Analysis of chemical constituents and bioavailable components in Yupingfeng Granules using ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry

Yupingfeng Granules， a classical and widely prescribed formula in traditional Chinese medicine， is extensively employed in clinical practice for its well-documented immunomodulatory and preventive activities. However， the highly complex chemical composition of this multi-herb formula and the specific material basis responsible for its therapeutic effects in vivo remain inadequately characterized and systematically elucidated. Research on its pharmacokinetically critical blood-entering components， which are indicative of systemic exposure and potential direct activity， particularly under representative pathological conditions， remains notably scarce and insufficient. This study therefore aims to systematically characterize the comprehensive chemical profile of Yupingfeng Granules by employing advanced analytical techniques such as HPLC-MS. Furthermore， it seeks to comprehensively investigate and characterize its blood-entering components specifically in cyclophosphamide-induced immunodeficient rat models， comparing them with normal physiological conditions where applicable. The overarching objective is to identify， quantify， and shed light on the formula′s potential active constituents that reach the systemic circulation. This work is expected to provide a more solid and detailed scientific basis for a deeper， molecular-level understanding of the pharmacodynamic mechanism of this important traditional Chinese medicine preparation and to inform its future rational clinical application and quality control. An ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF MS） technology was used to identify the chemical composition of Yupingfeng Granules and the plasma-entering components after administration to immunodeficient model rats. The prepared granule and serum samples were analyzed using a Waters CORTECS UPLC C18 column （150 mm×2.1 mm， 1.6 μm） with gradient elution of 0.1% formic acid aqueous solution and acetonitrile as the mobile phases. The flow rate was set at 0.3 mL/min， the column temperature was maintained at 35 ℃， and the injection volume was 3 µL. Data were acquired in both positive and negative ion modes using an electrospray ionization source. The identification of chemical constituents was performed by comparing retention times， accurate relative molecular masses， and MS/MS fragment ions with those of reference standards， along with reference to relevant literature. Through a systematic analysis involving comparison of retention times， accurate molecular weights， and MS/MS fragment ions， combined with cross-referencing against relevant literature and authenticated reference standards， the chemical profile of Yupingfeng Granules and the bioactive components absorbed in vivo were comprehensively characterized. The investigation successfully identified a total of 52 chemical constituents in Yupingfeng Granules. Furthermore， 11 bioavailable components were detected in rat plasma after administration. These compounds encompass a diverse range of chemical classes， specifically including 11 flavonoids， 13 organic acids， 13 phenylpropanoids， 12 terpenoids， and 3 additional components categorized as aldehydes， nucleosides， and amino acids. This detailed identification provides a thorough overview of the chemical composition of the granules and elucidates the key constituents that are systemically absorbed. This study systematically analyzed the blood-entering components of Yupingfeng Granules under immunodeficient pathological conditions. This distinction is critically important， as the altered internal environment in a disease model may significantly influence the absorption， metabolism， and distribution of herbal compounds. The research not only clarifies the comprehensive chemical material basis of the formula in vitro by establishing a detailed chromatographic fingerprint but also， more significantly， bridges the gap to in vivo pharmacology by identifying which specific compounds from the complex mixture actually enter the systemic circulation. By correlating the in vitro chemical profile with the in vivo exposure data， the study effectively pinpoints a refined set of potential direct-acting substances that are most likely to contribute to the observed therapeutic effects. Consequently， these findings provide crucial experimental clues and a reliable analytical foundation for subsequent， more targeted research. This includes in-depth investigations into efficacy correlation—linking specific blood components to immunomodulatory activities—and the detailed exploration of its molecular mechanisms of action， thereby significantly advancing the scientific understanding of this classic formula.