Determination Of Cyclosporin In Human Whole Blood By RP-HPLC And AxSYM And Study On Their Correlativity

Objectives1. To establish a solid phase extraction reverse phase high performance liquid chromatography (RP-HPLC) method for the determination of cyclosporin in human whole blood.2. To find the difference and correlation of the results between the determinations of RP-HPLC and AxSYM.Methods1. RP-HPLC method: the cyclosporin was extracted from whole blood and was analyzed by HPLC system. Chromatography conditions was as following: chromatography column: Shim-Pack C18 (250mm×4.6mm, 5μm); mobile phase: acetonitrile: water (76:24, v/v); temperature: 68℃; detecting wavelength: 210nm; flow rate: 1.0mL·min-1; Injection volume was 50μL.2. Determining cyclosporin concentration of each blood sample by RP-HPLC system and AxSYM analyzer simultaneously.3. Statistics were performed utilizing SPSS software ver10.0. Both measured value (RP-HPLC method and AxSYM method) was assessed by paired t-test. With RP-HPLC results as X-axis and AxSYM results as Y-axis, a linear regression model was established and a regression analyses was performed with these data for the evaluation of correlation.Results1. Determination of cyclosporin in whole blood by RP-HPLC method. Standard Calibration curve equation was: A=370.4C +4771.5 (r=0.9999,n=6); linear range was covered 50～1000ng·mL-1; the relative recovery rate of cyclosporin was 94.71﹪～98.52﹪with RSD less than 4﹪(n=5); the RSD of within-day less then 2﹪(n=5); the RSD of between-day less then 7﹪(n=5); the limit of detection was 30ng·mL-1 (S/N≥3).2. Total 100 blood samples of patients were measured by RP-HPLC assay and AxSYM assay.3. The results show that the measured value of AxSYM method were significantly higher than the measured value of RP-HPLC method. Both measured value were assessed by t-test, there was a significant difference between RP-HPLC method and AxSYM method, CAxSYM/CRP-HPLC=1.31±0.27 (n=100). With RP-HPLC results as independent variable and AxSYM results as dependent variable, The regression equation for the two methods was AxSYM =0.958X RP-HPLC+48.966, and the correlation coefficient was r=0.971, a good linear correlation relationship was obtained between RP-HPLC method and AxSYM method.Conclusions1. RP-HPLC method for determining cyclosporin in whole blood was accurate, sensitive. It was suitable for monitoring cyclosporin.2. AxSYM method for determination cyclosporin in whole boold was convenience, rapidity and sensitivity. It was suitable for routine monitoring concentration of cyclosporin.3. The RP-HPLC method gave significantly lower values than AxSYM method. It was a good correlation of RP-HPLC method with AxSYM method; you can obtain a estimated value of AxSYM method from the value of RP-HPLC method utilizing the regression model. The conversion formula between the result determined by RP-HPLC and that determined by AxSYM can help physician paying attention to the difference of the determination results based on different determination assay.