Determination of Methotrexate Concentration in Human Plasma by HPLC

OBJECTIVE To develop an HPLC method to determine the concentration of methotrexate(MTX) in human plasma. METHODS Theophyline was used as the internal standard. The analytical column was Agilent Eclipse XDB-C₁₈(4.6 mm×250 mm, 5 μm). The mobile phase was phosphate buffer solution with pH 6.6-methanol(82∶18). The UV determine wavelength of MTX was 306 nm and theophyline was 254 nm. The flow rate was 1.0 mL·min^-1, and the column temperature was 30 ℃. RESULTS The calibration curves of MTX were linear at a concentration range from 0.03 to 1.65 μmol·L^-1(r=0.999 9) and 1.65 to 66.08 μmol·L^-1(r=0.995 8). The intra-day and inte-day precision were both less than 5%. The average method recovery was 97.6% and the average extraction recovery was 68.5%, with a lowest limit of 0.03 μmol·L^-1. CONCLUSION The method is sensitive, accurate, wide linear range, suitable for plasma concentration monitoring of MTX.