目的:建立UPLC/Q-TOF-MS法鉴定市售凝血酶冻干粉中的蛋白组分并进行定量。方法:样品经胰蛋白酶消化后的多肽混合物,用超高效液相色谱进行分离,并在线进入ESI-Q-TOF-MS检测,获得的肽段一级母离子和碎片离子的高分辨准确质量数据(MSE方法),经ProteinLynx Global Server(PLGS 2.4)软件蛋白质谱库检索,确定其中的蛋白组分,再以Hi3非标记蛋白定量方法对各蛋白组分进行定量,得到质量百分比定量结果。结果:在以猪血为原料生产的26批凝血酶冻干粉中,鉴定出人血白蛋白和21种猪源蛋白,其中,有1家企业的3批样品中鉴定出猪和人血浆成分,其余23批次样品均为猪血浆成分;样品中的猪凝血酶含量在1.9%~43.7%之间。在以牛血为原料生产的3批凝血酶冻干粉中共鉴定出17种牛源蛋白;样品中的牛凝血酶含量在49.4%~77.6%之间。结论:UPLC/Q-TOF-MS法灵敏度高,选择性好,通过一次分析可对凝血酶冻干粉中蛋白组分同时进行定性和定量,并可获得有关工艺相关的数据信息如来源和种属等,可用于本品的工艺研究以及质量控制研究。该法可以作为同类生化药物质量研究和组分分析的方法。 OBJECTIVE: To establish a UPLC / Q-TOF-MS method for the identification of protein components in the commercial freeze-dried thrombin powder and quantification. METHODS: Samples were digested with trypsin and separated by ultra performance liquid chromatography (HPLC). The results showed that ESI-Q-TOF-MS was used to detect the high-resolution and accurate mass spectra of primary and secondary ions (MSE method). The proteins were searched by ProteinLynx Global Server (PLGS 2.4) software, and the protein components were determined. The protein components were quantified by Hi3 unlabeled protein quantitative method, and the mass percent quantitative results were obtained. Results: Among 26 batches of thrombin lyophilized powder produced from porcine blood, human serum albumin and 21 kinds of porcine source proteins were identified. Among them, pigs and human plasma components were identified in 3 batches of samples from 1 enterprise, The remaining 23 batches of samples were porcine plasma components; swine thrombin content in the sample was between 1.9% to 43.7%. Seventeen kinds of bovine protein were identified in 3 batches of thrombin lyophilized powder with bovine blood as raw material. The content of bovine thrombin in samples was between 49.4% and 77.6%. Conclusion: The UPLC / Q-TOF-MS method has high sensitivity and good selectivity. Through one analysis, the protein components in thrombin lyophilized powder can be qualitatively and quantitatively analyzed at the same time, and relevant process information such as source and species Is such as, can be used for the product of technology research and quality control research. The method can be used as a biochemical drug quality research and component analysis methods.