HPLC法测定术后疲劳综合征大鼠骨骼肌ATP,_ADP,_AMP的含量

目的: 建立改良的高效液相色谱(HPLC)法测定术后疲劳综合征(POFS)大鼠骨骼肌5’-三磷酸腺苷(ATP)、5’-二磷酸腺苷(ADP)、5’-磷酸腺苷(AMP)的含量。方法: 采用色谱柱Hypersil ODS2(4.6 mm×150 mm，5 μm)，柱温25 ℃；流动相为100 mmol·L^-1磷酸盐缓冲液(含12 mmol·L^-1的磷酸氢二钠和88 mmol·L^-1的磷酸二氢钠，pH=6.5)-甲醇(99.9∶0.1，体积比)；流速1.0 mL·min^-1；紫外波长为254 nm；进样量20 μL。结果: ATP、ADP、AMP色谱峰在10 min内得到了较好的分离，在测定范围内均呈良好的线性关系，r分别为1.000，0.9996，0.9994；精密度试验日内RSD≤4.1%，日间RSD≤3.5%；稳定性试验RSD≤4.8%；重复性试验RSD≤2.3%；回收率在97.1%～105.4%之间，RSD≤4.8%。应用改良的方法对模型组大鼠和对照组大鼠骨骼肌ATP、ADP、AMP的含量进行检测。结果显示，与对照组相比，模型组大鼠骨骼肌ATP含量在术后第1,3 d明显下降(P<0.05)，ADP含量在术后第7 d明显升高(P<0.05)，术后第10 d升高特别显著(P<0.01)，AMP含量在术后第10 d明显升高(P<0.05)。结论: 该方法操作简便，检测时间短，结果准确可靠,可以检测POFS大鼠骨骼肌ATP、ADP、AMP的含量，有助于POFS骨骼肌能量代谢的进一步研究。

Objective: To establish an improved HPLC method for determination of adenosine 5′-triphosphate (ATP)，adenosine 5′-diphosphate (ADP) and adenosine 5′-monophosphate (AMP) in skeletal muscle of rats of postoperative fatigue syndrome (POFS). Methods: A mobile phase containing 100 mmol·L^-1 phosphate buffer (consisting of 12 mmol·L^-1 Na₂HPO₄ and 88 mmol·L^-1 NaH₂PO₄, pH=6.5)and methanol(99.9∶0.1, v/v) was pumped through a Hypersil ODS2 (4.6 mm×150 mm，5 μm) column at temperature 25 ℃ at a flow rate of 1.0 mL·min^-1. The injection volume was 20 μL while the detection wavelength was set at 254 nm. Results: Optimum separation of the three compounds was achieved in <10 min. The range of standard concentrations was linear, which was proportional to the concentration of analytes in the interested samples. The correlation coefficients were 1.000, 0.9996 and 0.9994 for ATP，ADP and AMP,respectively. The method was precise (intra-day RSD≤4.1%, inter-day RSD≤3.5%), stable (RSD≤4.8%), repeatable (RSD≤2.3%) and accurate (recovery rates of 97.1%-105.4% with RSD≤4.8%). With this established method, a survey of ATP，ADP and AMP levels in samples of skeletal muscle was performed for rats for model group and control group. Sample results indicated that the content of ATP in skeletal muscle of rats of model group decreased significantly on postoperative day 1 and day 3 (P<0.05), while the content of ADP significantly increased on postoperative day 7 (P<0.05) and greatly significantly increased on postoperative day 10 (P<0.01), and AMP levels increased significantly on postoperative day 10 (P<0.05), compared with the control group. Conclusion: The established method is simple, quick, accurate and reliable, which can be used to determinate ATP，ADP and AMP in skeletal muscle of rats of POFS.It will be available to study the energy metabolism in skeletal muscle for POFS.