title: Development of an Assay for Methotrexate and Its Metabolites 7-Hydroxy Methotrexate and DAMPA in Serum by LC-MS/MS.
abstract: Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity. We describe a rapid and simple turbulent flow liquid chromatography (TFLC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of MTX, 7-hydroxymethotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 μL) after protein precipitation with a methanolic solution containing internal standard (MTX-D3) followed by centrifugation. The supernatant is injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFLC-ESI-MS/MS) and quantified using a six-point calibration curve. For MTX, 7-OH MTX, and DAMPA the assays were linear from 20 to 1000 nmol/L. Dilutions of 10-, 100-, and 1000-fold were validated giving a clinically reportable range of 20 to 1.0 × 10(6) nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Schofield
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.
authors.forename: Lakshmi V
authors.initials: LV
authors.lastname: Ramanathan
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Murata
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Fleisher
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Pessin
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA. carlowd@mskcc.org.
authors.forename: Dean C
authors.initials: DC
authors.lastname: Carlow
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000097:blood* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D016903:Drug Monitoring / Q000379:methods; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood* / Q000378:metabolism; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000097:blood* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000964:Antimetabolites, Antineoplastic; D007166:Immunosuppressive Agents; C011864:7-hydroxymethotrexate; D008727:Methotrexate
keywords: Carboxypeptidase-G2; Mass spectrometry; Methotrexate; Therapeutic drug monitoring; Turbulent flow liquid chromatography