Abstract:ABSTRACT:Objective To establish HPLC characteristic chromatogram and content determination methods for multi index components of Danggui Buxue decoction substance benchmark, providing reference for quality control and subsequent development and research of this famous classical prescriptions' substance benchmark. Methods The separation was performed on Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm) for gradient elution with acetonitrile-0.1% formic acid aqueous solution, and detection wavelength 260 nm. Then, HPLC fingerprint spectra of 15 batches of Danggui Buxue decoction substance standards were established, after that, analyzed similarities and assigned common peaks. Using cluster analysis (CA), principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) for quality analysis, and conducted quantitative analysis on 5 of the indicator components. Results The HPLC fingerprint of 15 batches of Danggui Buxue decoction identified 8 common peaks with a similarity of > 0.93. Cluster analysis and principal component analysis could divide the samples into three categories, and partial least squares discriminant analysis might screen out four quality difference biomarkers. The linear relationship between the five components of calycosin-7-O-β-D-glucosid, ferulic acid, ononin, calycosin, and formononetin was good within their respective injection volume ranges, with correlation coefficients>0.999. The average sample recovery rates were 98.62% - 103.49%, and the RSD was<2.00%, The contents of 5 components ranged from 0.357-1.286, 0.072-1.138, 0.115-0.684, 0.068-0.544, 0.026-0.297 mg/g, respectively. Conclusion The method established in this study has simple operation, strong specificity, good separation, and high sensitivity, can be used for the reference quality control of Danggui Buxue decoction substance benchmark.