OBJECTIVE To establish an ion-pair reversed-phase high performance liquid chromatography(HPLC) method and determine ATP,ADP and AMP in liver of the rats treated by electric acupuncture.METHODS Separation of ATP,ADP and AMP in the liver tissue samples of rats was performed on a Megres-C1 8 water resistant column(5 μm,250 mm×4.6 mm) by Shimadzu SIL-20AT.A mobile phase was 0.18 mol/L phosphate dihydrogen containing 5% methanol (pH =6.25).The detection wavelength was set at 254 nm and the flow rate was 0.8 mL/min.The injection volume was 20 μL and the column temperature was set at 25 ℃.RESULTS The linear ranges of ATP,ADP and AMP were 1.1~212.2 μg/mL(r =0.999 9), 0.9~180 μg/mL(r =0.999 9) and 0.9 ~ 181.8 μg/mL (r =0.999 7).The average recovery rates of ATP,ADP and AMP were 96.4%,98.3% and 100.8%;and RSD were 2.50%,2.88% and 4.14%,respectively.The RSD of intra-day and inter-day were 0.06%~0.40% and 0.06%~0.69%.The detection limits for ATP,ADP and AMP were 0.1 1,0.09 μg/mL and 0.045μg/mL,respectively.CONCLUSION The established method for the determination of ATP,ADP and AMP in the liver of rats is simple,quick,accurate and reliable,which will lay a foundation for the determination of small molecular bio-active substances in the animal tissues treated by acupuncture intervention.%目的：建立一种离子对反相高效液相色谱(IP-RPHPLC)法并将其用于检测电针对肥胖大鼠肝脏组织中 ATP、ADP 和AMP 含量的影响。方法应用岛津 SIL-20AT 高效液相色谱系统,Megres-C18(5μm,250 mm×4.6 mm)耐水性色谱柱,对大鼠肝脏组织中 ATP、ADP 和 AMP 进行分离,流动相为0.18 mol/L 的磷酸二氢钾缓冲液(含5%甲醇且 pH=6.25),检测波长为254 nm,流速为0.8 mL/min,进样体积为20μL,柱温为25℃。结果 ATP 在1.1~212.2μg/mL(r =0.9999),ADP 在0.9~180.0μg/mL(r =0.9999)及 AMP 在0.9~181.8μg/mL(r =0.9997)范围内线性关系良好。ATP、ADP 和 AMP 平均回收率分别为96.4%、98.3%、100.8%；RSD 分别为2.50%、2.88%、4.14%；日内和日间精密度 RSD 分别为0.06%~0.40%和0.06%~0.69%；准确度均在99.0%~108.6%之间；ATP、ADP 和 AMP 检测限为0.11、0.09、0.045μg/mL。结论建立的高效液相色谱法用于检测大鼠肝脏组织中 ATP、ADP 和 AMP 含量简便、快速并且准确可靠,为针刺干预治疗后组织中小分子生物活性物质浓度的检测等研究工作奠定了基础。
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