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The method is characterized by comprising the following steps: in an ultra-high performance liquid chromatograph, respectively mixing a mobile phase A and a mobile phase B according to different proportions to obtain a mobile phase, and carrying out gradient elution on a semaglutide sample, wherein the mobile phase A is selected from a mixed aqueous solution of one or more of trifluoroacetate, perchlorate and hexafluorophosphate, and the pH value is adjusted to be 2-4, and the mobile phase Bis selected from methanol, isopropanol and acetonitrile