Pharmacokinetic application of a bio‐analytical LC‐MS method developed for 5‐fluorouracil and methotrexate in mouse plasma,brain and urine

In the past we have reported significant cognitive deficits in mice receiving 5‐fluorouracil in combination with low‐dose methotrexate. To explain such interactions, a pharmacokinetic study was designed. A sensitive bio‐analytical method was therefore developed and validated for 5‐fluorouracil and methotrexate in mouse plasma, brain and urine with liquid chromatography coupled to a single quadrupole mass spectrometer. Chromatographic separation was accomplished by Agilent® Zorbax® SB‐C₁₈ column, with isocratic elution (5 mM ammonium acetate and methanol, 70:30, %v/v) at a flow rate of 300 μL/min. The limit of quantitation for both drugs was 15.6 ng/mL (plasma and brain) and 78.1 ng/mL (urine), with interday and intraday precision and accuracy ≤15% and a total run time of 6 min. This bio‐analytical method was used for the pharmacokinetic characterization of 5‐fluorouracil and methotrexate in mouse plasma, brain and urine over a period of 24 h. This method allowed characterization of the brain concentrations of 5‐fluorouracil over a period of 24 h. Copyright © 2013 John Wiley & Sons, Ltd.