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.
清骨散为第一批《古代经典名方目录》中的第69方，现代临床常用于治疗非感染性发热。但目前关于清骨散基准样品及其量值传递的研究较少，制约了其复方制剂的研发与上市，因此亟须建立一种全面、准确的质量控制方法，阐明关键质量属性。通过制备15批清骨散基准样品，明确干膏率范围，建立基准样品高效液相色谱（HPLC）定量指纹图谱测定方法并进行相似度评价，采用高效液相色谱-四极杆-飞行时间质谱（HPLC-Q-TOF-MS）技术对特征峰进行指认与归属，结合指标成分龙胆苦苷、芒果苷、胡黄连苷Ⅱ、胡黄连苷Ⅰ和甘草酸的含量范围及转移率范围进行量值传递分析。结果表明，15批清骨散基准样品的干膏率为24.10%~26.88%，指纹图谱相似度均大于0.95，对12个共有峰进行指认，包括6个环烯醚萜类、2个黄酮类、1个生物碱类、1个三萜皂苷类以及2个其他类，其中5个成分来源于胡黄连，3个成分来源于秦艽，2个成分来源于知母，1个成分来源于银柴胡，1个成分来源于甘草；15批基准样品中指标成分的含量范围分别为龙胆苦苷17.92~27.55 mg/g、芒果苷1.83~4.42 mg/g、胡黄连苷Ⅱ 23.08~36.44 mg/g、胡黄连苷Ⅰ 8.43~15.04 mg/g、甘草酸0.94~2.39 mg/g，从饮片到基准样品的转移率分别为龙胆苦苷47.91%~63.95%、芒果苷22.96%~59.39%、胡黄连苷Ⅱ 60.82%~77.82%、胡黄连苷Ⅰ 64.25%~99.53%、甘草酸15.30%~39.30%。该研究通过清骨散基准样品定量指纹图谱与质谱定性技术相结合，基本明确其化学组成，进行量值传递研究，阐明关键质量属性，为清骨散复方制剂的质量控制提供依据。
Keywords: Qinggusan; quality value transfer; quantitative fingerprint; reference sample.