UPLC法测定乌拉尔甘草与光果甘草中7个黄酮类成分的含量

目的：建立同时测定乌拉尔甘草与光果甘草药材中甘草素、异甘草素、甘草苷、异甘草苷、芹糖甘草苷、芹糖异甘草苷、甘草查尔酮A含量的高灵敏度、高效率分析方法。方法：以市售乌拉尔甘草、光果甘草为实验材料，采用UPLC法对样品中7个黄酮类成分进行测定。使用ACQUITY UPLC BEH C₁₈色谱柱（2.1 mm&#215;100 mm，1.7 μm），以乙腈（A）-0.05%磷酸水溶液（B）为流动相，梯度洗脱，流速为0.3 mL&#183;min^-1，检测波长分别为276 nm（检测芹糖甘草苷、甘草苷、甘草素）、360 nm（检测异甘草苷、芹糖异甘草苷）、370 nm（检测异甘草素）、380 nm（检测甘草查尔酮A），柱温40℃。结果：甘草素、异甘草素、甘草苷、异甘草苷、芹糖甘草苷、芹糖异甘草苷、甘草查尔酮A分离度良好，回归方程分别为Y=1.244 4&#215;10⁷X+2.511 4&#215;10²（r=0.999 9）、Y=2.676 2&#215;10⁷X+1.115 6&#215;10²（r=0.999 1）、Y=7.014 4&#215;10⁶X+8.672 4&#215;10³（r=0.996 7）、Y=1.532 8&#215;10⁷X+8.844 9&#215;10²（r=0.998 8）、Y=5.435 8&#215;10⁶X-2.554 9&#215;10³（r=0.998 7）、Y=1.227 8&#215;10⁷X-5.843 0&#215;10²（r=0.999 9）和Y=1.542 0&#215;10⁷X-2.888 4&#215;10³（r=0.996 4），线性范围分别为0.713~7.13 μg、0.078 2~0.782 μg、13.5~135 μg、2.08~20.8 μg、8.04~80.4 μg、3.15~31.4 μg、0.858~8.58 μg，检测下限和定量下限依次为1.43 ng和3.57 ng、0.019 4 ng和0.155 ng、0.172 ng和0.515 ng、0.078 3 ng和0.235 ng、0.211 ng和0.676 ng、0.120 ng和0.361 ng、0.182 ng和0.608 ng。本方法灵敏度、精密度、准确性、重复性、回收率、耐用性均良好。乌拉尔甘草中，除甘草查尔酮A含量为（0.171&#177;0.070）mg&#183;g^-1外，其他6个成分的含量[甘草素（0.399&#177;0.164）mg&#183;g^-1、异甘草素（0.131&#177;0.061）mg&#183;g^-1、甘草苷（7.116&#177;2.515）mg&#183;g^-1、异甘草苷（0.948&#177;0.366）mg&#183;g^-1、芹糖甘草苷（4.933&#177;1.873）mg&#183;g^-1、芹糖异甘草苷（1.193&#177;0.672）mg&#183;g^-1]均高于光果甘草样品中相应成分的含量[（0.276&#177;0.127）、（0.105&#177;0.041）、（4.342&#177;1.167）、（0.568&#177;0.262）、（4.706&#177;0.808）、（1.031&#177;0.437）]mg&#183;g^-1。甘草素与异甘草素、芹糖甘草苷与芹糖异甘草苷的含量在2种基原甘草样品中均有显著的相关关系（P<0.01）。结论：本文运用UPLC梯度洗脱方法建立了同时测定甘草药材中7个黄酮类成分含量的方法，为甘草药材质量标准的制定以及质量评价提供参考。

Objective:To establish an efficient and sensitive method for simultaneous determination of seven flavonoids including liquiritigenin, isoliquiritigenin, liquiritin, isoliquiritin, liquiritin apioside, isoliquiritin apioside and licochalcone A in Glycyrrhiza uralensis Fisch.and Glycyrrhiza glabra L.Methods:Licorice slices from two original plants were bought from markets and used as study materials.The contents of seven flavonoids were analyzed by UPLC method using chromatographic column ACQUITY UPLC BEH C₁₈ (2.1 mm&#215;100 mm, 1.7 μm) at a column temperature of 40℃.The mobile phase consisted of acetonitrile (A) and 0.05% phosphoric acid (B) with gradient elution at a flow rate of 0.3 mL&#183;min^-1.The UV detection wavelength was 276 nm for liquiritin apioside, liquiritin and liquiritigenin, 360 nm for isoliquiritin and isoliquiritin apioside, 370 nm for isoliquiritigenin and 380 nm for licochalcone A, respectively.Results:The calibration curves for liquiritigenin, isoliquiritigenin, liquiritin, isoliquiritin, liquiritin apioside, isoliquiritin apioside and licochalcone A were Y=1.244 4&#215;10⁷X+ 2.511 4&#215;10² (r=0.999 9), Y=2.676 2&#215;10⁷X+1.115 6&#215;10² (r=0.999 1), Y=7.014 4&#215;10⁶X+8.672 4&#215;10³ (r=0.996 7), Y=1.532 8&#215;10⁷X +8.844 9&#215;10² (r=0.998 8), Y=5.435 8&#215;10⁶X-2.554 9&#215;10³ (r=0.998 7), Y=1.227 8&#215;10⁷X-5.843 0&#215;10² (r=0.999 9), and Y=1.542 0&#215;10⁷X-2.888 4&#215;10³ (r=0.996 4) respectively, which reflected a good linearity in the range of 0.713-7.13 μg, 0.078 2-0.782 μg, 13.5-135 μg, 2.08-20.8 μg, 8.04-80.4 μg, 3.15-31.4 μg, and 0.858-8.58 μg.The LOD and LOQ were 1.43 ng and 3.57 ng, 0.019 4 ng and 0.155 ng, 0.172 ng and 0.515 ng, 0.078 3 ng and 0.235 ng, 0.211 ng and 0.676 ng, 0.120 ng and 0.361 ng, and 0.182 ng and 0.608 ng for liquiritigenin, isoliquiritigenin, liquiritin, isoliquiritin, liquiritin apioside, isoliquiritin apioside, and licochalcone A, respectively.The sensitivity, accuracy, precision, repeatability, durability and average recoveries all met the requirements.Except licochalcone A (0.171&#177;0.070 mg&#183;g^-1), the contents of liquiritigenin[(0.399&#177;0.164) mg&#183;g^-1], isoliquiritigenin[(0.131&#177;0.061) mg&#183;g^-1], liquiritin[(7.116&#177;2.515) mg&#183;g^-1], isoliquiritin[(0.948&#177;0.366) mg&#183;g^-1], liquiritin apioside[(4.933&#177;1.873) mg&#183;g^-1]and isoliquiritin apioside[(1.193&#177;0.672) mg&#183;g^-1]in Glycyrrhiza uralensis Fisch.were all higher than those in Glycyrrhiza glabra L.[(0.276&#177;0.127) mg&#183;g^-1, (0.105&#177;0.041) mg&#183;g^-1, (4.342&#177;1.167) mg&#183;g^-1, (0.568&#177;0.262) mg&#183;g^-1, (4.706&#177;0.808) mg&#183;g^-1 and (1.031&#177;0.437) mg&#183;g^-1].Furthermore, the content of liquiritin was significantly correlated with the content of isoliquiritin, and the content of liquiritin apioside was significantly correlated with the content of isoliquiritin apioside in all samples (P<0.01).Conclusion:UPLC with gradient elution is proved to be suitable for the simultaneous determination of seven flavonoids in licorice.It provides reference for development of the quality standard and evaluation of the quality of licorice.

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