Objective: To develop a HPLC method to determine the concentration of methotrexatate (MTX) in human plasma. Method: The ferulic acid was used as inter standard (I.S.) The analytical column was a Kromasil C18 column connected with the Agilent 1100 HPLC system. The isocratic mobile phase was the acetonitrile and buffer of phosphate acid (pH 6.8) with the rate of 15 ∶85.The detective UV wavelength was 306 nm and the column temperature was 30 ℃. The plasma samples

added ferulic acid as internal standard

were deposited down the protein with acetonitrile and the upper phase was evaporated by nitrogen at 60 ℃. The remains was dissolved with mobile phase

20 μL was injected into the sampler. Result: The chromatography was good and not interfered by the components of the plasma. The linear equation was Y=-1.65×10-2X+0.355

r=0.999 5. Conclusion: This is a accurate

sensitive and conventient method for the dertermination of MTX in the therapeutic drug monitoring.