Abstract:

Qinggusan is the 69th prescription in the first batch of “Catalogue of Ancient Chinese Classic Formulas”. In modern clinical practice, Qinggusan is mainly used to treat noninfectious fever. However, because few studies on Qinggusan reference samples and their quality value transfer are available, the development and promotion of its compound preparations are restricted. Therefore, establishing an accurate and comprehensive quality control method to clarify the critical quality attributes of Qinggusan reference samples is of great importance. In this study, 15 batches of Qinggusan reference samples were processed to determine the range of their dry extract ratios. Quantitative high-performance liquid chromatography (HPLC) fingerprint analysis was performed using a Waters Symmetry Shield RP18 column (250 mm×4.6 mm, 5 μm) with acetonitrile-0.1% (v/v) formic acid aqueous solution as the mobile phase in gradient elution mode. The flow rate was 1.0 mL/min, the column temperature was 30 ℃, and the detection wavelength was 254 nm. The HPLC fingerprints of the Qinggusan reference samples were established under these conditions to evaluate their similarity. The established method was systematically validated and found to demonstrate good precision, repeatability, and sample stability. Subsequently, characteristic peaks were identified and attributed by HPLC-quadrupole-time-of-flight-mass spectrometry (HPLC-Q-TOF-MS) analysis. MS was performed in electrospray ionization mode, the data were collected in both positive- and negative-ion modes, and the detection range was m/z 50-2000. The contents and transfer rate ranges of the index components, namely, gentiopicrin, mangiferin, picroside Ⅱ, picroside Ⅰ, and glycyrrhizic acid, were determined to analyze the quality value transfer of the samples. The results demonstrated that the dry extract rate of the 15 batches of Qinggusan reference samples ranged from 24.10% to 26.88% and that their fingerprint similarities were generally greater than 0.95. Twelve common peaks were identified by reference identification, literature comparison, and high-resolution MS analysis. Twelve compounds, including six iridoid glycosides, two flavonoids, one alkaloid, one triterpenoid saponin, and two others. Among them, L-picein, androsin, picroside Ⅳ, picroside Ⅱ and picroside Ⅰ were from Picrorhizae Rhizoma, loganin acid, swertiamarin and gentiopicrin were from Gentianae Macrophyllae Radix, neomangiferin and mangiferin were from Anemarrhenae Rhizoma, dichotomine B was from Stellariae Radix, and glycyrrhizic acid was from Glycyrrhizae Radix et Rhizoma. The five key components presented good linear relationships in their respective linear ranges, and all correlation coefficients were higher than 0.999. The relative standard deviations (RSDs) of precision, stability, and repeatability were less than 1.3%. The average recoveries varied between 95.92% and 102.5%, with RSDs less than 3.9%; these values meet the requirements defined in the 2020 edition of the Chinese Pharmacopoeia. The contents of gentiopicrin, mangiferin, picroside Ⅱ, picroside Ⅰ, and glycyrrhizic acid in the 15 batches of reference samples were in the range of 17.92-27.55, 1.83-4.42, 23.08-36.44, 8.43-15.04, and 0.94-2.39 mg/g, respectively, and their transfer rates from the decoction pieces to the reference samples were 47.91%-63.95%, 22.96%-59.39%, 60.82%-77.82%, 64.25%-99.53%, and 15.30%-39.30%, respectively. In this study, the chemical components of Qinggusan reference samples were comprehensively identified and their quality value transfer was studied through the combination of HPLC fingerprinting and MS. Clarification of the critical quality attributes of Qinggusan reference samples could provide a basis for the quality control of Qinggusan compound preparations.

Key words: quantitative fingerprint, quality value transfer, Qinggusan, reference sample