This paper presents an application of high performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry (HPLC-Q-Orbitrap HRMS) for the analysis of 27 β-blockers and metabolites in milk powder. Homogenized milk power samples were extracted by acetonitrile and purified by using Oasis PRiME HLB solid-phase extraction cartridges. The Ascentis^® C8 chromatographic column was used to separate the analytes. The quantification was achieved by using matrix-matched standard calibration curves with carazolol-d₇ and propranolol-d₇ as the internal standards. The results show an exceptional linear relationship with the concentrations of analytes over wide concentration ranges (0.5–500 μg kg⁻¹) as all the fitting coefficients of determination r² are > 0.995. All the limits of detection (LODs) and quantitation (LOQs) values were within the respective range of 0.2–1.5 μg kg⁻¹ and 0.5–5.0 μg kg⁻¹. Overall average recoveries were able to reach 66.1–100.4% with the intra- and inter-day variability under 10%. This method has been successfully applied to the screening of β-blockers and metabolites in commercial milk powders. At the same time, the corresponding characteristic fragmentation behavior of the 27 compounds was explored. The characteristic product ions were determined and applied to the actual samples screening.

Background/Objectives: There is high demand for Anshenbunao syrup (ABS) in Chinese medicine owing to its steady therapeutic efficacy for insomnia and neurasthenia. However, it contains a substantial proportion of Polygoni Multiflori Radix Praeparata (PMRP), which is associated with reported cases of drug-induced liver injury (DILI). Here, we aim to establish an integrated approach combining PK screening with a dual-model toxicity verification system to systematically identify liver injury components (from high to low concentrations and from direct to idiosyncratic hepatotoxicity) to accurately uncover diverse potential hepatotoxicity markers. Methods: A sensitive UPLC-MS/MS method was used to accurately quantify the components in plasma at the ng/mL level and conduct a pharmacokinetic analysis. Rat models were used to evaluate exposure levels of the eight active constituents and three major metabolites after a single oral gavage dose of 10 mL/kg ABS and identify the quality markers. The early-stage and high-throughput assessment of direct and idiosyncratic hepatotoxicity was conducted in vitro utilizing HepG2 cells. After the administration of the quality markers (0.01–80 μM), CCK-8 was used to detect cell viability on both normal and susceptible cells, and the latter was induced by lipopolysaccharide. Results: As a result, seven quality markers were screened based on their contents and exposure levels in rat plasma by UPLC–MS/MS, including emodin (EM), liquiritin (LI), 2,3,5,4′–Tetrahydroxystilbene–2–O–β–D–glucoside (TSG), icariin, emodin–8–O–β–D–glucoside, baohuoside I (BA), and 18β–glycyrrhetinic acid (GTA). Moreover, the half maximal inhibitory concentration values of both normal cells and the lipopolysaccharide-induced immune stress liver injury cells were fitted within the concentration range of 0.01–80 μM, based on which, EM, BA, and GTA were identified as the principal hepatotoxic constituents in ABS at elevated concentrations. This study is the first to demonstrate that TSG, EM, LI, and GTA exhibit synergistic cytotoxicity in LPS-sensitized hepatocytes at clinically relevant concentrations, whereas EM was also a direct hepatotoxic component. Given that TSG is one of the major ingredients in ABS, the underappreciated idiosyncratic hepatotoxicity could elevate the risk of adverse clinical outcomes. Conclusions: In conclusion, this study effectively identifies hepatotoxic constituents in ABS and evaluates their hazards under immune stress and toxicity profiles in clinical concentrations, which also provides a robust foundation for the awareness of PMRP-induced DILI due to ABS.

Background/Objectives: Anshenbunao Syrup (ABS) enjoys robust demand in the Chinese health application owing to its steady therapeutic efficacy for insomnia and neurasthenia. However, it contains a substantial proportion of Polygoni Multiflori Radix Praeparata (PMRP), which is associated with reported cases of drug–induced liver injury (DILI). Here, we aim to establish an integrated approach combining PK screening with a dual-model toxicity verification system to systematically identify liver injury components (from high to low concentrations, and from direct to idiosyncratic hepatotoxicity) for the accurate uncovering of diverse potential hepatotoxicity markers. Methods: A sensitive UPLC-MS/MS method was used to accurately quantify the components in plasma at ng/mL level and conduct pharmacokinetic analysis. Rat models were used to evaluate exposure levels of the eight active constituents and three major metabolites after a single oral gavage dose of 10 mL/kg ABS and identify the quality markers. The early-stage and high-throughput assessment of direct and idiosyncratic hepatotoxicity was conducted in vitro utilizing HepG2 cells. After administration of the quality markers (0.01-80 μM), CCK-8 was used to detect cell viability on both normal and susceptible cells, the latter was induced by lipopolysaccharide. Results: As a result, seven quality markers were screened based on their contents and exposure levels in rat plasma by UPLC–MS/MS, including emodin (EM), liquiritin (LI), 2,3,5,4′–Tetrahydroxystilbene–2–O–β–D–glucoside (TSG), icariin, emodin–8–O–β–D–glucoside, baohuoside I (BA) and 18β–glycyrrhetinic acid (GTA). Moreover, the half maximal inhibitory concentration values of both normal cells and the lipopolysaccharide–induced immune stress liver injury cells were fitted within the concentration range of 0.01–80 μM, based on which, EM, BA, and GTA were identified as the principal hepatotoxic constituents in ABS at elevated concentrations. This study is the first to demonstrate the idiosyncratic hepatotoxicity of TSG, EM, LI, and GTA in normal hepatocytes at low concentrations, whereas EM was also the direct hepatotoxic components. Given that TSG is one of the major ingredients in ABS, the underappreciated idiosyncratic hepatotoxicity could elevate the risk of adverse clinical outcomes. Conclusion: In conclusion, this study identifies effectively hepatotoxic constituents in ABS, and evaluates their hazards under the immune stress condition and the toxicity profiles in clinical concentrations, which also provides a robust foundation for the awareness of the PMRP–induced DILI of ABS.