基于HPLC波长切换联合梯度洗脱技术的越鞠片中9种主要成分定量研究

Quantitative study of nine main components in Yueju Tablets by HPLC coupled with wavelength switching and gradient elution method

目的建立HPLC波长切换联合梯度洗脱法(HPLC-DVD法)同时测定越鞠片中9种指标成分α-香附酮、京尼平龙胆二糖苷、栀子苷、西红花苷I、洋川芎内酯H、洋川芎内酯I、洋川芎内酯A、藁本内酯和苍术素的量。方法采用HPLC-DVD法,Zorbax Eclipse Plus C_(18)(250 mm×4.6 mm,5μm)色谱柱;甲醇-乙腈(2∶1,A)-0.2%冰醋酸溶液(B)为流动相,进行梯度洗脱,体积流量0.9 m L/min;α-香附酮、京尼平龙胆二糖苷和栀子苷的检测波长为240 nm,西红花苷I的检测波长为440 nm,洋川芎内酯H、洋川芎内酯I、洋川芎内酯A和藁本内酯的检测波长为280 nm,苍术素的检测波长为340 nm;进样量为10μL。结果 9种指标成分α-香附酮在2.58~51.60μg/m L(r=0.999 4)、京尼平龙胆二糖苷在11.99~239.80μg/m L(r=0.999 9)、栀子苷在17.96~359.20μg/m L(r=0.999 6)、西红花苷I在3.98~79.60μg/m L(r=0.999 7)、洋川芎内酯H在2.82~56.40μg/m L(r=0.999 9)、洋川芎内酯I在2.38~47.60μg/m L(r=0.999 9)、洋川芎内酯A在6.04~120.80μg/m L(r=0.999 5)、藁本内酯在7.98~159.60μg/m L(r=0.999 3)、苍术素在6.51~130.20μg/m L(r=0.999 2)质量浓度与峰面积具有较好的线性关系;精密度良好,RSD≤1.22%;重复性良好,RSD≤1.75%;供试品溶液在室温条件下_(18) h内稳定,RSD≤1.37%;平均加样回收率和相应的RSD分别为97.64%(0.98%)、99.09%(1.46%)、100.11%(1.03%)、97.87%(0.80%)、98.59%(1.19%)、96.89%(1.34%)、99.38%(0.58%)、98.50%(1.22%)、99.71%(0.85%)。10批次供试品中α-香附酮、京尼平龙胆二糖苷、栀子苷、西红花苷I、洋川芎内酯H、洋川芎内酯I、洋川芎内酯A、藁本内酯和苍术素量分别为0.282~0.344、2.099~2.445、3.628~4.225、0.758~0.913、0.241~0.286、0.217~0.266、1.077~1.291、1.386~1.623、1.137~1.434mg/片。结论建立的HPLC-DVD法同时测定越鞠片中的9种成分,方法操作简便、快速、准确,可为越鞠片质量控制提供科学依据。

Objective To establish HPLC coupled with wavelength switching and gradient elution method（HPLC-DVD） for simultaneous determination of nine main components（α-cyperone, genipin-1-β-D-gentiobioside, geniposide, crocin I, senkyunolide H, senkyunolide I, senkyunolide A, ligustilide, and atractylodin） in Yueju Tablets. Methods The chromatographic separation was achieved on Zorbax Eclipse Plus C_（18）（250 mm × 4.6 mm, 5 μm） column with methanol-acetonitrile（2∶1）（A）-0.2% glacial acetic acid solution（B） as mobile phases for gradient elution, at the flow rate of 0.9 m L/min; The detection wavelength was set at 240 nm for α-cyperone, genipin-1-β-D-gentiobioside and geniposide, 440 nm for crocin I, 280 nm for senkyunolide H, senkyunolide I, senkyunolide A and ligustilide, and 340 nm for atractylodin. The volume of sample injection was 10 μL. Results The nine active components were well separated and showed good linearity, such as α-cyperone 2.58—51.60 μg/m L（r = 0.999 4）, genipin-1-β-Dgentiobioside 11.99—239.80 μg/m L（r = 0.999 9）, geniposide 17.96—359.20 μg/m L（r = 0.999 6）, crocin-I 3.98—79.60 μg/m L（r = 0.999 7）, senkyunolide H 2.82—56.40 μg/m L（r = 0.999 9）, senkyunolide I 2.38—47.60 μg/m L（r = 0.999 9）, senkyunolide A 6.04—120.80 μg/m L（r = 0.999 5）, ligustilide 7.98—159.60 μg/m L（r = 0.999 3）, and atractylodin 6.51—130.20 μg/m L（r = 0.999 2）. The precision was good, and RSD was not more than 1.22%. The repeatability was good, and RSD was not more than 1.75%. The stability was good in _（18） h, and RSD was not more than 1.37%. The average recoveries and corresponding RSD values were 97.64%（0.98%）, 99.09%（1.46%）, 100.11%（1.03%）, 97.87%（0.80%）, 98.59%（1.19%）, 96.89%（1.34%）, 99.38%（0.58%）, 98.50%（1.22%）, and 99.71%（0.85%）, respectively. The contents of 10 batches of α-cyperone, genipin-1-β-D-gentiobioside, geniposide, crocin I, senkyunolide, H, senkyunolide I, senkyunolide A, ligustilide and atractylodin were 0.282—0.344, 2.099—2.445, 3.628—4.225, 0.758—0.913, 0.241—0.286, 0.217—0.266, 1.077—1.291, 1.386—1.623, and 1.137—1.434 mg/tablet. Conclusion HPLC coupled with wavelength switching and gradient elution method has been established for simultaneous determination of nine components in Yueju Tablets. The method is simple, quick, accurate, and it can be used for content determination and quality control of Yueju Tablets.