Lu Chen-Na, Ye Xiao, Liu Xiao-Qian, Feng Wei-Hong, Liang Yao-Hua, Li Chun, Wang Zhi-Min
National Engineering Laboratory of Quality Control Technology of Chinese Materia Medica, Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2022 Aug;47(15):4007-4014. doi: 10.19540/j.cnki.cjcmm.20220311.301.
A comprehensive quality control method was established to provide references for quality control and evaluation of substance benchmarks of Danggui Sini Decoction(DSD). The HPLC separation was performed on a Kromasil 100 C-8 column(4.6 mm×250 mm, 5 μm) with acetonitrile(A)-0.05% phosphoric acid in water(B) as mobile phase in a gradient elution mode at the flow rate of 1 mL·min(-1). The column temperature was 25 ℃ and the detection wavelength was set at 275 nm. Under these conditions, the content of seven components, including paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhetate, ligustilide, and asarinin was simultaneously determined. Under the same chromatographic conditions, the HPLC fingerprint method for analysis of 15 batches of DSD was established. The content determination of aristolochic acid I, using the same test solution as the content determination item, was performed on an ACQUITY UPLC BEH C_(18) column(2.1 mm×50 mm, 1.7 μm) with methanol(A)-water(including 0.1% formic acid and 5 mmol·L(-1) ammonium formate)(B) as the mobile phase in a gradient elution mode at the flow rate of 0.4 mL·min(-1) and the column temperature of 40 ℃ by LC-MS/MS. The MS conditions included electrospray ionization(ESI) as an ion source, positive ion ionization, selective reaction monitoring(SRM), the parent ion of 359.3, and the daughter ion of 297.8. The results of the methodological investigation all met the requirements of content determination/fingerprint determination. As a result, the content ranges of paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhetate, ligustilide, and asarinin were 5.419 8-11.267 3, 1.023-3.669 8, 0.145 6-0.444 1, 0.099 1-0.321 9, 3.159 1-7.731 9, 0.146 4-0.471 7, and 0.237 3-0.401 0 mg·g(-1), respectively. Twenty-two common peaks were selected and 10 of them were identified by the comparison with the reference substances. The fingerprint similarity of 15 batches of DSD was in the range of 0.91-0.996 and the content of aristolochic acid I in DSD was 300.03-638.13 ng·g~(-1). The method established in this study is reliable and easy to operate and has great practical value, which can be used for overall quality control of substance benchmarks for DSD.
建立了一种全面的质量控制方法，为当归四逆汤物质基准的质量控制和评价提供参考。采用Kromasil 100 C-8色谱柱（4.6 mm×250 mm，5 μm）进行高效液相色谱分离，以乙腈（A）-0.05%磷酸水溶液（B）为流动相，梯度洗脱，流速为1 mL·min⁻¹。柱温为25℃，检测波长设定为275 nm。在此条件下，同时测定了芍药苷、甘草苷、肉桂酸、肉桂醛、甘草酸铵、藁本内酯和细辛脂素7种成分的含量。在相同色谱条件下，建立了15批当归四逆汤的高效液相色谱指纹图谱分析方法。采用与含量测定项相同的供试品溶液，在ACQUITY UPLC BEH C₁₈色谱柱（2.1 mm×50 mm，1.7 μm）上，以甲醇（A）-水（含0.1%甲酸和5 mmol·L⁻¹甲酸铵）（B）为流动相，梯度洗脱，流速为0.4 mL·min⁻¹，柱温为40℃，采用液相色谱-串联质谱法测定马兜铃酸I的含量。质谱条件包括以电喷雾电离（ESI）为离子源，正离子模式电离，选择反应监测（SRM），母离子为359.3，子离子为297.8。方法学考察结果均符合含量测定/指纹图谱测定要求。结果显示，芍药苷、甘草苷、肉桂酸、肉桂醛、甘草酸铵、藁本内酯和细辛脂素的含量范围分别为5.419 8 - 11.267 3、1.023 - 3.669 8、0.145 6 - 0.444 1、0.099 1 - 0.321 9、3.159 1 - 7.731 9、0.146 4 - 0.471 7和0.237 3 - 0.401 0 mg·g⁻¹。共选择了22个共有峰，其中10个通过与对照品比较进行了鉴定。15批当归四逆汤的指纹图谱相似度在0.91 - 0.996之间，当归四逆汤中马兜铃酸I的含量为300.03 - 638.13 ng·g⁻¹。本研究建立的方法可靠、操作简便，具有较大的实用价值，可用于当归四逆汤物质基准的全面质量控制。
