HPLC法同时测定蟾酥中11个蟾毒配基成分的含量

Simultaneous determination of 11 bufogenins in Bufonis Venenum by HPLC

目的:建立HPLC法同时测定蟾酥中11个蟾毒配基成分(伪异沙蟾毒精、日蟾毒它灵、沙蟾毒精、蟾蜍它里定、远华蟾蜍精、蟾毒它灵、脂蟾毒精、南美蟾毒精、蟾毒灵、脂蟾毒配基和华蟾酥毒基)的含量。方法:采用Agilent InfinityLab Poroshell 120 EC-C18色谱柱(150 mm×3.0 mm 2.7μm),以0.3%乙酸水溶液-乙腈为流动相,梯度洗脱,流速为0.5 mL·min-1,柱温为30℃,检测波长为296 nm。结果:11个蟾毒配基成分的线性范围分别为2.024~20.24μg·mL^(-1)(r=0.9998)、18.44~184.4μg·mL^(-1)(r=0.9999)、12.40~124.0μg·mL^(-1)(r=0.9995)、3.720~37.20μg·mL^(-1)(r=0.9995)、10.28~102.8μg·mL^(-1)(r=0.9998)、22.16~221.6μg·mL^(-1)(r=0.9997)、3.280~32.80μg·mL^(-1)(r=0.9994)、5.440~54.40μg·mL^(-1)(r=0.9997)、28.84~288.4μg·mL^(-1)(r=0.9998)、32.76~327.6μg·mL^(-1)(r=0.9997)、71.28~712.8μg·mL^(-1)(r=0.9996),方法的精密度(RSD≤1.3%)、重复性(RSD≤4.4%)、稳定性(RSD≤3.3%)良好,平均加样回收率为95.6%~102.4%,RSD为0.98%~4.4%。18批次蟾酥样品中伪异沙蟾毒精、日蟾毒它灵、沙蟾毒精、蟾蜍它里定、远华蟾蜍精、蟾毒它灵、脂蟾毒精、南美蟾毒精、蟾毒灵、脂蟾毒配基和华蟾酥毒基的含量范围分别为0.2850~1.018、11.89~14.12、9.246~10.98、2.260~2.739、6.813~8.093、15.33~18.20、0.5280~1.178、1.492~2.403、17.79~26.17、18.75~29.94、43.12~60.81 mg·g^(-1)。聚类分析结果表明,18批次蟾酥样品可分为2类。结论:该方法简便易行,重复性好,灵敏度高,可用于蟾酥的质量控制和品质评价。

Objective:To establish an HPLC method for the simultaneous determination of eleven bufogenins,including pseudobufarenogin,gamabufotalin,arenobufagin,hellebrigenin,telocinobufagin,bufotaline,resibufagin,marinobufagenin,bufalin,resibufogenin and cinobufagin in Bufonis Venenum.Methods:The Agilent InfinityLab Poroshell 120 EC-C_(18)column(150 mm×3.0 mm,2.7μm)was used,and the mobile phase was water solution containing 0.3%acetic acid and acetonitrile with gradient elution,at the flow rate of 0.5 mL·min~(-1).The column temperature was at 30℃and the detection wavelength was set at 296 nm.Results:The linear ranges of eleven bufogenins were 2.024-20.24μg·mL^(-1)(r=0.9998),18.44-184.4μg·mL^(-1)(r=0.9999),12.40-124.0μg·mL^(-1)(r=0.9995),3.720-37.20μg·mL^(-1)(r=0.9995),10.28-102.8μg·mL^(-1)(r=0.9998),22.16-221.6μg·mL^(-1)(r=0.9997),3.280-32.80μg·mL^(-1)(r=0.9994),5.440-54.40μg·mL^(-1)(r=0.9997),28.84-288.4μg·mL^(-1)(r=0.9998),32.76-327.6μg·mL^(-1)(r=0.9997),71.28-712.8μg·mL^(-1)(r=0.9996),respectively.Results of precision(RSD≤1.3%),repeatability(RSD≤4.4%)and stability(RSD≤3.3%)were good.The average recoveries were 95.6%-102.4%with RSDs of 0.98%-4.4%.The contents of pseudobufarenogin,gamabufotalin,arenobufagin,hellebrigenin,telocinobufagin,bufotaline,resibufagin,marinobufagenin,bufalin,resibufogenin and cinobufagin in 18 batches of samples were 0.2850-1.018,11.89-14.12,9.246-10.98,2.260-2.739,6.813-8.093,15.33-18.20,0.5820-1.178,1.492-2.403,17.79-26.17,18.75-29.94 and 43.12-60.81 mg·g^(-1),respectively.The results of cluster analysis showed that 18 batches of samples could be divided into two categories.Conclusion:This method has the advantages of simple operation,good repeatability and high sensitivity,which can be used for quality control and quality evaluation of Bufonis Venenum.