多肽类非法添加物瑞他鲁肽的筛查测定

Screening and Determination of Illegally Added Polypeptide Retatrutide

关键词:  胰高血糖素样肽-1受体激动剂  四级杆飞行时间质谱  瑞他鲁肽  非法添加

Key words:  GLP-1RAs  HPLC-QTOF  Retatrutide  illegally added drugs

摘要:

目的 建立多肽类非法添加物瑞他鲁肽的筛查策略和流程。方法 采用Tricine-SDS-PAGE对样品进行初步筛查。然后利用HPLC-QTOF建立“自下而上”和“自上而下”的分析策略，分别在酶切肽段水平上进行结构的推测和在完整分子水平上的结构确认。以Agilent EC- C18(3.0 mm × 100 mm，2.7 μm)为色谱柱，流动相 A 为 0.1% 甲酸水溶液，流动相 B 为 0.1%甲酸乙腈溶液，梯度洗脱，流速0.3 ml/min；采用正离子 ESI+ 离子源进行数据采集。结果 通过凝胶电泳确定了多肽类物质的存在，利用“自下而上”的分析策略推测了瑞他鲁肽结构，并对修饰侧链位点进行定位。最终通过“自上而下”的分析策略，利用对照品对结构进行确认。结论 建立了瑞他鲁肽筛查方法,筛查流程准确、可靠，既能用于 GLP-1激动剂的一级结构的表征分析，也能用于未知多肽类非法添加物的筛查。

Abstract:

Objective To develop the strategy for screening and determining polypeptide Retatrutide using Tricine-SDS-PAGE and HPLC-QTOF. Method Tricine-SDS-PAGE was performed as the first rudimentary screening. Subsequently, "bottom-up" and "top-down" analytical strategies were established via HPLC-QTOF for identify the masses of the peptide fragments and the intact peptide. Separation was achieved on an Agilent EC-C18 column (3.0 mm × 100 mm, 2.7 μm) using a mobile phase consisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile with gradient elution at a flow rate of 0.3 mL/min. Data were acquired using a positive ion ESI+ ion source. Results The presence of the polypeptide substance was confirmed by Tricine-SDS-PAGE. The sequence of Retatrutide was deduced using the "bottom-up" strategy, which was also useful for identifying the fatty acid chain modification site. Full sequence coverage of retatrutide was accomplished by combining peptide maps generated using chymotrypsin. Ultimately, the structure was confirmed using a "top-down" analytical strategy with a reference standard, demonstrating strong specificity and good reproducibility. Conclusion The method makes up for the lack of test methods for Retatrutide.The workflow is accurate and reliable, and can be applied for the characterization of primary structures of GLP-1RAs analogues as well as for the screening of unknown illicit peptide additives.