The Application of IP-RPHPLC in Determination of ATP， ADP and AMP in Liver of the Rats Treated by Electric Acupuncture

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-C₁₈ 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 0.11，0.09μg/mL and 0.045μg/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.