Simultaneous determination of methotrexate and its polyglutamate metabolites in Caco-2 cells by liquid chromatography-tandem mass spectrometry.

In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu(n), n=2-5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu(n) in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2 x 10(6) cells) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the M+H ions of MTX and MTXGlu(n) to their common product ion at m/z 308.2 and of aminopterin at m/z 441.3-->294.2. Calibration curves for all analytes were linear in the range 2-250 nM (r(2)>0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4-15.1% and 4.3-18.4%, respectively with corresponding accuracies (as relative error) of -3.6 to +6.6% and -5.5 to +7.5%, respectively. Recoveries were in the range 60+/-4 to 108+/-13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24 h.