| Objectives: To establish a HPLC method for the determination of cefoselis sulfate in rats. To investigate the pharmacokinetic characteristics of cefoselis sulfate, after injected administration singly in rats or co-administration with foscarnet sodium, and to evaluate the influence of foscarnet sodium on the pharmacokinetics of cefoselis sulfate in rats.Methods:(1) Chromatographic conditions: A Diamonsil C18 column(250 mm×4.6 mm,5 μm) was selected and the mobile phase was acetonitrile and water(12:88, V/V) with a flow rate of 1 ml·min-1. The detection wavelength was set at 210 nm, and the column temperature was 30℃, the metronidazole was selected as internal standard, and the injection volume was 20 μl.(2) Plasma samples processing: 200 μl of plasma samples was added to a tube, 20 μl of internal standard and 4% perchloric acid were added and mixed thoroughly. 150 μl of supernatant was diluted with 600μl of purified water and 20 μl was injected into the chromatography systems.(3) Calibration curve: Calibration curve of plasma samples of cefocelis sulfate in series concentration of 5, 10, 25, 50, 100, 200, 400, 800, 1600 and 2000 mg·L-1, which were consist of Group Ⅰ(5~100 mg·L-1) and Group Ⅱ(100~2000 mg·L-1), were prepared, processed according to the method described above, and 20μl was injected into the chromatography systems. Calibration curve was drawn based on the peak area ratio of cefoselis sulfate to internal standard(Y) versus the plasma concentration of cefocelis sulfate(X), which was obtained two equation-Equation Ⅰ and Equation Ⅱ.(4) Recovery: Plasma samples contained cefoselis sulfate of 10, 40, and 80 mg·L-1 of Group Ⅰ, 200, 800, 1600 mg·L-1 of Group Ⅱ, were treated and injected into the chromatography systems. The absolute recovery of cefoselis sulfate was obtained by comparing the peak area of cefoselis sulfate in plasma samples with that of reference solutions of cefoselis sulfate. The absolute recovery of internal standard was obtained by comparing the peak area of internal standard of plasma samples with that of reference solutions of internal standard. The relative recovery was obtained by comparing the concentration of cefoselis sulfate calculated according to calibration curve with that of theory.(5) Precision: Plasma samples contained cefoselis sulfate of 10, 40, and 80 mg·L-1 of Group Ⅰ, 200, 800, 1600 mg·L-1 of Group Ⅱ, were treated and determined in different times within a day to obtain intra-day precision, or treated and determined in continuous 5 days to obtain inter-day precision.(6) Stability: Processed cefoselis sulfate plasma samples at concentration of 40 mg·L-1(Ⅰ) and 800 mg·L-1(Ⅱ) were determined at 0, 1, 2, 3, 4 h to investigate the stability of processed samples. Plasma samples of 40 mg·L-1(Ⅰ) and 800 mg·L-1(Ⅱ) of cefoselis sulfate were treated and determined immediately or freeze thaw for thrice to investigate the stability of plasma samples after repeated freeze thaw. Plasma samples of 40 mg·L-1(Ⅰ) and 800 mg·L-1(Ⅱ) of cefoselis sulfate were treated and determined immediately or after storage at-40℃ for 5days to investigate the stability of plasma samples stored at low temperature.(7) The pharmacokinetic experiment: Rats were randomly divided into two groups with 20 rats in each group. Each rat in control group was injected cefoselis sulfate(200 mg·kg-1) intravenously via vena caudalis, and each rat in test group was injected foscarnet sodium(360 mg·kg-1) intravenously, following a injected administration of cefoselis sulfate(200 mg·kg-1) immediately. The blood samples were collected at 0 min, 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h after administration and stored at-40 ℃.(8) Pharmacokinetic analysis: the plasma concentrations of cefoselis sulfate were determined by HPLC. The pharmacokinetic parameters of two groups were calculated with DAS2.1.1 pharmacokinetic software and were compared with SPSS13.1 statistic software.Results:(1) The retention times of cefoselis sulfate and internal standard were 6.3 min and 11.4 min, respectively. The endogenous substance in plasma didn’t affect the determination of cefoselis and internal standard.(2) Calibration curve: The standard curve equation Ⅰ was Y=0.0030X-0.0012(r=0.9994), and the linear range was 5~100 mg·L-1; The standard curve equation Ⅱ was Y=0.0031 X + 0.023(r=0.9997), and the linear range was 100~2000 mg·L-1.(3) Recovery: The absolute recoveries of plasma samples of standard curve equationⅠat concentrations of 10, 40 and 80 mg·L-1 were(96.07±2.10)%,(86.69±2.90)% and(85.46±5.26)%, respectively;The absolute recoveries of plasma samples standard curve equation Ⅱ at concentrations of 200, 800 and 1600 mg·L-1 were(82.70±2.10)%,(89.30±1.63)% and(97.05±0.46)%, respectively. The absolute recovery of internal standard was(80.33±2.85)%. The relative recoveries of plasma samples of standard curve equationⅠat concentrations of 10, 40 and 80 mg·L-1 were(108.64±2.08)%,(101.04±2.36)% and(100.48±3.88)%, respectively; The relative recoveries of plasma samples standard curve equation Ⅱ at concentrations of 200, 800 and 1600 mg·L-1 were(91.37±2.43)%,(99.77±2.03)% and(98.85±0.57)%, respectively.(3) Precision: The values of RSD of intra-day precision of plasma samplesⅠat three concentrations were 2.07%, 2.33% and 3.86%, respectively;the values of RSD of intra-day precision of plasma samplesⅡat three concentrations were 2.66%, 1.48% and 0.58%, respectively. The values of RSD of the inter-day precision Ⅰ at three concentrations were 4.61%, 2.65% and 3.53%, respectively; The values of RSD of the inter-day precision Ⅱ at three concentrations were 2.90%, 0.59% and 0.33% respectively.(4) Stability: The plasma samples were stable when stored at-40 ℃ for 5 days or freeze-thaw for thrice and the processed samples were stable within 4 h.(5) Pharmacokinetics: The parameters of test group and control group were obtained as below, t1/2 were(1.56±0.41) h and(1.20±0.49) h,Cmax were(1314.4±196.7) mg·L-1 and(1470.2±321.4) mg·L-1,AUC0-t were(853.6±187.4) mg·h·L-1 and(751.3±187.7) mg·h·L-1,AUC0-∞ were(874.8±212.3) mg·h·L-1 and(780.9±199.0) mg·h·L-1,V were(0.52±0.13) L·kg-1 and(0.45±0.16) L·kg-1,CL were(0.24±0.05) L·h-1·kg-1 and(0.27±0.07) L·h-1·kg-1, respectively.Conclusion: The HPLC method for determination of cefoselis sulfate concentration in plasma was simple, accurate, sensitive and reproducible. The cefoselis sulfate and internal standard could be separated completely, without interference of plasma endogenous substance. The plasma samples were stable under when stored at low temperature for 5 days and repeated freeze-thaw for thrice cycles. The processed plasma samples were stable within 4 h at room temperature.The main pharmacokinetics parameters of AUC0-t, AUC0-∞, Cmax, V and CL were insignificant changed(P>0.05), and the t1/2 of cefoselis sulfate was significant changed(P<0.05), which indicates that the excretion trend of cefoselis sulfate was became slower and the excretion mechanism was needed further research. |
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