Establishment of Fingerprints and Quantitative Analysis of Multi-Components in Zuoyu No.2 Prescription

OBJECTIVE To establish HPLC fingerprints of 15 batches of Zuoyu No.2 prescription decoction and to determine the contents of 4 components, in order to provide reference for its quality control and clinical rational application.

METHODS Amethyst C₁₈-H column(4.6 mm×250 mm, 5 μm) was used for separation, mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution; the injection volume was 10 μL, the detection wavelength was 214 nm, the column temperature was 30 ℃, and the flow rate was 1.0 mL·min⁻¹. The fingerprints of 15 batches of Zuoyu No. 2 prescription decoction were established using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine(Version 2012). Based on the similarity evaluation, cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis(OPLS-DA) were further performed, and the contents of gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin were simultaneously determined.

RESULTS The established HPLC fingerprints method met the methodological requirements. There were 14 common peaks in 15 batches of Zuoyu No.2 prescription decoction, and the similarity was greater than 0.900. Four components, namely gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin, were identified by comparison of reference substances. The 15 batches of samples could be clustered into 2 categories, and 4 main components affecting the classification of samples were gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin. The 4 main components had good linear relationships in their respective linear ranges(r≥0.9990). The average recoveries of gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin were 98.04%, 99.45%, 99.79% and 99.52%, respectively. The RSDs were 2.7%, 4.8%, 5.6% and 5.6%(n=6), respectively. The instrumental precision, sample stability, and method repeatability were all good. The content of gallic acid was 1.165−3.054 mg·mL⁻¹, the content of chlorogenic acid was 0.426−0.939 mg·mL⁻¹, the content of hydroxysafflor yellow A was 0.144−0.360 mg·mL⁻¹, and the content of linarin was 0.073−0.224 mg·mL⁻¹.

CONCLUSION The established HPLC fingerprints and the content determination method of the 4 components have good sensitivity, repeatability and stability, which can provide scientific basis for the quality control and clinical safety application of the Zuoyu No.2 prescription decoction.