title: 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.
abstract: 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.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.The analytes were extracted by using liquid-liquid extraction procedure and separated on a reverse phase C18 column (50 mm×3 mm i.d., 4.6 µ) using methanol: 2 mM 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.The method was validated over the concentration range of 1-1 000 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.The quantitation method was successfully applied for simultaneous estimation of methotrexate, dasatinib and N- deshydroxyethyl dasatinib in a pharmacokinetic study in Wistar rats.
authors.affiliation: Incozen Therapeutics Private Limited, Andhra Pradesh, India. satheesh.trs@gmail.com
authors.forename: S R S
authors.initials: SR
authors.lastname: Thappali
authors.forename: K V S
authors.initials: KV
authors.lastname: Varanasi
authors.forename: S
authors.initials: S
authors.lastname: Veeraraghavan
authors.forename: S K V S
authors.initials: SK
authors.lastname: Vakkalanka
authors.forename: M
authors.initials: M
authors.lastname: Khagga
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000097:blood* / Q000493:pharmacokinetics*; D019540:Area Under Curve; D001711:Biotransformation; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D000069439:Dasatinib; D004347:Drug Interactions; D006207:Half-Life; D007202:Indicators and Reagents; D008297:Male; D008727:Methotrexate / Q000097:blood* / Q000493:pharmacokinetics*; D047428:Protein Kinase Inhibitors / Q000097:blood* / Q000493:pharmacokinetics; D011743:Pyrimidines / Q000097:blood* / Q000493:pharmacokinetics*; D011786:Quality Control; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D013844:Thiazoles / Q000097:blood* / Q000493:pharmacokinetics*
chemical_list: D000964:Antimetabolites, Antineoplastic; D007202:Indicators and Reagents; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D000069439:Dasatinib; D008727:Methotrexate