title: Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum.
abstract: A rapid and simple turbulent flow liquid chromatography (TFC-LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC-LC-MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000nmol/L and for 7-OH MTX from 20 to 2000nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10nmol/L to 5×10(5)nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC-LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC-MS/MS assay.
authors: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors: Ryan C
authors: RC
authors: Schofield
authors: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors: Lakshmi V
authors: LV
authors: Ramanathan
authors: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors: Kazunori
authors: K
authors: Murata
authors: Department of Pathology and Laboratory Medicine, Mount Sinai School of Medicine, New York, NY, United States.
authors: Marie
authors: M
authors: Grace
authors: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors: Martin
authors: M
authors: Fleisher
authors: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors: Melissa S
authors: MS
authors: Pessin
authors: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States. Electronic address: carlowd@mskcc.org.
authors: Dean C
authors: DC
authors: Carlow
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000097:blood*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C000203:deoxyaminopteroic acid; C011864:7-hydroxymethotrexate; D008727:Methotrexate
keywords: 7-Hydroxy methotrexate; Carboxypeptidase-G2; DAMPA; Dihydrofolate reductase inhibition assay; Fluorescence polarization immunoassay; Mass spectrometry; Method correlation; Methotrexate; Therapeutic drug monitoring; Turbulent flow liquid chromatography