Analysis of Methotrexate Polyglutamate Derivatives in Vivo

This chapter focuses on the methodology for the extraction, separation, and quantitation of methotrexate polyglutamates, methotrexate (MTX)( glutamate, Glu_n) formed in vivo. The method is based on a radioligand binding assay for MTX (and polyglutamates) detection and either an isocratic phosphate buffer solution with a molecular sieve column or a newer methodology using a C₁₈ μ-Bondapak column to effect separation of MTX(Glu_n). These procedures have been used to quantitate MTX polyglutamates in clinical samples and to evaluate the pharmacodynamics of MTX given to laboratory animals for periods up to 1 year. The I-60 column is placed in line with the U6K injector and the M45 solvent delivery system with a guard column. The column is equilibrated in 0.1 M phosphate buffer (pH 7). Buffers are degassed and filtered prior to use. The flow rate is 1.0 ml/min (pressures 500–1000 psi are generated). The I-60 column appears to retain the lower chain length methotrexate derivatives. Thus, methotrexate elutes significantly later than where it should by strict molecular sieve chromatography. This allows more complete separation between the lower and higher methotrexate polyglutamates.