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There were no published reports of methods for detecting Enasidenib. In a word, this was the first paper to describe measurement of Enasidenib in rat plasma.Simple sample processing method and short run time.Linearity, precision and accuracy, extraction recovery, matrix effect and stability were validated and met the criteria established by FDA.This method can be used in further pharmacokinetic studies.Enasidenib, an oral product for treating Acute Myeloid Leukemia, has been approved by FDA in Aug, 2017. In this study, we set up an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for measuring Enasidenib and imatinib (internal standard, IS), simultaneously. Enasidenib and imatinib were separated on an ACQUITY UPLC BEH C18 Column (2.1mm×50mm, 1.7μm, 132Å). Mass detection was carried out by electrospray ionization in the position mode, and the multiple reaction monitoring transitions were m/z474.23→456.17 and m/z494.30→394.20 for Enasidenib and imatinib, respectively. Linearity (2−500ng·mL−1, R2>0.999), precision and accuracy (RE<±15%), extraction recovery (≥96.69%), matrix effect (≥96.47%) and stability (RE<±10%) were validated which demonstrated the robustness of our method. This rapid, efficient and reliable UPLC-MS/MS method shows specificity and repeatability of Enasidenib in rat plasma and can be used in further pharmacokinetic studies.