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目的建立二妙丸的HPLC指纹图谱质量评价方法,并对二妙丸中的化学成分进行UPLC-LTQ-Orbitrap定性分析。方法指纹图谱采用HPLC法,色谱柱为Shiseido C18柱(250 mm×4.6 mm,5μm),乙腈-0.1%甲酸水溶液为流动相进行梯度洗脱,体积流量为1.0 m L/min,检测波长为330 nm,以黄柏、苍术混合对照药材生成的色谱图为标准对照图谱,采用“中国药典委员会相似度评价软件(2012A)”对样品进行相似度评价;采用UPLC-LTQ-Orbitrap技术对二妙丸中化学成分进行定性分析,色谱柱为Thermo Scientific Syncronis C18柱(100 mm×2.1 mm,1.9μm),乙腈-0.1%甲酸水溶液梯度洗脱,经DAD检测器后采用LTQ-Orbitrap Elite质谱正离子模式采集,各成分的一级全扫描离子在离子阱内进行CID多级质谱、FT检测高分辨质谱数据分析。结果所建立的指纹图谱有10个共有色谱峰,20批样品的相似度在0.869~0.992。通过对照品指认、文献比对以及高分辨质谱数据解析,鉴定了21个化学成分,分别为neo-chlorogenic acid、木兰箭毒碱、竹叶椒碱、木兰花碱、3-O-feruloylquinic acid、蝙蝠葛碱、demethyleneberberine、oxyberberine、非洲防己碱、药根碱、小檗红碱、巴马汀、小檗碱、丁香酸、咖啡酸、(E)-4-(3-hydroxyprop-1-en1yl)-2-methoxyphenol、白术内酯II、acetosyringone、白术内酯I、selina-4(14),7(11)-dien-8-one、苍术素。结论建立的二妙丸HPLC指纹图谱专属性强,重现性好,结合液质联用技术的定性分析,可全面评价二妙丸的内在质量。
OBJECTIVE To establish a HPLC fingerprinting method for the quality evaluation of Ermiao Wan and analyze the chemical composition of Ermiao Wan qualitatively by UPLC-LTQ-Orbitrap. Methods The HPLC method was applied on a chromatographic column. The mobile phase consisted of a Shiseido C18 column (250 mm × 4.6 mm, 5 μm) with acetonitrile-0.1% formic acid as mobile phase. The volume flow rate was 1.0 m L / min and the detection wavelength was 330 nm. The chromatograms generated from the mixed herbs of Cortex phellodendri and Atractylodes were taken as standard control maps. The similarity of the samples was evaluated using the similarity evaluation software (2012A) of the Chinese Pharmacopoeia Commission. The UPLC-LTQ-Orbitrap Qualitative analysis of the chemical constituents in the pellets was carried out using a Thermo Scientific Syncronis C18 column (100 mm × 2.1 mm, 1.9 μm) eluting with a gradient of acetonitrile-0.1% formic acid in water. After DAD detector, LTQ-Orbitrap Elite Mode acquisition, the first-order full-scan ion of each component in the ion trap CID multi-stage mass spectrometry, FT high-resolution mass spectrometry data analysis. Results There were 10 common chromatographic peaks in the fingerprints established. The similarity of 20 batches of samples was 0.869 ~ 0.992. 21 chemical constituents were identified by comparison of reference substance, literature comparison and high-resolution mass spectrometry data, which are neo-chlorogenic acid, magnolol, pennyine, magnoliaine, 3-O-feruloylquinic acid, Dauricine, jatrorrhizine, berberine, palmatine, berberine, syringic acid, caffeic acid, (E) -4- (3-hydroxyprop-1- enyl) -2-methoxyphenol, atractylenolide II, acetosyringone, atracta lactone I, selina-4 (14), 7 (11) -dien-8-one, atractylodes. Conclusion The established HPLC fingerprint of Ermiao Pill possessed strong specificity and good reproducibility. Combined with the qualitative analysis of liquid chromatography - mass spectrometry, the intrinsic quality of Ermiao Pills could be fully evaluated.