Objective To establish the HPLC chromatograms of the classic prescription Huangqi Guizhi Wuwu Decoction (HGWD, 黄芪桂枝五物汤) of its decoction and freeze-dried powder, as well as quantitative determination methods for nine marker components, in order to characterize the law of quantitative transfer. Methods According to the “Technical Guidance for Pharmaceutical Research of Traditional Chinese Compound Preparations Managed According to Ancient Classic Formula Catalogue (Trial)”, HPLC finger printing profiles and a simultaneous quantitative method for eight active components, along with a separate assay for astragaloside A, were developed. Fifteen batches of HGWD decoction and freeze-dried powder were prepared using an optimized process. The value transfer process from medicinal materials to decoction pieces, decoctions, and finally freeze-dried powders was systematically characterized by evaluating the characteristic peak area of the unit mass compound yinpian (equivalent characteristic peak area) and/or quantitative levels of multiple constituents. Results The established HPLC methods both met the requirements stipulated in the 2025 edition of the Chinese Pharmacopoeia. The intra-batch similarity of the characteristic chromatograms for the 15 batches of HGWD and its freeze-dried powder was ≥ 0.983, while the inter-batch similarity was ≥ 0.909, indicating good consistency of sample quality both within and between batches. The 15 characteristic peaks in common covered all the herbal components of HGWD, among which seven peaks were identified. Among the 15 batches of HGWD decoctions, the contents of nine quality indicators-astragaloside IV, gallic acid, albiflorin, paeoniflorin, calycosin-7-O-β-D-glucoside, cinnamic acid, cinnamaldehyde, formononetin, 6-gingerol-were calculated based on the corresponding dosage of the herbs, ranging from 0.14 to 0.32, 1.33 to 1.84, 2.26 to 7.57, 11.57 to 15.01, 0.11 to 0.26, 0.17 to 0.32, 0.45 to 0.97, 0.01 to 0.02, and 0.21 to 0.32 mg/g respectively. The content ranges were mostly within the mean ± 30%. For the 15 equivalent characteristic peaks across the 15 batches of HGWD, the intra-batch transfer rates from decoction to freeze-dried powder ranged from 73% to 129% for 14 peaks, except for cinnamaldehyde in some batches, which showed a transfer rate of 45% to 71%. The mean inter-batch transfer rates ranged from 62% to 101%. Conclusion The decoction form is determined as the reference substance for HGWD. The established quality control indicators are comprehensive and reasonable. The method for characterizing the transfer of equivalent characteristic peak area is relatively scientific. It can provide new evaluation and quality control indicators for the research and development of benchmark samples of other classic prescriptions. These findings provide a basis for establishing quality standards for HGWD reference samples and for supporting the development of granule formulations.

Huangqi Guizhi Wuwu Decoction  /  reference sample  /  quantity value transfer  /  HPLC characteristic profile  /  astragaloside IV  /  gallic acid  /  albiflorin  /  paeoniflorin  /  calycosin-7-O-β-D-glucoside  /  cinnamic acid  /  cinnamaldehyde  /  formononetin  /  6-gingerol