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 of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Ryan C SchofieldDean C Carlow

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-...Continue Reading

References

Apr 1, 1980·Cancer Letters·J LankelmaF Ramaekers
Jun 24, 2006·The Oncologist·Brigitte C Widemann, Peter C Adamson
Apr 10, 2010·Analytical and Bioanalytical Chemistry·Alida A M StolkerCláudia P B Martins

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Citations

May 11, 2016·Clinical Biochemistry·Maria Shipkova, Dobrin Svinarov
Oct 21, 2016·Journal of Separation Science·Bruno Henrique FumesFernando Mauro Lanças
Jun 1, 2017·The Analyst·Nicolas GuichardSandrine Fleury-Souverain
Jun 20, 2019·Biomedical Chromatography : BMC·Merel van NulandJos H Beijnen
Aug 4, 2019·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Ryan C SchofieldDean C Carlow
Jan 19, 2021·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Yurong SongYuanyan Liu
Jun 30, 2021·Annals of Clinical Biochemistry·Malcolm P McTaggart, Brain G Keevil

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