A simplified fluorometric method for determination of plasma methotrexate.

A FLUOROMETRIC METHOD for the determination of pteroylglutamic acid (PGA) in nonbiologic material was reported by Alifrey et al (1). Freeman (2) adapted this method for the determination of amethopterm in plasma. Both procedures oxidized PGA or amethopterin with permanganate to yield a strongly fluorescent substance, 2-amino-4hydroxypteridine-6-carboxylic acid (3). The fluorescence due to the latter compound is much more intense than that of PGA (1-3). The method involved measuring fluorescence before oxidation and then after oxidation of the compound with permanganate. The difference between the two fluorescence readings was proportional to the concentration of PGA or amethopterin present. Our laboratory attempted to establish a relationship between chromosome aberration rates in leukocytes and the methotrexate (MTX) level in plasma of psoriatic patients maintained on a MTX regimen (4). For this study, we attempted to duplicate Freeman’s method (2) for the determination of plasma MTX levels. The results were not reproducible, especially at concentrations less than 20 mg/mI. However, when the trichloroacetic acid-treated plasma-MTX mixtures were heated at 1000 prior to filtering the precipitated plasma proteins, only a single fluorescence reading after oxidation was necessary to obtain linearity between concentration of MTX and fluorescence.