Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable significance in determining the efficacy and safety of traditional Chinese medicine (TCM) in vivo. However, this process is usually hindered by the insufficient characteristic fragments of metabolites, ubiquitous matrix interference, and complicated screening and identification procedures for metabolites. In this study, an effective strategy was established to systematically characterize the metabolites, deduce the metabolic pathways, and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo. The strategy was divided into five steps. First, the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry (MS) system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites. Second, an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites, which reduced approximately 39% of the interfering ions. Third, an acquisition model was developed and used to trigger more tandem MS (MS/MS) fragments of precursor ions based on the targeted ion list. The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode. Fourth, the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction. The main in vivo metabolic pathways of bufadienolides were elucidated. A total of 147 metabolites were characterized, and the main biotransformation reactions of bufadienolides were hydroxylation, dihydroxylation, and isomerization. Finally, the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS, and the metabolic profiles were clearly identified. This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.

血浆中代谢物和代谢谱的综合表征对于确定中药 (TCM) 在体内的疗效和安全性具有重要意义。然而，这一过程通常受到代谢物特征片段不足、普遍存在的基质干扰以及代谢物筛选和鉴定程序复杂等因素的阻碍。在这项研究中，建立了一种有效的策略来系统地表征代谢物，推断代谢途径，并描述从 Venenum Bufonis 体内分离的蟾蜍二烯内酯的代谢特征。该策略分为五个步骤。第一的，将空白和测试血浆样品以全扫描模式连续五次注入超高效液相色谱/线性阱四极杆-轨道阱-质谱（MS）系统，以筛选有效的基质化合物和代谢物。其次，建立了扩展质量缺陷过滤器模型，以获得蟾蜍二烯内酯代谢物列表中的目标前体离子，从而减少了大约 39% 的干扰离子。第三，开发了一种采集模型，并用于根据目标离子列表触发更多前体离子的串联 MS (MS/MS) 碎片。采集模式将采集能力提高了大约是常规数据相关采集模式的四倍。第四，将采集的数据导入 Compound Discoverer 软件，通过代谢网络预测识别代谢物。阐明了蟾蜍二烯内酯的主要体内代谢途径。共表征了 147 种代谢物，蟾蜍二烯内酯的主要生物转化反应为羟基化、二羟基化和异构化。最后，利用 LC-MS/MS 测定了血浆中不同时间点的主要原型蟾蜍二烯内酯，并明确了代谢谱。该策略可广泛用于阐明中药制剂或中成药的体内代谢谱，为合理用药提供关键数据。蟾蜍二烯内酯的主要生物转化反应是羟基化、二羟基化和异构化。最后，利用 LC-MS/MS 测定了血浆中不同时间点的主要原型蟾蜍二烯内酯，并明确了代谢谱。该策略可广泛用于阐明中药制剂或中成药的体内代谢谱，为合理用药提供关键数据。蟾蜍二烯内酯的主要生物转化反应是羟基化、二羟基化和异构化。最后，利用 LC-MS/MS 测定了血浆中不同时间点的主要原型蟾蜍二烯内酯，并明确了代谢谱。该策略可广泛用于阐明中药制剂或中成药的体内代谢谱，为合理用药提供关键数据。