活血止痛胶囊/片中5种皂苷类成分含量测定及确证非法添加方法的建立

Establishment of content determination of 5 saponins in Huoxue zhitong capsules/tablets and confirmation of illegal addition

目的建立同时测定活血止痛胶囊/片中5种指标性皂苷类成分的含量和确证人参、西洋参、三七茎叶非法添加的方法。方法采用超高效液相色谱-串联质谱法。以Agilent RRHD Eclipse Plus C18为色谱柱,以水(含0.1%甲酸)-乙腈为流动相进行梯度洗脱,流速为0.35 mL/min,柱温为35℃,进样量为2μL;采用电喷雾离子源,以多反应监测模式进行负离子扫描。结果三七皂苷R1、人参皂苷Rb1、人参皂苷Rg1、人参皂苷Rd、人参皂苷Re、人参皂苷R(f人参特有成分)、人参皂苷Rb(3三七茎叶特有成分)、拟人参皂苷F1(1西洋参特有成分)检测质量浓度的线性范围分别为9.99~1499.50、9.99~1499.50、10.01~1500.80、9.99~1499.10、10.00~1500.20、9.99~1499.50、10.01~1500.80、9.99~1499.00 ng/mL(R2>0.997);检测限、定量限分别不高于2.64、8.06 ng/mL;精密度、重复性、稳定性(24 h)试验的RSD均小于6%;胶囊和片剂中各皂苷类成分的平均加样回收率分别为98.72%~102.40%、95.18%~106.47%(RSD均小于5%,n=6)。18批活血止痛胶囊中,三七指标性成分人参皂苷Re、人参皂苷Rd、人参皂苷Rg1、三七皂苷R1、人参皂苷Rb1的含量分别为291.79~426.89、427.71~677.49、2294.28~3371.43、571.22~848.19、1841.33~2959.12μg/g,人参皂苷Rb3的含量均不高于45.02μg/g;22批活血止痛片中,三七指标性成分的含量分别为44.11~393.83、80.48~549.55、393.36~3548.57、79.83~872.60、288.64~2912.66μg/g,人参皂苷Rb3的含量不高于44.79μg/g;2种制剂均未检出人参皂苷Rf、拟人参皂苷F11。结论该方法可用于测定活血止痛制剂中主要指标性皂苷类成分的含量。40批制剂均未见人参、西洋参非法添加现象,但部分批次片剂样品的三七药材投料量较少。

OBJECTIVE To establish a method for simultaneous determination of 5 saponins in Huoxue zhitong capsules/tablets and to confirm the illegal addition of Panax ginseng,Panax quiquefolium and stems and leaves of Panax notoginseng.METHODS Ultra-high performance liquid chromatography tandem mass spectrometry was used.The Agilent RRHD Eclipse Plus C18 column was used with mobile phase of water(containing 0.1%formic acid)-acetonitrile for gradient elution at a flow rate of 0.35 mL/min.The column temperature was 35℃and the sample size was 2μL.Using electrospray ionization source,negative ion scanning was carried out in multi-reaction monitoring mode.RESULTS The linear ranges of notoginsenoside R1,ginsenoside Rb1,ginsenoside Rg1,ginsenoside Rd,ginsenoside Re,ginsenoside Rf(an unique ingredient of P.ginseng),ginsenoside Rb(3 an unique ingredient of stems and leaves of P.notoginseng)and pseudo-ginsenoside F11(an unique ingredient of P.quiquefolium)were 9.99-1499.50,9.99-1499.50,10.01-1500.80,9.99-1499.10,10.00-1500.20,9.99-1499.50,10.01-1500.80,9.99-1499.00 ng/mL(R2>0.997);the detection limits and the quantitative limits were not higher than 2.64 and 8.06 ng/mL,respectively.RSDs of precision,repeatability and stability(24 h)tests were all less than 6%.The average recoveries of saponins in capsules and tablets were 98.72%-102.40%and 95.18%-106.47%,respectively(all RSDs<5%,n=6).In 18 batches of Huoxue zhitong capsules,the contents of ginsenoside Re,ginsenoside Rd,ginsenoside Rg1,notoginsenoside R1 and ginsenoside Rb1 were 291.79-426.89,427.71-677.49,2294.28-3371.43,571.22-848.19 and 1841.33-2959.12μg/g,respectively;the contents of ginsenoside Rb3 were no more than 45.02μg/g.In 22 batches of Huoxue zhitong tablets,the contents of above indicators of P.notoginseng were 44.11-393.83,80.48-549.55,393.36-3548.57,79.83-872.60,and 288.64-2912.66μg/g,respectively;the contents of ginsenoside Rb3 were no more than 44.79μg/g.Ginsenoside Rf and pseudo-ginsenoside F11 were not detected in the two preparations.CONCLUSIONS The method can be used to determine the contents of saponins in Huoxue zhitong preparations.No illegal addition of P.ginseng and P.quiquefolium are found in 40 batches of preparations,but the input of P.notoginseng in some batches of tablet samples is less.