HPLC法同时测定钩吻茎中胡蔓藤碱丙等6个生物碱的含量

Simultaneous determination of 6 major alkaloids including humantenidine in the stem of Gelsemium elegans Benth.by HPLC

目的:建立HPLC法同时测定钩吻茎中胡蔓藤碱丙、钩吻素甲、钩吻素子、钩吻素己、钩吻绿碱和胡蔓藤碱乙的含量,并比较评价各产地钩吻生物碱含量的差异。方法:采用外标法进行测定,色谱柱为ZORBAX Bonus-RP Analytical(4.6 mm×250 mm,5μm),流动相为甲醇-0.1%甲酸水梯度洗脱,流速1.0 m L·min^(-1),检测波长254 nm,柱温30℃。分别测定32批钩吻茎,并进行聚类分析。结果:胡蔓藤碱丙、钩吻素甲、钩吻素子、钩吻素己、钩吻绿碱、胡蔓藤碱乙6个成分的线性范围分别为1.00~50.06μg·mL^(-1)(r=1.000 0)、1.13~50.85μg·mL^(-1)(r=0.999 8)、1.16~52.20μg·mL^(-1)(r=1.000 0)、1.16~52.20μg·mL^(-1)(r=0.999 7)、1.06~50.88μg·mL^(-1)(r=0.999 7)和1.01~50.50μg·mL^(-1)(r=1.000 0),平均加样回收率为93.1%~103.8%(RSD为2.0%~2.9%)。不同产地样品中钩吻茎中胡蔓藤碱丙、钩吻素甲、钩吻素子、钩吻素己、钩吻绿碱和胡蔓藤碱乙含量范围分别为0.136~2.202、0.564~2.686、0.718~3.553、0.160~2.025、0.151~3.493、0.288~4.700 mg·g^(-1)。结果显示不同产地的钩吻6个成分含量均有差异,32批不同产地钩吻药材可聚为6大类。结论:该法可为钩吻多指标成分的质量评价提供参考。不同产地钩吻的各个化学成分的含量呈现明显分类特点,为钩吻地区鉴定提供依据。

Objective： To establish an HPLC method for the simultaneous determination of 6 major alkaloids including humantenidine, gelsemine, koumine, gelsenicine, gelsevirine and humantenine in the stem of Gelsemium elegans Benth., and to compare the difference of the contents of these alkaloids in samples from different habitats. Methods： The external standard method was performed on a ZORBAX Bonus-RP Analytical column （ 4.6 mm × 250 mm, 5 μm ） with a gradient elution using methanol and 0.1% formic acid. The column temperature was 30 ℃. The flow rate was 1.0 mL· min^-1 and the detection wavelength was set at 254 nm. 32 batches of samples were determined and the results were analyzed by cluster analysis. Results： The linear ranges of humantenidine, gelsemine, koumine, gelsenicine, gelsevirine and humantenine were 1.00-50.06μg·m L^-1 （ r=1.000 0 ）, 1.13-50.85 μg·mL^-1 （ r=0.999 8 ）, 1.16-52.20μg·mL^-1 （ r=1.000 0 ）, 1.16-52.20 μg·mL^-1 （ r=0.999 7 ）, 1.06-50.88μg·mL^-1（ r=0.999 7 ）, 1.01-50.50 μg·mL^-1（ r=1.000 0 ）,respectively. The average recoveries were 93.1%-103.8% with the RSD ranging from 2.0% to 2.9%. The content ranges of humantenidine, gelsemine, koumine, gelsenicine, gelsevirine and humantenine in samples from different habitats were 0.136-2.202 , 0.564-2.686, 0.718-3.553,0.160-2.025, 0.151-3.493 and 0.288-4.700 mg· g^-1, respectively. 6 alkaloids in samples varied in habitats and 32 batches of samples could be clustered into 6 cassifications. Conclusion： The proposed method offered reference for multicomponent determination of G. elegans. Obvious classification was found in contents of 6 compounds in G. elegans from different habitats, which provided basis for habitat identification of the drug.