HPLC法测定大鼠离体肝微粒体中氯胺酮和去甲氯胺酮的含量及其性别差异研究

目的:建立同时测定大鼠离体肝微粒体中氯胺酮和去甲氯胺酮含量的方法,比较氯胺酮在雌雄大鼠肝微粒体中代谢的性别差异。方法:采用高效液相色谱法,以非那西丁为内标,色谱柱为WelchromC18,流动相为乙腈-0·015mol·L-1乙酸胺(加醋酸调pH5·85)=40:60,流速为1mL·min-1,检测波长为215nm,柱温为30℃,进样量为20μL。比较5～1000μg·mL-1氯胺酮在雌雄大鼠离体肝微粒体(1·0mg·mL-1)中孵育15min的代谢情况。结果:氯胺酮检测浓度的线性范围为0·1～20·0μg·mL-1(r=0·9999)和20·0～1000·0μg·mL-1(r=0·9994),去甲氯胺酮的线性范围为0·1～20·0μg·mL-1(r=0·9998),最低检测限均为2ng·mL-1;日内RSD均小于10·8%,日间RSD均小于12·7%;方法回收率分别为(89·6±8·5)%～(102·5±6·4)%、(86·5±3·6)%～(101·7±4·8)%,提取回收率分别为(90·6±6·3)%～(92·8±4·2)%、(84·5±7·9)%～(87·7±5·5)%。加药量在5～1000μg·mL-1时,氯胺酮消除速率和去甲氯胺酮生成速率在雄大鼠肝微粒体中均明显高于雌大鼠。结论:本方法灵敏、简便、准确,可用于大鼠肝微粒体中氯胺酮和去甲氯胺酮的含量测定。氯胺酮在大鼠离体肝微粒体中的代谢存在性别差异。

Determination of Ketamine and Norketamine in Rats Liver Microsome in Vitro by HPLC and Its Gender Difference

OBJECTIVE:To develop a method for the determination of ketamine and norketamine in rat liver microsome in vitro,and to compare the gender difference of ketamine metabolism in liver microsome between male rat and female rat.METHODS:HPLC method was adopted using phenacetin as internal standard.The determination was performed on Welchrome C18 column with acetonitrile-0.015 mol·L-1 ammonium acetate(pH value adjusted to 5.85 by adding triethylamine,40:60) as mobile phase at the flow rate of 1 mL·min-1.The detection wavelength was 215 nm,the column temperature was 30 ℃ and the injection volume was 20 μL.The metabolism of 5～1 000 μg·mL-1 ketamince cultured in isolated liver microsome(1.0 mg·mL-1) between male rat and female rat for 15 min was compared.RESULTS:The linear ranges of ketamine were 0.1～20.0 μg·mL-1(r=0.999 9) and 20.0～1 000.0 μg·mL-1(r=0.999 4).The linear range of norketamine was 0.1～20.0 μg·mL-1(r=0.999 8).The lowest limit of detection was 2 ng·mL-1.The intra-day and inter-day RSD were all less than 10.8% and 12.7%.And the method recoveries were(89.6±8.5)%～(102.5±6.4)%,(86.5±3.6)%～(101.7±4.8)%;extration recoveries were(90.6±6.3)%～(92.8±4.2)%、(84.5±7.9)%～(87.7±5.5)%.The elimination velocity of ketamine and generation rate of norketamine in male rats microsome were higher than those of female rats in the adding dose of 5～1 000 μg·mL-1.CONCLUSION:The method is sensitive,simple and accurate,and it can be used for the content determination of ketamine and norketamine.The results of velocity indicate the manner of ketamine metabolism in vitro is of gender difference.