High-performance Liquid Chromatographic Analysis of MTX, 7-OH-MTX and MTX Derivatives: Application to Intracellular Metabolism in Tumor Cells (HT 29)

In order to study the intracellular metabolism of Methotrexate (MTX) and the cytotoxicity of the anti-folates, a specific paired-ion HPLC method has been developed which permits the simultaneous determination of DAMPA, MTX, 7-OH-MTX, MTX-G1, MTX-G2 and MTX-G3. Cells were incubated with H-3-MTX. The MTX metabolites were extracted, purified on SEP-PAK cartridges and further analysed by high-performance liquid chromatography (HPLC). The stationary phase was constituted by a C-18 mu Bondapak and the mobile phase by 5 mM phosphate buffer (pH 7.4) containing 2.5 mM tetrabutylammonium nitrate. The elution was performed with a linear methanol gradient (20-30%). HPLC fractions were collected and radioactivity evaluated by beta counting (retention times: DAMPA = 12.93 min; MTX = 18.29 min; 7-OH-MTX = 21.13 min; MTX-G1 = 22.69 min; MTX-G2 = 26.81 min; MTX-G3 = 30.61 min). This analytical procedure was applied to separate and characterize multiple forms of MTX polyglutamate derivatives in HT 29, a human adenocarcinoma cell line varying the incubation time (4-18 h) and MTX concentration (0.6-10.6 PM). The incorporation process seems to be characteristic of a cell line resistant to MTX. The incorporation were very low and after a 4-h exposure time only 5% of the MTX was converted to polyglutamates. Between 1.6 and 10.6 mu M MTX, no difference was observed in the polyglutamization. The defect in the incorporation of the drug and in the metabolization process in vitro could partially explain the failure of the MTX treatment in colorectal cancer.