Plasma levels of methotrexate in cancer patients as studied by an improved spectrophotofluorometric method

Abstract An improved spectrophotofluorometric method for the determination of methotrexate (MTX) in biological samples has been developed with special emphasis on the analysis of human plasma. A major feature of the procedure is the extraction of MTX into n -butanol in the presence of TCA. 3 Following reextraction into an aqueous phase, the drug was oxidized using potassium permanganate to yield products whose fluorescence was linear over a wide range of plasma concentrations. Conditions such as pH, length of oxidation, stability of fluorescence, and concentration of appropriate reagents were studied to optimize partition coefficients and RFI. 3 Plasma recoveries ranged from 85 to 97% with a usual value of 93%. Drugs which interfered with previous methods (sodium hypaque, EDTA, sodium tungstate, heparin) did not interfere in the present method. MTX could be determined in the presence of Ara-C and triamterene. Compared to previous spectrofluorometric methods we obtained a more than 10-fold higher fluorescence using equal aliquots of plasma. Readings were based on a direct measurement rather than an increment and the lower limit of reliable measurement was 0.1 mg/liter of MTX in plasma. Only readings with a sample: “blank” ratio of at least 2 were considered significant. This method was employed to study plasma levels of MTX in cancer patients.