Determination of plasma concentration of phenytoin and phenobarbital by HPLC

Objective:To determine the method for plasma concentration of phenytoin and phenobarbital by HPLC.Methods:After vortex shaking and high-speed centrifugation,20 μl serum was determined by HPLC.The column was ZORBAX SB-C18(4.6 nm×250 nm,5 μl),the mobile phase was potassium dihydrogen phosphate buffer(pH=3.5)-methanol(40:60)(V/V);the detection wavelength was λ=210 nm,the flow rate was 1 ml·min-1 and the column temperature was 25 ℃.Results:Blood concentrations of phenytoin and phenobarbital were within the detection ranges of 2.62-23.58 μg·ml-1 and 6.24-56.12 μg·ml-1(r=0.9991,0.9992),with all in good linear relationship.The ratios of average recovery were 98.7% and 98.6% respectively,and both precisions within a day and among days were less than 4.0%.Conclusion:This method is accurate,reliable,simple,fast and suitable for determination of plasma concentration of phenytoin and phenobarbital.