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With the rising interest in covalent drugs, the likelihood of covalent protein binding has significantly increased. We established a workflow forMetID in proteinresidues, including sample preparation, LC-MS analysis,and data processing. Using Osimertinib as a model, we found that low extraction recovery rates in plasma, liver microsomes, and hepatocytes were due to covalent binding with matrix proteins. Protein-bound products of Osimertinib were detected in these matrices. This method supports the study of drug covalent binding, offering a more comprehensive metabolite profile without the need for radiolabeled compounds.
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