目的 建立对照品非依赖性的20种花青素类成分定量分析方法,并利用该法对市售欧洲越橘Vaccinium myrtillus提取物进行质量控制,同时筛选出代表性抗氧化活性成分。方法 以欧洲越橘标准对照提取物替代对照品,采用超高效液相色谱-三重四极杆串联质谱(UPLC-QQQ-MS/MS)联用技术,建立一种针对20种花青素类成分的定量方法,并利用该法对10批市售欧洲越橘提取物中的花青素类成分含量进行研究。采用DPPH自由基清除法和总抗氧化能力测试法评价不同欧洲越橘提取物的抗氧化能力。结果 方法学验证结果显示,20种花青素类成分线性良好,r为0.999 4~1.000 0,精密度RSD为0.05%~4.23%,稳定性RSD为0.29%~3.38%,平均加样回收率为98.1%~102.1%,RSD为0.50%~4.12%。样品分析结果显示,不同样品中部分花青素类成分含量存在显著差异,其中锦葵素变化最大,RSD为18.23%。欧洲越橘提取物具有较好的DPPH自由基清除能力和总抗氧化能力。其中矢车菊素与DPPH自由基率相关性为0.94,芍药素-3-O-半乳糖苷与总抗氧化活性相关性为0.93。二者有望作为欧洲越橘提取物清除DPPH自由基活性和总抗氧化活性的潜在质控成分。结论 基于UPLC-QQQ-MS/MS所建立的对照品非依赖性的欧洲越橘提取物中20种花青素类成分定量分析方法,灵敏度高,准确性好,方法简单,适用于欧洲越橘提取物中20种花青素类成分的含量测定,为中药对照提取物替代单体对照品在中药质量控制中的应用提供了数据支撑,为中药材质量控制研究提供新的研究思路。

Objective To establish a method for the quantitative analysis of 20 anthocyanin components based on the reference extract of Ouzhouyueju (Vaccinium myrtillus) and to utilize the method for the quality control of commercialized V. myrtillus macrocarpon extracts, and screen out a representative antioxidant active ingredient. Methods An ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QQQ-MS/MS) method was developed for the quantification of 20 anthocyanin components using reference extract of V. myrtillus macrocarpon as the standard, and the method was utilized to investigate 10 batches of commercially available V. myrtillus macrocarpon extracts. The antioxidant capacity of different V. myrtillus extracts was evaluated using the DPPH free radical scavenging method and the total antioxidant test. Results A UPLC-QQQ-MS/MS method was developed for the quantitative analysis of 20 anthocyanin compounds in the extract of V. myrtillus with reference extract. Methodological validation demonstrated good linearity (r:0.999 4-1.000 0), precision RSD (0.05%-4.23%), stability (0.29%-3.38%), and average recovery rate (98.1%-102.1%) of the 20 anthocyanin components. The results of the sample analysis showed that there were significant differences in the content of some components in different samples, with the greatest variation being malvidin with an RSD of 18.23%. V. myrtillus extract has better DPPH radical scavenging and total antioxidant capacity. Cyanidin and peonidin-3-O-galactoside could be potential quality control components for assessing the DPPH radical scavenging and total antioxidant activity of V. myrtillus extracts, respectively. Conclusion A quantitative analysis method of 20 anthocyanin components in V. myrtillus extract was established in this study based on UPLC-QQQ-MS/MS. The method is sensitive, accurate, simple, and suitable for the determination of the content of 20 anthocyanin components in the V. myrtillus extract, which provides data support for the application of the reference extract of traditional Chinese medicine to replace the single standard in the quality control of traditional Chinese medicine. The method provides a new research idea for the quality control study of traditional Chinese medicine.

中央级公益性科研院所基本业务费专项资金资助（ZZ13-YQ-45）；中国中医科学院科技创新工程项目（CI2021A04902）；中央级公益性科研院所基本业务费专项资金资助（ZXKT18015）