HPLC fingerprint of Ermiao Pills and chemical constituents analysis by UPLC-LTQ-Orbitrap / 中草药

ABSTRACT

Objective To establish a quality assessment method for Ermiao Pills (EP) based on HPLC fingerprint and qualitatively analyze the chemical constituents. Methods The chromatographic column Shiseido C18 (250 mm × 4.6 mm, 5 μm) was used, acetonitrile-0.1% formic acid as mobile phase with gradient elution at the flow rate of 1.0 mL/min, and the detection wavelength was 330 nm. The standard chromatographic fingerprint was synthesized from chromatogram of the mixed standard herbs of Phellodendron Rupr. and Atractylodes DC, and the similarity evaluation of Ermiao Pills samples was carried out by the Similarity Evaluation System for Chromatographic Fingerprints of TCM (Chinese Pharmacopoeia Commission, version 2012A). Ultra high-performance liquid chromatography coupled with linear ion trap-Orbitrap Elite mass spectrometer (UPLC-LTQ-Orbitrap) was used to characterize the chemical constituents of Ermiao Pills. A Thermo Scientific Syncronis C18 (100 mm × 2.1 mm, 1.9 μm) column and a gradient elution of acetonitrile-0.1% formic acid were used for UPLC separation. The combination of ESI-LTQ-Orbitrap mass analyzer with a linear ion trap was applied for high resolution mass spectrometry and collision-induced dissociation (CID). Results The chromatographic fingerprints were generated with 10 common peaks. The similarity scores of 20 samples between each material batch and the reference fingerprint ranged from 0.869-0.992. Twenty-one components were identified via referring to reference components and literatures and analyzing MS data, they were neo-chlorogenic acid, magnocurarine, xanthoplanine, magnoflorine, 3-O-feruloylquinic acid, menisperine, demethyleneberberine, oxyberberine, columbamine, jatrorrhizine, berberubine, palmatine, berberine, syringic acid, caffeic acid, (E)-4-(3-hydroxyprop-1-en1yl)-2-methoxyphenol, atractylenolide II, acetosyringone, atractylenolide I, selina-4(14), 7(11)-dien-8-one, and atractylodin. Conclusion The established method of fingerprint is specific, combined with LC-MS qualitative analysis, can be used for the quality evaluation of Ermiao Pills, giving support to quality control comprehensively.