金生源胶囊中5种化学成分HPLC及HPLC-MS/MS测定方法研究

Study on HPLC and HPLC-MS/MS Determination Methods of Five Chemical Components in Jinshengyuan Capsules

关键词:  金生源胶囊  高效液相色谱法  高效液相色谱-串联质谱法  甘草苷  甘草酸  丹酚酸B  华蟾酥毒基  酯蟾毒配基

Key words:  Jinshengyuan capsules  HPLC  HPLC-MS/MS  liquiritin  glycyrrhizic acid  salvianolic acid B  cinobufagin  resibufogenin

摘要:

目的 建立HPLC和HPLC-MS/MS测定金生源胶囊中甘草苷、甘草酸、丹酚酸B、华蟾酥毒基和酯蟾毒配基5种成分的方法。方法 采用HPLC测定甘草苷、甘草酸和丹酚酸B，采用岛津C₁₈ ODS色谱柱（250 mm×4.6 mm，5 μm），流动相乙腈（A）-0.05%磷酸溶液（B），梯度洗脱，二极管阵列检测器检测，检测波长分别为276，250，286 nm，流速1.0 mL·min^-1。华蟾酥毒基和酯蟾毒配基测定，采用HPLC-MS/MS，ESI源为离子源，正离子多反应监测模式，ZORBAXSB-C₁₈色谱柱（250 mm×4.6 mm，5 μm），流动相为乙腈-0.1%甲酸（41：59），流速1.0 mL·min^-1，柱温30℃。结果 甘草苷、甘草酸、丹酚酸B、华蟾酥毒基和酯蟾毒配基分别在3.14~62.8，4.18~83.6，7.00~140.0，0.495~9.90，0.520~10.4 μg·mL^-1内线性关系良好，平均加样回收率分别为99.13%，98.96%，97.96%，99.51%，96.22%，RSD分别为1.43%，2.76%，1.68%，2.54%，1.39%。结论 该方法分离度好、准确可靠、灵敏度高，可为复方中药中干蟾皮微量成分检测及质量控制提供实验依据。

Abstract:

OBJECTIVE To establish a method for the determination of five constituents(liquiritin, glycyrrhizic acid, salvianolic acid B, cinobufagin and resibufogenin) in Jinshengyuan capsules by HPLC and HPLC-MS/MS. METHODS HPLC was used for imultaneous determination of liquiritin, glycyrrhizic acid and salvianolic acid B. The analysis was performed on SHIMADZU C₁₈ ODS(250 mm×4.6 mm, 5 μm), with the mobile phase comprising of acetonitrile(A)-0.05% phosphoric acid(B) flowing at 1.0 mL·min^-1 in a gradient elution manner, and the detection wavelength was set at 276, 250 and 286 nm by diode array detector. Cinobufagin and resibufogenin was analyzed by HPLC-MS/MS method, ESI source as ion source, positive ion multiple reaction monitoring mode, which performed on 30 ℃ thermostatic ZORBAXSB-C₁₈(250 mm×4.6 mm, 5 μm), with the mobile phase comprising of acetonitrile(A)-0.1% formic acid(B) flowing at 1.0 mL·min^-1. RESULTS The liner ranges of liquiritin, glycyrrhizic acid, salvianolic acid B, cinobufagin and resibufogenin were within 3.14-62.8, 4.18-83.6, 7.00-140.0, 0.495-9.90, 0.520-10.4 μg·mL^-1. The average recovery rates were 99.13%, 98.96%, 97.96%, 99.51% and 96.22% with the RSD of 1.43%, 2.76%, 1.68%, 2.54% and 1.39%. CONCLUSION The method has good separation, accuracy and reliability, high specificity and sensitivity, which can provide experimental basis for the detection and quality control of the trace components of dried toad skin in compound traditional Chinese medicine.