基于高效液相色谱法测定长白猪肌肉组织和肠道内容物中腺苷酸含量
Determination of adenosines in muscle and intestinal digesta of Landrace pigs by high preformance liquid chromatography
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摘要: 腺苷酸是能量代谢的载体，为了更好地研究长白猪肌肉组织和肠道内容物中能量代谢的差异，建立了一种快速检测猪肌肉组织和肠道内容物中腺苷酸含量的高效液相色谱（HPLC）分析方法。色谱条件为：Waters Symmetry C18 反相色谱柱（250 mm×4.6 mm，5 μm）；流动相为甲醇-50 mmol/L 磷酸二氢钾缓冲液（pH6.5），其体积比为9∶91；流速0.5 mL/min；柱温30（±1）℃；紫外检测波长259 nm，进样体积10μL 时，在0.1~100 mg/L 浓度范围内，ATP、ADP 和AMP 质量浓度与峰面积呈良好的线性关系，线性相关系数分别为0.999、0.998 和0.996，检出限（LOD，S/N ≥ 3）分别为0.0001、0.004、0.004 mg/L，定量限（LOQ，S/N ≥ 10）分别为0.002、0.02、0.02 mg/L，且平均回收率分别为92.00%~99.05%、100.77%~104.48% 和110.84%~113.72%，相对标准偏差（RSD）为0.29%~1.02%、0.33%~1.65% 和0.69%~1.92%，整个过程可在10min 内完成。该方法具有快速、准确、灵敏性高、重复性好等特点，可用于猪肌肉组织和肠道内容物中ATP、ADP 和AMP 含量的检测以及能量代谢研究。Abstract: Adenosines are the vector of energy metabolism. The aim of this work was to establish a method for the differences of energy metabolism from the pig muscle and intestinal digesta by high preformance liquid chromatography（HPLC）.When the analytic condition of HPLC in feed was a reverse phase Waters C18 column （250 mm×4.6 mm，5 μm），mobile phase was methanol（V）/ 50mmol/L KH2PO4 buffer solution（pH6.5） （V 9∶91） with the flow rate of 0.5 mL/min，the column temperature was at 30（±1）℃，the detection wavelength was operated at 259 nm，and the injection volume was 10 μL，the calibration curves had good linearity over concentration range of 0.1-100 mg/L. The linear relative coefficient for ATP，ADP and AMP over concentration range of 0.1-100 mg/L were 0.999，0.998 and 0.996，respectively. The limit of detections（LOD， S/N ≥ 3）of ATP，ADP and AMP were 0.0001，0.004 and 0.004 mg/L，and their limit of quantifications （LOQ，S/N ≥ 10）were 0.002，0.02 and 0.02 mg/L，respectively. The mean recovery ratio of ATP，ADP and AMP were 92.00%-99.05%，100.77%-104.48% and 110.84%-113.72% with RSDs of 0.29%-1.02%， 0.33%-1.65% and 0.69%-1.92%，respectively. The whole time of analysis lasted for 10 min.This method has properties of rapidity，accuracy，sensitivity and repeatability，which can be applied in detecting the concentration of ATP，ADP and AMP and analyzing the energy metabolism.
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