The measurement of methotrexate polyglutamate metabolites in red blood cells has potential to aid in individualization of methotrexate therapy in rheumatoid arthritis and juvenile idiopathic arthritis. In this report a method is presented for rapid analysis of these metabolites in human red blood cells. The analytical procedure is a simple “one pot” pre-column reaction involving the addition of sodium dithionite as a reducing agent followed by 15 minutes of boiling. After centrifugation the supernatant is introduced into a conventional HPLC system equipped with a fluorescence detector, without the need for further workup. By performing the derivatization reaction pre-column, a time consuming (6–14 h) commonly used deglutamation procedure utilizing blank human plasma, becomes obsolete. Using the described procedure the total sample preparation time for a 50 sample run should not exceed 1–1.5 hours. The chromatographic run time per sample is 7 minutes using isocratic elution conditions. The method was found to be linear over the clinical relevant concentration range of 10–500 nM of intra-cellular methotrexate polyglutamates. The intra-run mean accuracy of the target value was between 98.1% and 106.0%. The intra-run precision was between 1.2% and 8.8%.
