柱前荧光衍生化RP-HPLC测定环维黄杨星D含量

DETERMINATION OF CYCLOVIROBUXINED BY RP-HPLC WITH PRECOLUMN FLUORESCENCE DERIVATIZATION

目的 建立环维黄杨星D含量测定方法。方法 环维黄杨星D同异氰酸萘酯反应后 ,RP HPLC荧光法测定。色谱柱为C1 8柱 ;流动相为甲醇 水 (85∶15 ) ;流速 :1mL·min- 1 ;荧光检测激发波长 30 5nm ,发射波长 385nm ;柱温35℃。结果 衍生化反应重复性好 ,RSD为 1 2 1% ,主峰与相关物质分离良好。结论 本法简单、准确 ,可用于环维黄杨星D及其相关物质含量测定。

AIM To establish a RP HPLC method for determination of cyclovirobuxine D. METHODS Cyclovirobuxine D reacted with a derivative reagent 1 naphthyl isocyanate in ch loroform to form fluorescence derivatives, stopped the reaction by adding the mo bile phase and then directly injected the solution into the chromatograph to sep erate it by RP HPLC. The analysis was carried out on C 18 column, the mobi le phase is methanol water (85∶15), the excitation wavelength was set at 305 n m, emission at wavelength 385 nm, and the flow rate was 1 mL·min -1 . T he effect of several factors including the reaction medium, temperature, time an d amount of 1 naphthyl isocyanate on the yield of the derivatization was also i nvestigated systematically. RESULTS A simple and rapid RP HPLC method for the simultaneous isolation and analysis o f cyclovirobuxine D and its related substances was developed, and the absence of interference between the derivative peak responses of cyclovirobuxine D and its related substances were verified by UV diode array detecter and MS. The lineari ty was obtained from 0 75 μg·mL -1 to 2 5 μg·mL -1 of cy clovirobuxine D derivatives with a correlation coefficient of 0 9991. The detec tion limit of cyclovirobuxine D derivative was 1 ng·mL -1 , the repeata bility of derivatization was good with relative standard derivation no more than 1 2% and derivative was stable within 48 h. The method described conforms to t he validation of China Pharmacopiea compendial methods used for pharmaceutical p roducts in general. CONCLUSION The established method is proved to be reliable quantitative method for the qual ity control of cyclovirobuxine D.