An improved high-performance liquid chromatography method for quantification of methotrexate polyglutamates in red blood cells of children with juvenile idiopathic arthritis

Abstract

Simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation/emission wavelengths of 370/463 nm) was developed for quantition of the sum of methotrexate polyglutamates in erythrocytes. Polyglutamates are determined as sum of all forms after their enzymatic conversion with gamma-glutamylhydrolase to methotrexate.

Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6 ml/min and consisting of a mixture (110:890:0.25 v/v) of acetonitrile, ammonium acetate (0.05 M, pH=5.5) and hydrogen peroxide 30% (w/w). The method is linear over the concentration range of 25-400 nmol/l.

Its intra- and inter-day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9 nmol/l, respectively