Simultaneous Determination of Methotrexate, Dasatinib and its Active Metabolite N- Deshydroxyethyl Dasatinib in Rat Plasma by LC-MS/MS: Method Validation and Application to Pharmacokinetic Study

Background: Dasatinib is a multi-kinase inhibitor that potently inhibits Bcr-Abl, Src family and platelet-derived growth factor receptor kinases. Methotrexate is an antimetabolite and antifolate drug. Clinical trials utilizing a combination of dasatinib and methotrexate in patients with Philadelphia chromosome positive and/or Bcr-Abl positive acute lymphoblastic leukemia are currently ongoing. A need therefore exists to develop a sensitive analytical method for determination of dasatinib and methotrexate in plasma. Objective: To estimate methotrexate, dasatinib and its active metabolite N-deshydroxyethyl dasatinib simultaneously using liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in Wistar rat plasma. Method: The analytes were extracted by using liquid-liquid extraction procedure and separated on a reverse phase C18 column (50 mmx3 mm i.d., 4.6 mu) using methanol: 2mM ammonium acetate buffer, pH 4.0 as mobile phase at a flow rate 1 mL/min in gradient mode. Selective reaction monitoring was performed using the transitions m/z 455.0>175.0, 488.1 >401.0, 444.26>401.0, and 271.1 >- 155.0 to quantify methotrexate, dasatinib, N-deshydroxyethyl dasatinib and tolbutamide respectively. Results: The method was validated over the concentration range of 1-1000 ng/mL and the lower limit of quantitation was 1 ng/mL. The recoveries from spiked control samples were >79% for all analytes and internal standard Intra- and Interday accuracy and precision of validated method were within the acceptable limits of <15% at all concentration. Conclusion: The quantitation method was successfully applied for simultaneous estimation of methotrexate, dasatinib and N- deshydroxyethyl dasatinib in a pharmacokinetic study in Wistar rats.