厄他培南对米卡芬净大鼠体内药动学的影响

发布时间：2018-04-22 17:40

本文选题：HPLC-UV + UPLC-UV　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:建立大鼠血浆中米卡芬净HPLC及游离米卡芬净的UPLC测定方法,并考察厄他培南对米卡芬净在大鼠体内药代动力学的影响,完善两药合用的药代动力学研究。方法:色谱条件:HPLC,色谱柱为Diamonsil C18柱(150 mm×4.6 mm,5μm),流动相为乙腈:0.01 mol/L醋酸铵缓冲液(以冰醋酸调至p H4.5)(42.5:57.5,V/V),检测波长269 nm,流速1.0 m L/min,柱温30℃,进样量10μL,选择艾瑞昔布为内标;UPLC,色谱柱为Acquity UPLC?BEH C18柱(2.1 mm×50 mm,1.7μm),流动相为乙腈:0.01 mol/L醋酸铵缓冲液(以冰醋酸调至p H 4.5)(45:55,V/V),检测波长269 nm,流速0.2 m L/min,柱温30℃,进样量2μL。动物实验方法:将健康雄性SD大鼠40只,随机分为2组,每组20只,对照组大鼠尾静脉注射给予米卡芬净15 mg·kg~(-1),实验组大鼠尾静脉注射给予厄他培南100 mg·kg~(-1)和米卡芬净15 mg·kg~(-1),分别于给药后2、10、20、30 min和1、2、3、4、6、12、24 h于眼内眦取血0.5 m L,置于含肝素钠的抗凝离心管中,离心(10000 r·min-1)5 min后取上层血浆,置-40℃冰箱保存待测。血浆处理方法:血浆药物浓度测定,取大鼠血浆100μL,置1.5 m L离心管中,加乙腈400μL以及内标溶液10μL,涡旋混合1 min,离心(10000r·min-1)5 min,取上清液100μL于进样瓶中,10μL进样并测定;游离药物测定,取大鼠血浆300μL,置玻璃细管中,加入中空纤维,离心(6000r·min-1)10 min,取中空纤维内超滤液,2μL进样并测定。药动学参数处理方法:用DAS 2.1.1软件对每只大鼠的系列血药浓度进行统计矩自动拟合得到药动学参数,用SPSS 13.1软件对2组的主要药动学参数进行统计学分析。若进行比较的2组数据均服从正态分布,用两个独立样本的t检验进行分析,若其中任意一组不服从正态分布,用非参数检验进行分析。当检验的P0.05时则认为两组间差异有统计学意义。结果:HPLC中,米卡芬净的保留时间为7 min,艾瑞昔布的保留时间为11 min,血浆内源性杂质对其浓度的测定无干扰;标准曲线为Y=0.0443X-0.0025,(r2=0.9994),线性范围1~200μg·m L-1;米卡芬净低(1.5μg·m L-1)、中(25μg·m L-1)、高(160μg·m L-1)三个浓度的绝对回收率依次为(99.10±3.96)%、(90.10±4.43)%和(95.00±3.49)%,相对回收率分别为(101.7±6.1)%、(108.4±7.3)%和(103.8±8.0)%,艾瑞昔布的绝对回收率为(97.70±4.18)%;米卡芬净低、中、高三个浓度的日内RSD分别为6.3%,7.9%和7.7%,日间RSD分别为8.4%,8.9%和8.6%;米卡芬净血浆样品于-40℃低温保存14天及反复冻融三次后性质稳定,且处理后的血浆样品于4℃放置24 h对测定无影响。UPLC中,米卡芬净保留时间为2.33 min,无血浆内源性杂质干扰;标准曲线为Y=9259X-26.721,(r2=0.9999),线性范围20~640μg·m L-1;米卡芬净低(40μg·L-1)、中(160μg·L-1)、高(512μg·L-1)三个浓度的绝对回收率依次为(91.81±6.17)%、(96.83±1.59)%和(90.61±3.29)%,相对回收率分别为(94.05±4.12)%、(99.52±2.10)%和(96.97±3.64)%;米卡芬净低、中、高三个浓度的日内RSD分别为2.5%,1.4%和0.6%,日间RSD分别为3.8%,3.5%和3.4%。药动学参数:血浆浓度测定部分,实验组和对照组主要药动学参数如下,AUC0-24h分别为(294.9±25.6)mg·h·L-1和(301.2±41.0)mg·h·L-1;AUC0-∞分别为(349.4±28.0)mg·h·L-1和(359.5±53.9)mg·h·L-1;t1/2分别为(9.261±0.935)h和(9.295±1.133)h;V分别为(0.577±0.077)L·kg~(-1)和(0.568±0.087)L·kg~(-1);CL分别为(0.043±0.003)L·h-1·kg~(-1)和(0.043±0.007)L·h-1·kg~(-1);Cmax分别为(65.95±9.71)mg·L-1和(84.13±12.98)mg·L-1。实验组的AUC0-24h、AUC0-∞、t1/2、CL和V与对照组相比均无显著性差异(P0.05),实验组的Cmax与对照组相比有统计学差异(P0.05)。结论:本文所建立法测定大鼠血浆中米卡芬净浓度的HPLC-UV及UPLC-UV法,方法新颖、分析速度快、灵敏度高。全面进行方法学验证后,证明所建立的方法专属性、灵敏度、精密度、准确度较好,适用于厄他培南对米卡芬净大鼠体药动学变化的研究。对照组和实验组相比,体内药动学参数除了Cmax其余AUC0-24h,AUC0-∞,t1/2,CL和V均无统计学差异,提示厄他培南可能影响米卡芬净在大鼠体内的分布,初步推断大鼠体内厄他培南对米卡芬净的药动学有影响。游离药物浓度测定结果显示,提示厄他培南影响米卡芬净的部分药动学参数,由此推断米卡芬净在大鼠体内的药动学受到一定影响。
[Abstract]:Objective: to establish a method for the determination of Mikafin net HPLC and free Mika Finn Jing in rat plasma, and to investigate the effect of UPLC on the pharmacokinetics of Mika Finn Jing in rats and improve the pharmacokinetics of two drugs. Methods: the chromatographic conditions: HPLC, the column of Diamonsil C18 (150 mm * 4.6 mm, 5 u m), and the mobile phase of acetonitrile: 0 .01 mol/L ammonium acetate buffer (with glacial acetic acid to P H4.5) (42.5:57.5, V/V), detection wavelength 269 nm, velocity 1 m L/min, column temperature 30 degrees C, sample quantity 10 mu L, select alicoxib as the internal standard; UPLC, the column is Acquity, 0.01 ammonium acetate buffer (4.5 acetic acid to 4.5). 45:55, V/V), the detection wavelength 269 nm, the flow rate 0.2 m L/min, the column temperature 30 degrees C, the sample volume 2 micron animal experiment method: the healthy male SD rat 40, randomly divided into 2 groups, 20 in each group, the control group rats tail vein injection to Mika Finn Jing 15 mg. Kg~ (-1), experimental group rats tail vein injection of 100 mg kg~ (100) and Mika Finn Jing 15 15. Kg~ (-1), after the administration of 2,10,20,30 min and 1,2,3,4,6,12,24 h respectively after the administration of 2,10,20,30 min and 1,2,3,4,6,12,24 h in the inner canthus, they were placed in the anticoagulant centrifuge tube containing heparin sodium. After the centrifugation (10000 r min-1) 5 min, the upper plasma was taken and stored in the refrigerator to be preserved. Plasma treatment method: plasma drug concentration was measured, the plasma of rats was 100 micron, and 1.5 acetonitrile was added to the centrifugation tube, plus acetonitrile 4. 00 mu L and internal standard solution 10 u L, vortex mixture 1 min, centrifuge (10000r. Min-1) 5 min, take the supernatant 100 um L in the sample bottle, 10 u L into sample and determine; free drug determination, take the rat plasma 300 micron, insert hollow fiber, centrifuge (6000r min-1) 10 min, take the hollow fiber ultrafiltration fluid, 2 micron intake sample and determine the pharmacokinetic parameters. Treatment method: the pharmacokinetic parameters were obtained by DAS 2.1.1 software, and the major pharmacokinetic parameters of the 2 groups were statistically analyzed with SPSS 13.1 software. If the 2 groups of data were compared to normal distribution, the t test of two independent samples was used for analysis, if any one of them was analyzed. The group was not subjected to normal distribution and was analyzed by nonparametric test. When P0.05 was tested, the difference between the two groups was statistically significant. Results: in HPLC, the retention time of Mika Finn Jing was 7 min, and the retention time of alisoxib was 11 min, and the plasma endogenous impurities were not interfered with the determination of its concentration; the standard curve was Y=0.0443X-0.0025, (r2=0.99 94), the linear range 1~200 mu g. M L-1, mecamen net low (1.5 mu g. M L-1), (25 mu g. M L-1), high (160 mu g m) the absolute recovery rate is (99.10 + 3.96)%, (90.10 + 4.43)% and (95 + 3.49)%, and the relative recovery rate is (101.7 + 6.1)%, (101.7 + three)%, and the absolute recovery rate of erioxib is RSD was 6.3%, 7.9% and 7.7% in the low, middle and high concentrations of M, 7.9% and 7.7%, respectively, 8.4%, 8.9% and 8.6% in daytime, respectively, for 14 days at -40 C for 14 days and three times by repeated freezing and thawing, and the treated plasma samples were placed at 4, 24 h for.UPLC, and the retention time of Mika Finn Jing was 2.33. Min, no plasma endogenous impurity interference; the standard curve is Y=9259X-26.721, (r2=0.9999), the linear range 20~640 G. M L-1; the net low (40 mu g. L-1), 160 micron G. L-1), the absolute recovery rate of three concentrations (91.81 + 6.17)%, (96.83 + 1.59)% and (90.61 + 3.29)%, and (99.52)%, respectively (99.52). (+ 2.10)% and (96.97 + 3.64)%; the intraday RSD of the middle and high three concentrations were 2.5%, 1.4% and 0.6% respectively, and the day RSD was 3.8%, 3.5% and 3.4%. pharmacokinetic parameters: the plasma concentration measurement part, the main pharmacokinetic parameters of the experimental and control groups were as follows, AUC0-24h was (294.9 + 25.6) Mg, H. L-1, and (301.2 + 41) mg. H L-1; AUC0- infinity Respectively (349.4 + 28) mg / h / L-1 and (359.5 + 53.9) mg. H. L-1, t1/2 respectively (9.261 + 0.935) H and (9.295 + 1.133) h respectively, V respectively (0.577 + 0.077) L kg~ (0.568 + 0.087), respectively. There was no significant difference in AUC0-24h, AUC0-, t1/2, CL and V between the experimental group and the control group (P0.05). The Cmax in the experimental group was statistically different from that of the control group (P0.05). Conclusion: the method established in this paper is a novel method for the determination of Mika Finn Jing concentration in rat plasma, the method is novel, the analytical speed is fast and the sensitivity is high. A comprehensive methodology is carried out. After verification, it was proved that the established method was specific, sensitive, precise and accurate. It was suitable for the study of the pharmacokinetic changes of miccen in rats. Compared with the experimental group, the pharmacokinetic parameters of the control group were not statistically different from the other Cmax AUC0-24h, AUC0-, t1/2, CL and V, suggesting that it might affect rice. The distribution of Kafin in rats preliminarily deduced that the pharmacokinetics of Mika Finn Jing had an influence on the pharmacokinetics of Mika Finn Jing in the rat. The results of the determination of the concentration of free drug showed that the effects of the pharmacokinetic parameters of Mika Finn Jing on the pharmacokinetic parameters of Mika Finn Jing were influenced by the result.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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