反相高效液相色谱法测定千层纸素的含量和有关物质

Determination of related substances and content of oroxylin A by RP-HPLC

目的建立测定千层纸素的含量和有关物质的RP-HPLC方法。方法采用RP-HPLC法,色谱柱为ODS-2(4.6 mm×150 mm,5μm),甲醇-乙腈-10 mmol·L^(-1)磷酸二氢钾(用磷酸调节pH值至2.5)为流动相梯度洗脱,流速1.0 mL·min-1,柱温30℃,检测波长271 nm。结果在含量测定色谱条件下,千层纸素浓度在2.02~20.23μg·m L^(-1)内与峰面积呈良好线性关系(r=1.000,n=6);在有关物质测定色谱条件下,有关物质黄芩素、汉黄芩素、杂质Ⅰ分别在质量浓度0.0197~0.7872μg·m L^(-1)(r=1.000,n=6)、0.0313~0.6250μg·m L^(-1)(r=0.9997,n=6)、0.0818~2.0440μg·m L^(-1)(r=0.9999,n=5)内与峰面积呈良好的线性关系;黄芩素、杂质Ⅰ和各杂质总含量分别为0.11%、0.39%、0.57%。结论本方法灵敏度高、重现性好、准确可靠,可用于千层纸素有关物质的检测分析及质量控制。

Objective To establish an RP-HPLC method to determine the related substances and content of oroxylin A. Methods RP-HPLC was adopted. ODS-2 C18 column(4.6 mm×150 mm, 5 μm) was used with a gradient elution system of acetonitrile-methanol-10 mmol·L-1 phosphate buffer. The column temperature was 30℃, the flow rate was 1.0 mL·min^-1, and the detection wavelength was 271 nm. Results A linearity between the area of oroxylin A and its concentration was found at 2.02-20.23 μg·mL^-1(r = 1, n = 6). For the determination of impurities, the standard curves of baicalein, wogonin and impurity Ⅰ were linear at 0.0197-0.7872 μg·mL^-1(r = 1.0000, n = 6), 0.0313-0.6250 μg·mL^-1(r = 0.9997, n = 6), and 0.0818-2.0440 μg·mL^-1(r = 0.9999, n = 5), respectively. The contents of baicalein, impurity Ⅰ and the total content of the impurities were 0.11%, 0.39%, and 0.57%, respectively. Conclusion The method is sensitive, reproducible, accurate and reliable in the determination of related substances and quality control of oroxylin A in the raw material.