As a common and severe microvascular complication of diabetes mellitus and a cause of end-stage kidney disease, diabetic nephropathy (DN) has high morbidity and mortality worldwide [1]. The current interventions for DN mainly concentrate on early diagnosis, renin–angiotensin–aldosterone system blockade, and blood glucose level control, which can postpone the onset and advancement of DN [2]. With the benefit of its multi-target function, traditional Chinese medicine (TCM) can act as a promising avenue for primary or alternative therapy for DN [3]. Qizhi Yishen Capsule (QYC), manufactured by Shandong Phoenix Pharmaceutical Co., Ltd. (Shandong, China), was approved by the China Food and Drug Administration (CFDA) in November 2021 as a Class I new drug [4]. It was designed to be applicable to TCM syndromes characterized by qi and yin deficiency with blood stasis, including symptoms such as fatigue, dry mouth and throat, decreased appetite, pale complexion, and numbness of the limbs [5]. The launch of QYC fills the gap in the treatment of early-stage diabetic nephropathy with Chinese medicine, providing a new treatment option for patients [6].

QYC was developed from clinical experience with Qihuang Decoction [7], which was composed of ten herbs, including Astragali Radix (AR), Rehmanniae Radix (RR), Ligustri Lucidi Fructus (LLF), Hirudo (HR), Bombyx Batryticatus (BB), Eupolyphaga Steleophaga (ES), Rhei Radix et Rhizoma (RRR), Folium Gymnemae (FG), Sinomenii Caulis (SC), and Plantaginis Semen (PS), along with pharmaceutical excipients (Fig. 1A). In this formula, AR serves as the monarch drug (Jun Yao), promoting blood circulation, while RR and LLF function as minister drugs (Chen Yao), nourishing yin in accordance with TCM theory. Due to its relatively short time on the market, there are few research reports on QYC. Although 23 components of QYC have been identified in rat serum [5], the chemical composition characteristics of QYC in vitro remain unclear. According to drug quality standards, astragaloside IV is used as a content-limiting ingredient, while oleanolic acid, emodin, sinomenine, and silychristin C are used for thin-layer identification. However, the synergistic effects of multiple compounds and multiple targets, as well as the advantages in comprehensively treating and broadly regulating diseases involving multiple sites and targets, are the cornerstones of TCM’s superior therapeutic outcomes [8,9]. The lack of chemical composition information is bound to hinder the promotion and application of QYC and may pose potential risks to its medication safety. Thus, it is necessary to clarify the chemical composition characteristics of QYC, which can support more comprehensive strategies for its quality control.

As the preferred tool for qualitative analysis of chemical components in TCM, ultra-performance liquid chromatography-Q trap-tandem mass spectrometry (UPLC-Q Trap-MS/MS) offers advantages of speed, accuracy, and high sensitivity [10]. However, there are still limitations to UPLC-Q Trap-MS/MS. Although the Multiple Reaction Monitoring (MRM) scanning mode can filter out peak overlap interference and does not affect the mass spectrometry results [11,12], the resulting spectrum (total ion chromatogram) usually cannot fully represent the overall chemical characteristics of the sample. Fingerprint profiling, which adequately reflects the types and quantities of chemical constituents in TCM, is considered a convenient and efficient technology for quality inspection [13]. HPLC fingerprint analysis, which can address most non-volatile components by matching different types of detectors [11,12,14], has undoubtedly become one of the most successful technologies applied in plant medicine research [15,16]. In addition, when combined with multivariate statistical analysis methods, fingerprint spectra can highlight the characteristic and representative information of fingerprint regions [17], further enhancing its application value in the quality evaluation of TCM [18].

In this study, we clarified the chemical components of QYC for the first time and established a new method based on qualitative and quantitative analyses combined with chemometric methods for the quality evaluation of QYC. First, a total of 103 compounds, including 24 phenolic acids, 15 alkaloids, 15 flavonoids, 15 quinones, 12 terpenoids, and 22 other phytochemicals, were putatively identified by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS (Fig. 1B). Afterwards, a photodiode array (PDA) detector and an evaporative light scattering detector (ELSD) were connected in series to the HPLC system (HPLC-PDA-ELSD) to collect as much chromatographic information as possible (Fig. 1C). Subsequently, a new and fully validated HPLC fingerprint method was developed, and two fingerprints for 19 batches of QYC were obtained in PDA mode and ELSD mode, respectively. The characteristic peaks were identified and attributed to their corresponding herbal sources in the prescription. Meanwhile, the contents of ten chemical constituents, including the qualitative markers, in 19 batches of samples were simultaneously determined by HPLC. This research will provide a reference for exploring the material basis and quality control methods of QYC.