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

• Published in: Journal of pharmaceutical and biomedical analysis 2009-09, Vol.50 (2), p.262-266
• Main Authors: Chen, Gong, Fawcett, J. Paul, Mikov, Momir, Tucker, Ian G.
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Caco-2 Cells; Chromatography, High Pressure Liquid - methods; Fundamental and applied biological sciences. Psychology; General pharmacology; Glutamic Acid - analysis; Humans; LC–MS/MS; Medical sciences; Methotrexate - analysis; Methotrexate - metabolism; Methotrexate polyglutamates; Pharmacology. Drug treatments; Reproducibility of Results; Tandem Mass Spectrometry - methods
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2009.04.025

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 × 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.