黄杨宁原料及片中环维黄杨星D含量及含量均匀度测定___

目的：筛选并建立黄杨宁原料及片中环维黄杨星D的含量和含量均匀度测定方法。方法：对正相高效液相色谱末端检测法和正相高效液相色谱ELSD检测法以及反相离子对高效液相色谱末端检测法进行了比较。并建立了反相离子对高效液相色谱法，以C18柱分离，柱温30 ℃；流动相为0.0 1 mol•L-1庚烷磺酸钠与0.01 mol•L-1磷酸二氢钾等量混合溶液（含0.2%三乙胺，用磷酸调pH 3.5)-乙腈（77∶23），流速1.0 mL•min-1，检测波长为206 nm。结果：4种方法中，综合通用性、合理性、耐用性、色谱行为、准确性和灵敏度各方面，以新建立的反相离子对高效液相色谱末端检测法为最佳，黄杨宁原料及片中的环维黄杨星D可与所含的其他相同骨架结构同系生物碱组分完全分离。方法经3根不同填料色谱柱验证，分离度均在1.5以上，且峰形较对称；片剂和原料的平均回收率分别为98.8%和98.0%(n=9）；不同仪器的中间精密度分别为片剂4.2%（n=3）和原料4.7% (n=3)；柱间耐用性片剂0.2% (n=3)和原料1.4% (n=3)；检测限为0.052 μg（RSD 3.4%，n=5)；定量限为0.200 μg（RSD 6.6%，n=5)。结论：经比较，新建立的反相离子对高效液相色谱末端检测法简单合理，通用性和耐用性强，准确性和重现性俱佳，方法灵敏度能满足小规格黄杨宁片的含量均匀度检测要求，完全适宜作为药典方法。

Objective:To screen and develop a pharmacopeias method for the determination and uniformity examination of cyclovirobuxine D in materials and Huangyangning tablets.Method:The NP-HPLC method with end-wavelength detection and RP-HPLC method with ELSD as well as the ion pair RP-HPLC method with end-wavelength detection were compared,and a new ion pair RP-HPLC method was developed,using C18 as a column with a temperature at 30 ℃,the mixed solution (containing 0.2% triethylamine and adjusted pH to 3.5 with phosphoric acid) composing of 0.01 mol•L-1 sodium-heptanesulfonate solution and equal quantity of 0.01 mol•L-1 potassium dihydrogen phosphate solution-acetonitrile (77∶23) as a mobile phase,the flow rate was 1.0 mL•min-1,the detection wavelength was at 206 nm.Results:Among the 4 methods,in terms of universality,rationality,robustness,chromatographic feature,accuracy and sensitivity,the newly developed ion pair RP-HPLC method was the best,by which cyclovirobuxine D was separated completely with the other alkaloids with similar structure abtaining above 1.5 resolution on three different packed C18 columns,and the relatively asymmetric peaks in all instances.Moreover,the average recoveries of the developed method was 98.8% and 98.0% (n=9) for tablets and materials respectively.The intermediate precision between instruments was 4.2%（n=3）for tablets and 4.7% (n=3) for materials；the robustness between columns was 0.2% (n=3) for tablets and 1.4% (n=3) for materials.The average LOD was 0.052 μg（RSD 3.4%,n=5) and LOQ was 0.200 μg（RSD 6.6%,n=5).Conclusions:By comparison,the newly developed ion pair RP-HPLC method is simple and reasonable,which has a good universality and robustness,and displays a perfect performance in terms of accuracy and reproducibility.The LOQ can meet the need of uniformity examination for the tablets with minimum specification,therefore it is qualified for being as a pharmacopeia method.