Identification and quantitation of methotrexate and methotrexate metabolites in clinical high-dose therapy by high pressure liquid chromatography and field desorption mass spectrometry.

Abstract

High-pressure liquid chromatography in combination with field desorption mass spectrometry as techniques of high specificity and sensitivity have been applied to the identification and quantitation of the anticancer drug methotrexate and its metabolites which occur in clinical high-dose therapy. Field desorption mass spectra of methotrexate and several methotrexate and folic acid derivatives, when investigated as free acids or ammonium salts, yield abundant protonated molecular ions and a consistent pattern of structurally significant fragments. High-pressure liquid chromatographic separation of methotrexate metabolites was performed on reverse-phase, C-18 columns using a volatile, ammonium bicarbonate/acetate containing mobile phase that was especially suited for the field desorption mass spectral analysis of isolated metabolites, and provided the definite identification of 7-hydroxymethotrexate and 4-[[2,4-diamino-6-pteridinyl]methyl]methylamino]-benzoic acid in serum and urine of patients treated with high-dose methotrexate. The high intensity and stability of the [MH]+ ions was found suitable for the quantitation of methotrexate and related folate analogues by field desorption mass spectrometry. A synthetic methotrexate derivative, methotrexate-gamma-(2-hydroxy)ethyl-amide was used as internal standard for the quantitative determination of methotrexate in serum and urine. In a study to comparatively assess the potential of specific quantitation methods, serum and urine levels of methotrexate and its major metabolite, 7-hydroxymethotrexate were determined by (i) an enzyme immunoassay, (ii) reverse phase high-pressure liquid chromatography and (iii) field desorption mass spectrometry. Results obtained from four patients with osteogenic sarcoma receiving high-dose methotrexate/leucovorin rescue therapy consistently show the sustained elimination of 7-hydroxymethotrexate over several days, thus indicating the utility of specifically monitoring this nephrotoxic metabolite, at massive methotrexate doses.