高效液相色谱法测定骨骼肌ATP、ADP、AMP、NAD^+、NADH含量

Determination of ATP, ADP, AMP, NAD^+,NADH in Skeletal Muscle by HPLC

采用高效液相色谱法测定骨骼肌腺嘌呤核苷酸和辅酶Ⅰ含量。高压液相系 统包括：惠普公司ＺＯＢＡＸ－Ｃ１８反相柱５ｍｍ×１２ｃｍ，５９０ＵＶ检测器。流动相为２２０ｍｍｏｌ／Ｌ磷酸钾缓冲液，内含１０％甲醇，用四丁基氢氧化胺ＴＢＡＨ调ｐＨ至６５。等比洗脱，流速为０８ｍｌ／ｍｉｎ。检测波长为２５４ｎｍ。所有样品和标准品在进样前均用０４５μｍ孔径的虑膜过滤后进样５μｌ作色谱分析。结果表明：ＨＰＬＣ可以同时测定ＡＴＰ、ＡＤＰ、ＡＭＰ、ＮＡＤ＋、ＮＡＤＨ，分离效率高，操作简便，快速每个样品仅需１５分钟，而且，重现性好，定性可靠，定量准确。

ATP, ADP, AMP, NAD^+,NADH in skeletal muscle were assessed by an isocratic HPLC separation with UV detection. The HPLC system consisted of a ZOBAXò■-Ｃ18 reversed- phase column(Smmxl2cm) and a 590 UV detector. The separation was carried out with 22Ommol/ L sodium phosphate buffer containing 10% methanol adjusted by TBAH to pH6.5. Flow rate was 0.8m1/min. Indirect CV detection was operated at 254nm and reference wavelength at 500, 1OOnm for all experiments. The injection volume was 5μl. This method proved to be reliable and reproducible.