小時內皆趨於穩定。結果顯示此方法可較簡易且準確地分析人類血漿與痰液中之Colistin。 Colistin, the common name is Polymyxin E. It is a constricted and polypeptide antibiotic. The Colistin was separated from Bacillus polymyxa var. Colistinus by Mr. Koyama in 1950s. They could be administered to multidrug-resistant (MDR) patients to inhibit the development of the Gram-negative bacteria, but don't to Gram-positive bacteria. There are many antibiotics on Gram-negative bacteria with multidrug-resistant, the most important one is Colistin. In order to detect the Colistin concentrations in human plasma and sputum, a simple and accurate method to assay Colistin concentrations in human plasma and sputum is necessary. The purpose of this study is using HPLC with fluorescence detector to analysis Colistin in human plasma and sputum. The human plasma and sputum are derivatized with FMOC-Cl at pH = 9.5. Following 1 min of reaction, the derivatives are mixed with 30% trichloroacetic acid for protein precipitation, and then centrifuged at 10,000g for 10 min at 4℃. The supernatant is collected for HPLC analysis. The mobile phase is acetonitrile/tetrahydrofuran/water (90:4:6). The column is used a Supelco Ascentis® C18 (250×4.6 mm, 5μm). The flow rate is set at 1.0 mL/min. The run time is 25 min. A good linear regression (R2> 0.999) were obtained for calibration curves with concentration range from 0.5 to 20.0 mg/L. The Colistin B and Colistin A retention times were 15 and 18 min and their resolving power (Rs) was at least 1.5. The recoveries of Colistin were at least 98%. The intra-day and inter-day precisions of less than 10% were found for Colistin. The FMOC derivatives of Colistin were found to be stable for at least 8 hours. The application of this method to determine Colistin in human plasma and sputum would be simple and accurate.
關聯:	校內校外均不公開，學年度：99，86頁
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