柱前衍生化高效液相色谱法测定莲子心中去甲乌药碱含量

Determination of higenamine in Nelumbo nucif eraby HPLC with precolumn derivatization

以9-芴甲基-N-琥珀酰亚胺基碳酸酯(Fmoc-OSu)为荧光衍生化试剂,建立了柱前荧光衍生化反相高效液相色谱法分析去甲乌药碱的高灵敏分析方法.在硼酸缓冲溶液(pH=8.5)中,去甲乌药碱与Fmoc-OSu在温和的反应条件下反应生成具有荧光性的去甲乌药碱衍生物.采用UltimateR○XB-C18(5μm,150mm×4.6mm i.d.)色谱柱,用乙腈-水(体积分数比为85∶15)等溶液强度洗脱,以λ=265nm为激发波长,λ=315nm为发射波长,对去甲乌药碱衍生物进行了检测.实验结果显示:去甲乌药碱在0.05～20μg/mL范围内呈良好的线性关系(r=0.999 8),其最低检出限(S/N=3)为5.0ng/mL,加标回收率为94.1%～105.9%,RSD为2.74%(n=5).本方法灵敏度高、选择性好、准确度高,能够满足含去甲乌药碱中药材的质量控制.

A sensitive method for determination of higenamine based on a derivatization reaction with a fluores- cent tagging reagent, 9-fluorenylmethoxycarbonylsuccinimide （Fmoc-OSu） is developed. The Fmoc-OSu pref- erably reacts with higenamine under mild reaction conditions in the presence of borate buffer （pH = 8. 5） to produce the corresponding fluorescent derivative with an excitation maximum at 265 nm and an emission maxi- mum at 315 nm. The derivative of higenamine are efficiently resolved on a Ultimate XB-C18 （5μm, 150 mm ×4.6 mm i. d. ） column by an isocratic elution with water-acetonitrile （85 ： 15） mobile phase. The linear range is 0. 05-20 μg/mL （r=0. 999 8） for higenamine. The average recovery of the higenamine is 94. 1% 105.9% （RSD 2.74%）. The limits of detection （S/N=3） per injection is 5.0ng/mL. The developed method is applied successfully to the determination of higenamine in embryo of Nelumbo nucifera, a Chinese herbal medicine.