Analysis of Specific Chromatogram of Wenjingtang Reference Sample / 中国实验方剂学杂志

Objective:To control the quality of the reference sample of Wenjingtang by establishing the specific chromatograms. Method:On the basis of analyzing 15 batches of Wenjingtang freeze-dried powder samples， a high performance liquid chromatography （HPLC） specific chromatogram analysis method of Wenjingtang was established. The system adaptability was investigated and the retention time， relative retention value and deviation caused by different chromatographic columns and instruments were calculated by using the same brand of chromatographic columns， four different brands of chromatographic columns and instruments from three different manufacturers. The precision， repeatability and stability of this method was further completed. The possible chemical components of the freeze-dried powders were speculated and identified by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF-MS<italic>ⁿ</italic>）. Chromatographic separation was performed on ACQUITY UPLC BEH C₁₈ column （2.1 mm×100 mm， 1.7 μm） with acetonitrile （A）-0.1% formic acid aqueous solution （B） as mobile phase for gradient elution （0-2.8 min， 10%A； 2.8-8.0 min， 10%-18%A； 8.0-12.2 min， 18%-25%A； 12.2-15.3 min， 25%-40%A； 15.3-17.4 min， 40%A； 17.4-20.5 min， 40%-90%A）， and column temperature was set at 30 ℃ with flow rate of 0.4 mL·min^-1. Mass spectrometry was performed on electrospray ionization， data were collected under positive and negative ion modes， and the detection range was <italic>m</italic>/<italic>z</italic> 50-1 600. Result:Ten characteristic peaks were selected as the distinguishing features in this specific chromatograms， and eight of them were identified by comparing with the reference standards， including paeoniflorin （peak 1）， liquiritin apioside （peak 2）， liquiritin （peak 3）， ferulic acid （peak 4）， iquiritigenin （peak 6）， cinnamaldehyde （peak 8）， paeonol （peak 9）and glycyrrhizic acid （peak 10）. By mass spectrometry analysis， 30 compounds were identified， and the source of medicinal materials were assigned. It mainly contained triterpenoid saponins and flavonoids from Glycyrrhizae Radix et Rhizoma， ginsenosides from Ginseng Radix et Rhizoma， monoterpenoid glycosides and tannins from Paeoniae Radix Alba， steroids in Achyranthis Bidentatae Radix， phenolic acids in Angelicae Sinensis Radix. Conclusion:The established characteristic chromatographic analysis method of Wenjingtang is simple， stable and repeatable. The chemical composition of the freeze-dried powder of Wenjingtang is basically defined by mass spectrometry identification and source attribution， which can provide reference for the development and quality control of Wenjingtang in the future.