单步萃取-反相高效液相色谱法测定人全血中环孢菌素A及其药动学参数

Determination of cyclosporin A in human whole blood by reversed phase liquid chromatography with single step extraction and its pharmacokinetics

目的：建立单步萃取反相高效液相色谱法测定人全血中环孢菌素Ａ（ＣｙＡ）浓度，研究该药物在人体内的药动学和生物利用度。方法：血样经乙醚单步萃取后，以Ｃ８硅胶键合相为固定相，乙腈甲醇水异丙醇（５７∶１８∶２５∶１．５）为流动相，６５℃柱温下进行色谱分离。采用环孢菌素Ｄ为内标，在２０８ｎｍ处紫外定量检测。所得数据采用ＰＫＢＰＮ１药动学程序拟合计算，求出有关药动学参数。结果：ＣｙＡ血浓度在４０～１２００ｎｇ·ｍｌ－１范围内与色谱峰面积比值呈良好的线性关系，全血中ＣｙＡ最低检测浓度为２０ｎｇ·ｍｌ－１，日内（ｎ＝７）及日间（ｎ＝６）测定ＲＳＤ为４．８％及７．８％，，方法回收率为９８．６％～９９．５％。对１０名青年男性正常者单剂量ｐｏＣｙＡ胶囊后研究表明，其药时曲线符合一级吸收的双室模型。血药浓度约在１．２ｈ达到峰值８７９．９ｎｇ·ｍｌ－１，消除半衰期约为５ｈ。结论：本方法简便，准确，能较好地满足ＣｙＡ临床常规血药浓度监测及人体药动学过程研究。

OBJECTIVE:To develop a reversed phase liquid chromatographic method utilizing a single step extraction procedure for the determination of cyclosporin A(CyA) in human whole blood and the study of pharmacokinetics and bioavailability in humans.METHOD:The drug was extracted from whole blood with diethyl ether and separated isocratically using an octyl bonded silica column and a mobile phase of acetonitrile methanol deionized water and isopropanol(57∶18∶25∶1.5).The column temperature was maintained at 65℃.CyA was quantified by absorbance at 208 nm,with cyclosporine D as internal standard.The data obtained were fitted with PKBP N1 program on a computer.RESULTS:A linear relationship was obtained between the peak area ratio and the amount of CyA from 40 ng·ml -1 to 1 200 ng·ml -1 and the lower limit of detection of CyA was 20 ng·ml -1 .Within-day and between-day coefficients of variation for assaying of CyA at the 200 ng·ml -1 level were 4.8%(n=7) and 7.8%(n=6),respectively.The recoveries of CyA were 98.6%～99.5% for whole blood.The method was used to determine CyA in the whole blood samples from ten volunteers and provided data on the pharmacokinetics of the drug.The results showed that the dispostion of CyA was conformed to a two compartment model with t 1/2α =(0.37±0.23) h,t 1/2β =(4.98±1.05) h,t max =(1.24±0.33) ng·ml -1 ,c max =(879.9±186.8) ng·ml -1 and AUC 0→∞ =(3 993.8±796.3) ng·h·ml -1 .CONCLUSION:The method provides excellent recovery and good precision,and is simple and accurate.It is very suitable for routine determination of a large number of samples.