The
folate antagonist
methotrexate is commonly used in low dose for treatment of
rheumatoid arthritis and
juvenile idiopathic arthritis.
Therapeutic effects are attributed to intracellular levels of various
methotrexate polyglutamates. The present methodology, combining a simple preparation step with ion-pairing reversed-phase liquid chromatography and electrospray ionization mass spectrometry, is suitable for the measurement of
methotrexate and its polyglutamates(2-7), in human red blood cells. Sample preparation consists of
perchloric acid protein precipitation followed by solid-phase extraction. Baseline separation of all analytes was achieved within 10 min using a Phenomenex Synergy C18 column together with a gradient
solvent program obtained from blending
acetonitrile with pH 7.5, 5 mM aqueous dimethylhexylamine. Seven
methotrexate polyglutamates were detected using multiple reaction monitoring, with the mass spectrometer operating in positive ion mode. Using 20 microL injection volumes, limits of detection were 2.5 nM for individual
methotrexate polyglutamates, while large volume (100 microL)
injections led to detection limits of 0.5 nM and linear calibration from 0.5 to 100 nM for individual analytes. Finally, the presented methodology was applied for the analysis of
methotrexate and its polyglutamates in red blood cells obtained from patients being treated for
juvenile idiopathic arthritis with
methotrexate. Significantly, the methodology proved suitable for determination of long-chain
methotrexate polyglutamates(5-7) and further, appears to be superior with respect to sensitivity, selectivity and speed as compared to all previously described approaches.