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 Journal Article
| Authors: | Schofield, R. C.; Ramanathan, L. V.; Murata, K.; Grace, M.; Fleisher, M.; Pessin, M. S.; Carlow, D. C. |
| Article 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-N10-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-D<inf>3</inf>) 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 5×105nmol/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. © 2015 Elsevier B.V. |
| Keywords: | controlled study; unclassified drug; methotrexate; mass spectrometry; accuracy; fluorescence; patient monitoring; standard; immunology; quantitative analysis; tandem mass spectrometry; body fluids; drug metabolite; turbulent flow; dihydrofolate reductase; liquid chromatography; polarization; assays; carboxypeptidase; methanol; electrospray; drug products; ionization; spectrometry; fluorescence polarization immunoassay; chromatography; precipitation; liquids; priority journal; article; formic acid; therapeutic drug monitoring; 7-hydroxy methotrexate; carboxypeptidase-g2; dampa; dihydrofolate reductase inhibition assay; method correlation; turbulent flow liquid chromatography; electrospray ionization; high pressure liquid chromatography; ionization of gases; ionization of liquids; inhibition assays; 4 amino 4 deoxy n 10 methylpteroic acid; 7 hydroxy methotrexate; enzyme inhibition assay; turbulent flow chromatography |
| Journal Title: | Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences |
| Volume: | 1002 |
| ISSN: | 1570-0232 |
| Publisher: | Elsevier Science BV |
| Date Published: | 2015-10-01 |
| Start Page: | 169 |
| End Page: | 175 |
| Language: | English |
| DOI: | 10.1016/j.jchromb.2015.08.025 |
| PROVIDER: | scopus |
| PUBMED: | 26322588 |
| PMCID: | PMC4982141 |
| DOI/URL: | |
| Notes: | Export Date: 2 October 2015 -- Source: Scopus |
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33Murata -
312Fleisher -
77Ramanathan -
29Schofield -
42Carlow
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