Objective  To establish HPLC fingerprint of Tongchang Qingyi mixture, and to determine the content of 5 components.

Methods  An Agilent ZORBAX SB-C₁₈ chromatographic column (250 mm×4.6 mm, 5 μm) was used. Acetonitrile-pH 5.0 phosphate buffer solution was taken as the mobile phase with gradient elution. The detection wavelengths were set at 230 nm and 250 nm. The flow rate was 1.0 mL/min, the column temperature was 30 °C, and the injection volume was 10 μL. The HPLC fingerprints of 10 batches of Tongchang Qingyi mixture were established by the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 version)", and the similarity evaluation was conducted simultaneously. Cluster analysis and principal component analysis were carried out in combination with the software SPSS 27.0 and OriginPro 2024b.

Results  Baicalin, paeoniflorin, rhein, magnolol and honokiol all had good linear relationships within certain concentration ranges respectively (r≥0.999 8). The average recovery rates and RSDs were between 96.39%-97.96% and 1.54%-2.37% (n=6), respectively. The fingerprint chromatograms of 10 batches of Tongchang Qingyi mixture showed that there were 12 common peaks, with the similarities ranging from 0.984 to 1.000. The 10 batches of samples could be clustered into 3 categories. Among them, S1-S7 belonged to the first category, S10 belonged to the second category, and S8 and S9 belonged to the third category. Five components had been identified, namely rhein, paeoniflorin, baicalin, magnolol and honokiol.

Conclusion  The established HPLC fingerprint and component content measurement method for Tongchang Qingyi mixture in the research are accurate, reliable, and highly reproducible, and provide a scientific basis for the quality control of Tongchang Qingyi mixture.

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