Objective: To establish a HPLC method for determination of methotrexate concentration in plasma using higher doses. Method: The separation was performed in a Gold column(4.6 mm×150 mm

5 μm) with a mobile phase of methanol: phosphate buffer (19∶81

pH 7.2) and determinated at 306 nm.Result: The measurable lowest concentration was 0.002 μmol·L-1. The experiment showed that the recovery from plasma was 90.2%

94.4%

101.2%. The RSD of within day and between day was less than 9.0%. A good linearity was found in the range of 0.01～1.38 μmol·L-1 (r=0.999 7) and in the range of 1.38～103.5 μmol·L-1 (r=0.999 5).Conclusion: The method is simple

sensitive and accurate with wide linear dynamic range.