Liquid chromatography and tandem mass spectrometry method for the quantitative determination of saxagliptin and its major pharmacologically active 5-monohydroxy metabolite in human plasma: Method validation and overcoming specific and non-specific binding

► A liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin and its major active metabolite, 5-hydroxy saxagliptin. ► The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. ► Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. ► The assay has been used to support multiple clinical studies and regulatory approvals.