Animal-derived medicines have been a vital component for traditional Chinese medicine. However, their quality control remains challenging due to the large polarity of the contained endogenous small molecules (ESMs) that are difficult to separate by reversed-phase HPLC. Herein, an intelligentized strategy by ultra-high performance hydrophilic interaction chromatography/quadrupole time-of-flight mass spectrometry (HILIC/QTOF-MS(E)) is presented, and used for the ESMs characterization and differentiation of two geographic origins of earthworm (Guang Di-long, GD; Hu Di-long, HD) as a case study. Chromatographic separation was performed on a BEH Amide column (2.1 × 100 mm, 1.7 μm). The MS(E) data in both negative and positive ion modes were acquired to record the high-accuracy MS and MS/MS data of all precursor ions. Automatic data processing was enabled by use of Progenesis QI software. As a consequence, 926 metabolites among 4705 features and 761 among 3418 features were characterized in the negative and positive modes, respectively, by searching the human metabolome database (HMDB). To reduce the false positive identifications, structural confirmation was conducted by comparison with the reference standards (tR and MS, MS/MS data) or matching with theoretical data or commercial library. Principal component analysis (PCA) of the GD and HD samples showed distinct classifications. Further orthogonal partial least squares discriminant analysis (OPLS-DA) and variable importance in projection (VIP) plot revealed the potential discriminatory markers between GD and HD. The present study provides a powerful and practical strategy that facilitates the primary metabolites characterization and quality evaluation of animal-derived medicines more efficiently. Graphical Abstract A general flowchart illustrating the application of ultra-high performance HILIC/QTOFMS(E) coupled with data processing by Progenesis QI to characterization of the endogenous small molecules and discriminatory analysis of animal-derived traditional medicine: earthworm as a case study.

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Sepsis-related acute kidney injury (AKI) is known to be caused by inflammation. We explored the renal protective effects of aerosol inhalation of a hydrogen-rich solution (HRS; hydrogen gas dissolved to saturation in saline) in a mouse model of septic AKI. Septic AKI was induced through 18 hours of cecal ligation and puncture. AKI occurred during the early stage of sepsis, as evidenced by increased blood urea nitrogen and serum creatinine levels, pathological changes, renal fibrosis and renal tubular epithelial cell apoptosis, accompanied by macrophage infiltration and M1 macrophage-associated pro-inflammatory cytokine (Il-6 and Tnf-α) generation in renal tissues. Aerosol inhalation of the HRS increased anti-inflammatory cytokine (Il-4 and Il-13) mRNA levels in renal tissues and promoted macrophage polarization to the M2 type, which generated additional anti-inflammatory cytokines (Il-10 and Tgf-β). Ultimately, aerosol inhalation of HRS protected the kidneys and increased survival among septic mice. HRS was confirmed to promote M2 macrophage polarization in lipopolysaccharide-stimulated RAW 264.7 cells. The TGF-β1 receptor inhibitor SB-431542 partly reversed the effects of HRS on renal function, fibrosis, tubular epithelial cell apoptosis and senescence in mice. Thus, HRS aerosol inhalation appears highly useful for renal protection and inflammation reduction in septic AKI.