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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 at low concentrations

Authors
Journal
Journal of Chromatography B
1570-0232
Publisher
Elsevier
Publication Date
Identifiers
DOI: 10.1016/j.jchromb.2012.01.033
Keywords
  • Antidiabetic Agent
  • Onglyza™
  • Saxagliptin
  • Bms-477118
  • 5-Hydroxy Saxagliptin Metabolite
  • Dipeptide-Peptidase
  • Dpp4
  • Lc–Ms/Ms
  • Liquid Chromatography–Tandem Mass Spectrometry
  • Diastereomer
  • Protein Precipitation
  • Human Plasma
  • Cross-Validation
Disciplines
  • Pharmacology

Abstract

Abstract A liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1–50ng/mL for saxagliptin and 0.2–100ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis® dC18 column (50mm×2.1mm, 5μm) for LC–MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at −20°C was demonstrated. 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.

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