| Cefprozil(CPZ), a second-generation semi-synthetic cephalosporin antibiotic, is widely used in clinic. It is composed of cis-cefprozil and trans-cefprozil at the ratio of approximately 9:1. Currently, there are a lot of reports on HPLC method for the determination of cefprozil in human plasma, with low sensitivity, long analysis time, complex mobile phase composition and interference of endogenous substances.Cis-cefprozil and trans-cefprozil could be easily separated under achiral conditions. In this study, a simple, rapid and sensitive LC-MS/MS method was developed for determination of cefprozil diastereomers in plasma samples for the first time. To compared the bioequivalence of tablet which made in Sino-us Shanghai Squibb Pharmaceutical company(refrence formulation) and Beijing Jingfeng Pharmaceutical company(test formulation) in human body and to explored the stereoselective of pharmacokinetics of cis-cefprozil and trans-cefprozil.Part one Establishment of LC-MS/MS method for the determination ofcis-cefprozil and trans-cefprozil in human plasmaObjective: To develop a highly sensitive and rapid method for the determination of cis-cefprozil and trans-cefprozil in human plasma by liquid chromatography-mass spectrometry(LC-MS/MS)Methods: In an ice bath condition, using cephalexin as internal standard, the samples were prepared by protein precipitation using acetonitrile and then separated on Synergi 4u Hydro-RP 80 A column using methanol-0.2% formic acid(25:75, v/v) as mobile phase. Detection was performed by tandem mass spectrometry using an electrosprasy source(ESI) in the negative ion mode, operating in the multiple reaction monitoring(MRM) of the transitions of m/z 388.0 ® m/z 205.0 for cefprozil diastereomers and m/z 346.1 ®m/z 268.1 for cephalexin.Results: The calibration curves of cis-cefprozil showed good linearity in the range of 0.125~16.0 μg/m L in plasma, r>0.998. The calibration curves trans-cefprozil showed good linearity in the range of 0.0403~1.72 μg/m L in plasma, r>0.998. The lower limit of quantification for cis-cefprozil and trans-cefprozil was 0.125 μg/m L and 0.0403 μg/m L in plasma, respectively. The intra- and inter-day precisions(RSD) for cis-cefprozil from QC samples were all less than 9.7%, and the accuracy(RE) ranged from 99.2% to 104.7%. The intra- and inter-day precisions(RSD) for trans-cefprozil from QC samples were all less than 6.5%, and the accuracy(RE) ranged from 100.6% to 102.2%. The average matrix effect and the average recoveries for cis-cefprozil were(109.7 ± 3.2)% and(80.5 ± 1.9)%, respectively. The average matrix effect and the average recoveries for trans-cefprozil were(104.5 ± 3.3)% and(80.2 ± 2.8)%, respectively. The samples were stable in ice water for 6 h, in the autosampler at room temperature for 10 h, in three freeze-thaw cycles test, and in storage at-80 for ℃ 35 days.Conclusion: A highly selective, sensitive, rapid and reproducibility method for the determination of cefprozil in human plasma by LC-MS/MS has been developed and validated in this study. According to the biological samples analysis requirements, the method can be employed in the bioequivalence study of cefprozil tablet.Part two Study on bioequivalence of cefprozil tablet in healthy volunteersObjective: The study aimed to determine the concentrations of cis-cefprozil and trans-cefprozil in human plasma by developed LC-MS/MS method after following a single oral dose of test and reference preparations, and to evaluate the bioequivalence of test and reference formulations of cefprozil.Mehods: The study was a single oral dose, two-way randomized crossover design with a 5-day washout period between the doses. 24 healthy Chinese males were randomly assigned to receive 500 mg of either the test or reference formulation orally. The blood samples were collected over an 11-hour interval. The LC-MS/MS method was used to determine the plasma concentrations of cis-cefprozil and trans-cefprozil. Win Nonlin 6.3 software was employed to process data and calculate the pharmacokinetic parameters. Analysis of variance were finished after the values of AUC0-t,AUC0-∞,Cmax were logarithmically transformed. Analysis of the t-test and 90% confidence interval were conducted to evaluate the bioequivalence of test and reference formulation of cefprozil.Results: The major pharmacokinetic parameters of the cis-cefprozil of test and reference formulations were as follows: AUC0-t were(28.91 ± 3.76) μg×h/m L and(29.47 ± 3.43) μg×h/m L; AUC0-∞ were(28.91 ± 3.76) μg×h/m L and(29.47 ± 3.43) μg×h/m L; Cmax were(8.73 ± 1.22) μg/m L and(9.00 ± 1.30) μg/m L; Tmax were 1.38(1.00~4.00) h and 1.50(1.00~3.00) h; t1/2 were(1.30 ± 0.14) h and(1.28 ± 0.14) h, respectively. The major pharmacokinetic parameters of trans-cefprozil of test and reference formulations were as follows: AUC0-t were(2.70 ± 0.38) μg×h/m L and(2.83 ± 0.36) μg×h/m L; AUC0-∞ were(2.73 ± 0.38) μg×h/m L and(2.86 ± 0.37) μg×h/m L; Cmax were(0.906 ± 0.143) μg/m L and(0.956 ± 0.13) μg/m L; Tmax were 1.50(1.00~4.00) h and 1.50(1.00~3.00) h; t1/2 were(1.05 ± 0.19) h and(1.08 ± 0.17) h, respectively. The major pharmacokinetic parameters of the cis- and trans-cefprozil of test and reference formulations were as follows: AUC0-t were(31.57 ± 4.09) μg×h/m L and(32.29 ± 3.72) μg×h/m L; AUC0-∞ were(32.00 ± 4.14) μg×h/m L and(32.69 ± 3.79) μg×h/m L; Cmax were(9.64 ± 1.36) μg/m L and(9.94 ± 1.43) μg/m L; Tmax were 1.38(1.00~4.00) h and 1.50(1.00~3.00) h; t1/2 were(1.28 ± 0.12) h and(1.25 ± 0.15) h, respectively.Cmax, AUC0-t, AUC0-∞ by the logarithmic conversion after analysis of variance of the test and reference formulation are no markable difference(P>0.05). The results of two one-side t-test and 90% confidence interval analysis for cis-cefprozil were shown as follows: The 90% confidence interval of AUC0-t and AUC0-∞ were 95.92%~100.03% and 95.91%~99.98%, respectively; The 90% confidence interval of Cmax were 92.31%~102.33%. The results of two one-side t-test and 90% confidence interval analysis for trans-cefprozil were shown as follows: The 90% confidence interval of AUC0-t and AUC0-∞ were 92.57%~98.10% and 92.63%~98.09%, respectively; The 90% confidence interval of Cmax were 89.11%~100.45%. The results of two one-side t-test and 90% confidence interval analysis for cis- and trans-cefprozil were shown as follows: The 90% confidence interval of AUC0-t and AUC0-∞ were 95.56%~ 99.73% and 95.71%~99.81%; The 90% confidence interval of Cmax were 92.16%~102.20%.Conclusion: In this study, we evaluated the bioequivalence of cefprozil formulations using the measurement of cis-cefprozil, trans-cefprozil and total cefprozil, respectively. The results of the study showed that the test formulation was bioequivalence to the reference formulation by any of measurement indicators. |
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