目的: 建立小儿感冒颗粒的超高效液相色谱(UPLC)指纹图谱、多指标成分含量测定和化学计量学相结合的质量评价方法。方法: 采用Waters Acquity UPLC BEH C18色谱柱 (100 mm×2.1 mm,1.7 μm),以乙腈-0.2%磷酸水溶液为流动相,梯度洗脱,流速为0.3 mL·min-1,柱温为30 ℃,多波长切换检测。使用中药色谱指纹图谱相似度评价软件(2012A版)对8家生产企业共30批次小儿感冒颗粒建立指纹图谱,确定共有峰并对其进行相似度评价。测定其中绿原酸、对羟基苯乙酮、木犀草苷、连翘酯苷A、毛蕊花糖苷、4,5-O-二咖啡酰奎宁酸、连翘苷的含量并进行方法学验证。基于指纹图谱共有峰峰面积的测定结果,采用聚类分析、主成分分析、多元统计等方法对不同生产企业的小儿感冒颗粒进行质量评价。结果: 建立了小儿感冒颗粒的UPLC指纹图谱,30批样品的相似度均>0.9。确定了17个共有峰,通过与对照品比较指认了其中的8个色谱峰,并对其中7个进行了含量测定。7个成分在各自质量浓度范围内线性关系良好(r≥0.999 8),精密度(RSD≤1.1%),重复性(RSD≤1.5%)均良好,供试品在24 h内稳定,平均加样回收率为97.9%~102.6%,RSD为0.71%~1.4%。30批次样品中绿原酸、对羟基苯乙酮、木犀草苷、连翘酯苷A、毛蕊花糖苷、4,5-O-二咖啡酰奎宁酸、连翘苷的含量分别为77.22~160.75、11.04~163.47、26.73~214.65、128.30~1291.02、17.52~120.02、26.51~124.71、57.76~401.96 μg·g-1。当欧式距离为5时,8家生产企业可以聚为三类;主成分1~4是影响小儿感冒颗粒生产企业间差异性的主要因子。多元统计分析表明同一生产企业生产的样品,有的生产工艺和原药材的质量控制较为稳定,批间差异性较小;而有的批间差异性较大。其中峰9、1、3、15(连翘苷)、6和11(毛蕊花糖苷)这6个色谱峰所代表的化学成分是引起样品差异性的主要因素。结论: UPLC指纹图谱结合多指标成分定量及化学计量学的小儿感冒颗粒质量评价方法快速、高效、准确,特征性强,重复性和稳定性较好,可用于小儿感冒颗粒的质量评价。
Objective: To establish a quality evaluation method of Xiao'er Ganmao granules based on UPLC fingerprint, multi-component determination, and chemometric analysis. Methods: The Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) was adopted, the mobile phase was acetonitrile-0.2% phosphoric acid aqueous solution with gradient elution. The flow rate was 0.3 mL·min-1, the column temperature was 30 ℃, and multi wavelength switching detection was used. The TCM chromatographic fingerprint similarity evaluation software (2012A version) was used to establish fingerprints of 30 batches of Xiao'er Ganmao granules from 8 manufacturers, the common peaks were identified and the similarity were calculated. The content determination methods for chlorogenic acid, 4'-hydroxyacetophenone, luteolin-7-O-glucoside, forsythoside A, verbascoside, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and forsythin were validated. Based on the determination results of the common peak area of the fingerprint, the quality of Xiao'er Ganmao granules from different manufacturers were evaluated by cluster analysis, principal component analysis and multivariate statistics. Results: The UPLC fingerprint of Xiao'er Ganmao granules was established, and the similarities of 30 batches of samples were all above 0.9. Seventeen common peaks were identified, eight of which were identified by comparison with the reference substance, and the contents of seven common peaks were determined. The seven components had good linearity (r≥0.999 8), precision (RSD≤1.1%) and repeatability (RSD≤1.5%) within their respective concentration ranges. The samples were stable within 24 h, and the average recoveries were 97.9%-102.6%, with the RSDs of 0.71%-1.4%. The contents of chlorogenic acid, 4'-hydroxyacetophenone, luteolin-7-O-glucoside, forsythoside A, verbascoside, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid and forsythin in 30 batches of samples were 77.22-160.75, 11.04-163.47, 26.73-214.65, 128.30-1291.02, 17.52-120.02, 26.51-124.71 and 57.76-401.96 μg·g-1 respectively。The eight manufacturers could be grouped into three categories when the European distance was set at 5. The principal components 1-4 were the main factors affecting the difference between the manufacturers. Multivariate statistical analysis showed that some samples produced by the same manufacturer could be grouped into one group, owing to their stable quality control of processes and original medicinal materials. However, some samples produced by the same manufacturers had differences among batches, and the chemical components of peak 9, peak 1, peak 3, peak 15 (forsythin), peak 6 and peak 11 (verbascoside) were the main factors causing the differences among samples. Conclusion: The method based on UPLC fingerprint, multi-components determination, and chemometrics analysis of Xiao'er Ganmao granules is fast, efficient, accurate, characteristic, repeatable and stable. It can be used for the quality evaluation of Xiao'er Ganmao granules.
[1] 中华人民共和国药典2020年版. 一部[S]. 2020: 589
ChP 2020. Vol Ⅰ[S]. 2020: 589
[2] 朱海燕,岑利峰,史训龙,等.小儿感冒颗粒对H1N1流感病毒诱导小鼠急性肺损伤的保护作用[J]. 中成药,2020, 42(3):758
ZHU HY, CENG LF, SHI XL, et al. Protective effect of Xiao'er Ganmao Granules on acute lung injury induced by H1N1 influenza virus in mice [J]. Chin Tradit Pat Med, 2020, 42(3):758
[3] 雷静. 小儿感冒颗粒的镇咳祛痰作用[J]. 世界最新医学信息文摘,2015, 15(37):92
LEI J. The antitussive and expectorant effects of Xiao'er Ganmao Keli. [J] World Lat Med Inf (Elec Ver), 2015, 15(37):92
[4] 李一民,赵长江. 小儿感冒颗粒联合中药沐浴治疗小儿急性上呼吸道感染风热型100例疗效观察[J]. 中医儿科杂志,2015, 11(4):22
LI YM, ZHAO CJ. Xiao'er Ganmao Keli combined with TCM bath in the treatment of 100 cases of children with acute upper respiratory tract infection of wind heat type [J]. J Ped TCM, 2015, 11(4):22
[5] 朱月梅,董海芳,李静. 中药治疗小儿急性上呼吸道腺病毒感染的临床效果研究[J]. 中华医院感染学杂志,2014, 24(18):4466
ZHU YM, DONG HF, LI J. Clinical effect of Chinese medicine on treatment of children with acute upper respiratory tract infection caused by adenovirus [J]. Chin J Nosocomiol, 2014, 24(18):4466
[6] 吴平平,李桂龙,靳文仙,等. 小儿感冒颗粒中绿原酸测定方法的改进[J]. 中草药,2011, 42(6):1135
WU PP, LI GL, JIN WX, et al. Improvement in determination of chlorogenic acid in Xiao'er Ganmao Granula [J]. Chin Tradit Herb Drug, 2011, 42(6):1135
[7] 张锦,毛庆,郑小平,等. RP-HPLC法测定小儿感冒颗粒中连翘苷的含量[J]. 药物分析杂志,2007, 27(7):1065
ZHANG J, MAO Q, ZHENG XP, et al. RP-HPLC determination of forsythin in Xiao'er Ganmao granules [J]. Chin J Pharm Anal, 2007, 27(7):1065
[8] 田雅琴. HPLC测定小儿感冒颗粒中绿原酸及连翘苷含量[J]. 中成药,2009, 31(12):1965
TIAN YQ. Determination of chlorogenic acid and forsythiaside in Xiao'er Ganmao Granules by HPLC [J]. Chin Tradit Pat Med, 2009, 31(12):1965
[9] 任菲菲,张静,刘德丽,等. HPLC法测定小儿感冒颗粒中绿原酸、木犀草苷、3,5-O-二咖啡酰奎宁酸、连翘苷的含量[J]. 中国药品标准,2019, 20(4):325
REN FF, ZHANG J, LIU DL, et al. Simultaneous determination of chlorogenic acid, luteolin, 3,5-O-dicaffeoyl quinic acid and forsythin in Xiao′er Ganmao granules by HPLC [J]. Drug Stand Chin, 2019, 20(4):325
[10] 陈国宝,郑艳萍,严国俊,等. HPLC法同时测定小儿感冒颗粒中4种成分的含量[J].中国药房,2016, 27(27):3836
CHEN GB, ZHENG YP, YAN GJ, et al. Simultaneous determination of 4 components in Xiaoer Ganmao granule by HPLC [J]. Chin Pharm, 2016, 27(27):3836
[11] 任菲菲,郑艳青,马斐,等.小儿感冒颗粒HPLC指纹图谱研究[J]. 药学研究,2020, 39(10):575
REN FF, ZHENG YQ, MA F, et al. HPLC fingerprint of Xiaoerganmao granules [J]. J Pharm Res, 2020, 39(10):575
[12] 赵桉熠,倪凤燕,陈彭月,等.以一致性为核心的中成药质量研究现状及优质评价新模式探讨[J]. 中国中药杂志,2020, 45(15):3740
ZHAO AY, NI FY, CHEN PY, et al. Current studies on quality of Chinese patent drugs with consistency as core and discussions on new high-quality evaluation model [J]. China J Chin Mater Med, 2020, 45(15):3740
[13] 王娟,周植星,杨莉,等.补骨脂药材UPLC指纹图谱建立及12种主要成分含量测定[J]. 中草药,2021, 52(2):552
WANG J, ZHOU ZX, YANG L, et al. Establishment of UPLC fingerprint and determination of 12 main components in Psoraleae Fructus [J]. Chin Tradit Herb Drugs. 2021, 52(2):552
[14] 徐鑫,戚华文,高德嵩,等.基于化学识别的栀子UPLC定量指纹图谱研究[J].中国中药杂志,2020, 45(18):4416
XU X, QI HW, GAO DS, et al. UPLC quantitative fingerprint research on Gardeniae Fructus based on chemical pattern recognition technology [J]. China J Chin Mater Med, 2020, 45(18):4416
[15] 许丽丽,汪冰,周广涛,等.基于HPLC指纹图谱、多指标成分含量测定及化学计量学的槐芩软膏质量评价[J]. 药物分析杂志,2022, 42(3):501
XU LL, WANG B, ZHOU GT, et al. Quality evaluation of Huaiqin ointment based on combinative methods of HPLC fingerprint, quantitative analysis of multi-components and chemometrics analysis [J]. Chin J Pharm Anal, 2022, 42(3):501
[16] 刘庆焕,白建疆,张岩,等.基于指纹图谱、模式识别及多成分定量的金花清感颗粒质量评价研究[J]. 中国现代应用药学,2021, 38(21):2701
LIU QH, BAI JJ, ZHANG Y, et al. Study on quality evaluation of Jinhua Qinggan granules based on fingerprint, pattern recognition and multi-components determination [J]. Chin J Mod Appl Pharm, 2021, 38(21):2701
