OBJECTIVE To establish an automatic,rapid and accurate method for the determination of methotrexate concentration in human blood.METHODS A double-temary liquid chromatography system with ultraviolet detector in series was used.Athena C18-WP(4 mm×20 mm,5 μm)column was used as the on-line SPE column,and Athena C18-WP(4.6 mm×150 mm,5 μm)column was used as the analysis column.The mobile phase was 10 mmol·L-1 ammonium acetate,the stationary phase was acetonitrile,and the flow rate was 1 mL·min-1.The detection wavelength was 306 nm.The column temperature was 40 C.The injection volume was 100 μL.Serum samples were injected after protein precipitation with a precipitating reagent(5％ trichloroacetic acid+10％zinc sulfate).The specificity,standard curve and lower limit of quantitation,precision and accuracy,recovery rate and stability of the method were investigated.RESULTS Methotrexate had good linearity in the range of 0.078-8.000 μmol·L-1(R2=0.999 4).The recoveries of methotrexate were 86.41％to 87.09％,RSD of intra-day and intra-day were lower than 7％.CONCLUSION This method is accurate,reproducible,simplified the steps of sample pretreatment and improved the analysis efficiency.It is suitable for the detection of blood drug concentration in high dose methotrexate treatment.
维普
万方数据