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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。
OBJECTIVE: To establish a method for the simultaneous determination of ketamine and norepinephrine in isolated rat liver microsomes, and to compare the gender differences of ketamine metabolism in liver microsomes between male and female rats. METHODS: Phenacetin was used as internal standard in HPLC. The column was Welchrom C18. The mobile phase consisted of acetonitrile-O-015mol·L-1 ammonium acetate (pH5 85 with acetic acid) = 40:60, The flow rate was 1 mL · min-1, the detection wavelength was 215 nm, the column temperature was 30 ℃ and the injection volume was 20 μL. Metabolism of 5 ~ 1000μg · mL-1 ketamine incubated in isolated rat liver microsomes (1.0 mg · mL-1) for 15 min was compared. Results: The linear range of ketamine was 0.1-1.20 μg · mL-1 (r = 0.9999) and 20.0-1000.0 μg · mL-1 (r = 0.999 4) The linear range was 0.1-1.20 μg · mL-1 (r = 0.9998). The minimum detectable limit was 2 ng · mL-1. The intra-day RSD was less than 10.8% and the daytime RSD was less than 12. The recovery rates were (89.6 ± 8.5)% ~ (102.5 ± 6.4)%, (86.5 ± 3.6)% ~ (101.7 ± 4.8) %, The recoveries were (90.6 ± 6.3)% ~ (92.8 ± 4.2)%, (84.5 ± 7.9)% ~ (87.7 ± 5.5)% . Ketamine elimination rate and norepinephrine production rate were significantly higher in the male rat liver microsomes than those in the female rats at the dosage of 5 ~ 1000μg · mL-1. Conclusion: The method is sensitive, simple and accurate and can be used for the determination of ketamine and norepinephrine in rat liver microsomes. Sex differences in the metabolism of ketamine in isolated rat liver microsomes.