利用乌头类双酯型生物碱水解规律测定醇胺型乌头碱的含量

Determination of Alcohol Amine-diterpene Alkaloids by Hydrolysis Rule of Diester Aconitum Alkaloids

目的:研究乌头类双酯型生物碱水解转化为醇胺型生物碱的转化率,水解乌头类双酯型生物碱对照品,利用水解转化率计算出水解原液中醇胺型生物碱的量,以此量作为醇胺型生物碱对照品的量,建立测定制川乌中乌头原碱、次乌头原碱、新乌头原碱含量的方法。方法:通过控制乌头碱、次乌头碱、新乌头碱3种双酯型生物碱的水解条件,得到乌头原碱、次乌头原碱、新乌头原碱。采用高效液相色谱四级杆飞行时间串联质谱(HPLC-QTOF-MS)法,使用Agilent ZORBAX Extend-C18RRHT(2.1 mm×50 mm,1.8μm)色谱柱,流速为0.21 ml·min^(-1),柱温为30℃,流动相为甲醇-水(含0.1%甲酸和2.5 mmol·L^(-1)醋酸铵)系统梯度洗脱,采用电喷雾离子(ESI)源,正离子方式检测。结果:在本文的水解条件下,乌头碱对于乌头原碱的转化率为99.64%;次乌头碱对于次乌头原碱的转化率为99.94%;新乌头碱对于新乌头原碱的转化率为99.57%。HPLC-QTOF-MS方法学表明,乌头原碱、次乌头原碱、新乌头原碱在一定范围内具有较好的线性关系,r值均大于0.999 1,精密度、重复性和稳定性的RSD都小于5%,加样回收率均在99.43%~100.10%之间。结论:本方法操作简便、专属性和重复性较好,在缺乏对照品的情况下,可以为乌头类中药材质量控制提供依据。

Objective： To investigate the hydrolysis conversion rate of alcohol amine-diterpene alkaloids from aconitum alkaloids, hydrolyze aconitum alkaloids reference substance, calculate the amount of alcohol amine-diterpene alkaloids in the hydrolysis solution by the hydrolysis conversion rate, which is used as the amount of alcohol amine-diterpene alkaloids reference substance, and establish a content determination method for aconine, hypaconitine and aconine in Acotriti radix cocta. Methods： Through controlling the hydrol- ysis conditions of aconitine, hypaconitine and mesaconitine, aeonine, hypaconitine and aconine were obtained. The determination was performed on an Agilent ZORBAX Extend-C18 RRHT（2.1 mmx 50 ram, 1.8 μm） column with the mobile phase consisting of methanol （A）-water（B containing 0.1% formic acid and 2.5 mmol · L-1 ammonium acetate） with gradient elution by HPLC-QTOF-MS. The flow rate was 0.21 ml · min-1. The column temperature was 30 ℃. MS instrument was equipped with an ESI ＋ ion source. Results： Under the hydrolysis conditions of this study, the conversion rate of aconine from aconitine was 99.64% ; the conversion rate of hyp- aeonitine from hypaconine was 99.94% ; the conversion rate of mesaconitine from mesaconine was 99.57%. The HPLC-QTOF-MS methodological investigation showed the 3 kinds of alcohol amine-diterpene alkaloids were with good linearity （ r 〉 0. 999 1 ）. The RSD of the precision, repeatability and stability tests were less than 5%. The average recoveries were within the range of 99.43%- 10（3.10%. Conclusion： The validated method is simple, specific, reliable and reproducible. In the absence of reference substance, it can be used for the quality control of the herbs of Aconitum L. species.