Determination of N-Phenyl-phthalamic Acid in Biological Samples by HPLC
WEI Si-hua;DAI Ling-ling;ZHOU Feng;LI Long;Department of Health Toxicology,Huazhong University of Science and Technology;
[Aims] The HPLC method for determination of N-phenylphthalamic acid in murine serum and tissue samples was established to provide reference for further study. [Methods] The blank biological samples which contained N-phenylphthalamic acid were detected by HPLC-UV, meanwhile the chromatographic condition was optimized to realize the separation of the drug chromatographic peak and impurity peak. The chromatographic conditions were: chromatographic column was XB- NH_2 column, wavelength was 254 nm, column temperature was 40 ℃, sample size was 10 μL and flow rate was 1 mL/min. Serum and tissue samples were eluted with different mobile phase. The mobile phase for serum samples was 65% of acetonitrile and 35% of water. The water phase contained 0.4% of formic acid. The mobile phase for tissue samples was 80% of acetonitrile and 20% of water, the water phase contained 0.2% of formic acid. According to the chromatographic condition, the accuracy, reproducibility and recovery were tested.[Results] The extra-day and intra-day accuracy variable coefficient was below 2%, and the reproducibility was below5%. The recovery rates was between 85.2 and 97.5%. [Conclusions] The method has high stability and reproducibility.It could conform to demand of the study and reference for the analogous experiment. The results of the preliminary experiment suggested that with increasing the volume of the organic phase, the N-phenylphthalamic acid would retain more long in the chromatographic column. In addition, pH value of water phase can influence the shape of chromatographic peak of the drug.