高效液相色谱法测定更昔洛韦在血液及脑脊液中的浓度

近些年来 ,一些抗病毒药物开始用于临床 ,更昔洛韦为广谱抗病毒药之一 ,对多种ＤＮＡ病毒有显著作用 ,是目前临床治疗病毒性感染应用较为广泛的药物［１～３］。但是 ,抗病毒药物的测定方法 ,尤其在儿科临床监测方面报道甚少。为此 ,笔者建立了更昔洛韦的高效液相色谱测定法 ,并对病毒性脑炎患儿用该药后进行血液和脑脊液药物浓度测定 ,以观察该药的脑膜通透性 ,为临床合理用药奠定基础。１材料与方法１．１材料（１）仪器与试剂 :仪器采用美国Ｗａｔｅｒｓ公司６００Ｅ系列高效液相色谱仪。更昔洛韦标准品由丽珠集团湖北科益药业有限公司提…     

高效液相色谱法测定更昔洛韦滴眼液的含量

目的 :建立测定更昔洛韦滴眼液含量的高效液相色谱法。方法 :以HypersilC18为色谱柱 ,甲醇 水 (10∶90 )为流动相 ,检测波长 2 5 2nm。结果 :更昔洛韦在 2 .5～ 4 0mg·L-1浓度范围内线性关系良好 (r =0 .9998) ,平均回收率为 10 0 .7% ,RSD为0 .33%。结论 :本方法简便、快速、灵敏 ,可作为该滴眼液的质量控制。     

高效液相色谱法测定更昔洛韦凝胶的含量

目的：建立测定更昔洛韦凝胶含量的高效液相色谱法。方法：以迪马C18为色谱柱．甲醇-水（10：90）为流动相，检测波长252nm。结果：更昔洛韦在2．5～40mg·L^-1浓度范围内线性关系良好（r=0．9998），平均回收率为100．5％，RSD为0．21％。结论：本方法简便、快速、灵敏，可作为该制刺的质量控制。     

反相高效液相色谱法测定更昔洛韦血药浓度

目的建立反相高效液相色谱法测定血清中更昔洛韦血药浓度的方法。方法C^18色谱柱（250．0ramX4．6mm,10μm）,流动相为甲醇一水（5：95,V／V）,流速1．0ml／min,紫外检测波长254nm。结果药物峰和各血液杂质峰均达到基线分离且线性关系良好。其线性范围为0．05～l0μg／ml。回归方程为：C=-0．0959＋1．57×10^-5A（r=0．9992）。结论本法简便、快速、重现性好、回收率高,适用于更昔洛韦的含量测定。     

反相高效液相色谱法测定更昔洛韦血药浓度

目的 建立反相高效液相色谱法测定血清中更昔洛韦血药浓度的方法.方法 C18色谱柱(250.0mm×4.6mm,10μm),流动相为甲醇-水(5:95,V/V),流速1.0ml/min,紫外检测波长254nm.结果 药物峰和各血液杂质峰均达到基线分离且线性关系良好.其线性范围为0.05～10μg/ml.回归方程为:C=-0.0959+1.57×10-5 A(r=0.9992).结论 本法简便、快速、重现性好、回收率高,适用于更昔洛韦的含量测定.     

高效液相色谱法测定更昔洛韦凝胶剂中更昔洛韦的含量

目的：建立高效液相色谱法测定更昔洛韦含量的方法。方法：采用Hypersil ODS色谱柱（125mm×4．0mm,5μm）;以甲醇-水（7：93）为流动相;流速：1．0ml·min^-1,柱温：25℃;检测波长：252nm。结果：更昔洛韦的线性范围为2．0～90．0μg·ml^-1,r=0．9999。平均回收率为100．0％,RSD为0．5％（n=9）。结论：HPLC法简便,准确,快速,重复性好,可作为更昔洛韦凝胶剂的质量控制。     

高效液相色谱法测定更昔洛韦葡萄糖注射液的含量

目的建立更昔洛韦葡萄糖注射液中更昔洛韦的含量测定方法。方法采用高效液相色谱（HPLC）法测定更昔洛韦的含量。色谱柱为Phenomenex C18柱（250mm×4．6mm,5μm）,流动相为水-甲醇（95：5）,检测波长为254nm,流速1．0mL／min。结果更昔洛韦进样量在0．5986～1．5962μg范围内与峰面积呈良好的线性关系（r=0．9998）,平均回收率为99．86％,RSD=0．27％（n=9）。结论该法简便、准确、重现性好,可用于测定更昔洛韦葡萄糖注射液中更昔洛韦的含量。     

高效液相色谱法测定更昔洛韦葡萄糖注射液的含量

目的:建立HPLC法测定更昔洛韦葡萄糖注射液含量中更昔洛韦的方法。方法:采用DikmaC18色谱柱(200mm×4.6mm,5μm),以甲醇-水(8∶92)为流动相;流速为1.0mL.min-1;检测波长为252nm。结果:更昔洛韦在10.16～60.96mg.L-1范围内呈良好线性关系(r=0.9999),进样重复性RSD为0.40%(n=5),平均回收率为99.9%。结论:本法简便、快速、结果准确可靠,可用于更昔洛韦葡萄糖注射液中更昔洛韦的含量测定。     

高效液相色谱法测定更昔洛韦血药浓度的方法改进

目的建立高效液相色谱法(HPLC)测定更昔洛韦血药浓度.方法以阿昔洛韦为内标,Ultrasphere-ODS(4.6 mm×250 mm,5μm)色谱柱为分离柱,流动相甲醇-水(595,V/V),检测波长252 nm.结果血中更昔洛韦浓度在0.2～16.0mg·L-1范围内与峰面积呈良好的线性关系,平均回收率为98.9%,日内及日间RSD均小于5%.结论本法简便,灵敏.适合于更昔洛韦血药浓度测定及药动学研究.     

利用Caco-2细胞单层模型评价P-糖蛋白对安妥沙星转运的影响

目的 评价P-糖蛋白抑制剂酮康唑对安妥沙星转运的影响.方法 利用人源结肠腺癌系Caco-2细胞单层模型对安妥沙星进行双向转运,采用高效液相色谱法对安妥沙星进行定量分析,计算其表观渗透系数(Papp).结果 加入酮康唑后,低浓度安妥沙星(100μmol/L)Papp(B-A)/Papp(A-B)由4.12降至1.23(P＜0.01),高浓度安妥沙星(500μmol/L)Papp(B-A)/Papp(A-B)由3.54降至1.22(P＜0.01).结论 安妥沙星在Caco-2细胞单层模型中的转运机制可能以主动转运为主,P-糖蛋白参与其从细胞基底侧向管腔面的分泌.     

肠道转运Caco-2细胞单层模型的建立及验证评价

目的建立Caco-2细胞单层模型用于药物转运研究。方法按照常规的细胞培养方法,将Caco-2细胞接种到Millicell小室内(接种密度1×10⁶个·mL^-1),培养21 d。定期用细胞电位仪监测跨上皮细胞电阻（TEER）,评价细胞单层的紧密性与完整性;通过荧光黄转运实验检查Caco-2细胞单层模型细胞旁路转运通透性;通过普萘洛尔转运实验验证Caco-2细胞单层模型跨细胞被动转运通透性。结果培养21 d后,TEER值达到（981±123）Ω·cm²,荧光黄和普萘洛尔的表观通透系数分别为0.33×10及16.7×10^-6cm·s^-1。结论本研究建立的Caco-2细胞单层模型紧密、完整,具有良好通透性,可用于药物转运研究。     

Characterization of Caco-2 cell monolayer drug transport properties by cassette dosing using UV/fluorescence HPLC.

Cassette dosing is commonly used in pharmacokinetic studies to decrease time, labor and expenditures. Cassette dosing (having several compounds in a mixture) has been used to screen in vitro permeabilities in Caco-2 cell monolayers. The cassette dosing method has accelerated both transport experiments and sample analyses, which are both part of Caco-2 permeability screening. In this study, a cassette dosing method was used for a mixture of heterogeneous test drugs, which are transported by various mechanisms across the Caco-2 cell monolayer. To characterize the Caco-2 cell monolayer absorption properties, we developed a new UV/fluorescence HPLC method for nine heterogeneous drugs. This new analytical method is fast and specific for high throughput analysis. Fluorescence detection was used to analyze the low concentration of drugs while UV detection was suitable for higher concentrations. The permeability results of single drugs and the mixture of drugs showed a high degree of similarity for each individual compound. All drugs can be applied to the Caco-2 cell culture as a mixture, and the cassette dosing method is suitable for permeability studies.     

脉君安片中主要成分对氢氯噻嗪在Caco-2细胞模型中转运的影响

利用人源结肠腺癌细胞系Caco-2细胞单层模型, 研究了脉君安片中主要降压活性成分葛根素、钩藤碱与氢氯噻嗪配伍使用前后对氢氯噻嗪体外吸收与转运的影响, 探讨了氢氯噻嗪与葛根素、钩藤碱配伍使用时在吸收阶段的药物相互作用。结果表明, 氢氯噻嗪在Caco-2单层细胞模型中的吸收可能存在由载体介导的主动转运, 葛根素能减少P-糖蛋白 (P-gp), 对氢氯噻嗪的外排, 钩藤碱对氢氯噻嗪的吸收转运无显著影响, 葛根素、钩藤碱与氢氯噻嗪合用能增强氢氯噻嗪的吸收。同时, 实验还发现氢氯噻嗪可能是P-gp的作用底物, 且pH值对氢氯噻嗪的转运有重要影响, 在弱酸性条件下, 氢氯噻嗪的吸收比在中性条件下好。     

千层纸素A在Caco-2细胞模型中的吸收机制研究

目的研究千层纸素A在Caco-2细胞模型中的吸收机制。方法 MTT实验考察千层纸素A在Caco-2细胞中的安全浓度范围,再利用Caco-2细胞单层模型研究千层纸素A的双向转运机制,以转运量及表观渗透系数(Papp)为指标,考察时间、浓度、pH和P-gp抑制药维拉帕米对其吸收的影响。结果千层纸素A在Caco-2细胞模型中的转运与时间和浓度呈正相关;并受pH值影响,P-gp抑制药维拉帕米对其转运无影响,从单层细胞层顶端(AP)到基底端(BL)的转运与基底端到顶端的转运大致相同。结论千层纸素A在Caco-2细胞模型中的吸收是被动转运。     

Intestinal efflux transport kinetics of green tea catechins in Caco-2 monolayer model

The bioavailability of green tea catechins (GTCs), including epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC) is low in both animals and humans. The contribution of intestinal efflux to this low bioavailability has been suggested by previous studies. The objective of the present study was to investigate the kinetics of efflux transport of the four major GTCs in Caco-2 cell lines, to provide comparison on the efflux transport between each GTC. The basal-to-apical transport of each GTC at concentrations ranging from 15 to 265 microM was examined using the Caco-2 cell monolayer model. Transported amount of GTC was measured by high-performance liquid chromatography with electrochemical detection. Kinetic parameters, V(max), K(m) and V(max)/K(m) were determined and compared among the four studied GTCs. The extent of basal-to-apical transport was, in descending order, EC > EGC > ECG approximately EGCG. Kinetic studies indicated that active and saturable efflux transport of EC took place in Caco-2 cells, with a K(m) of 131 microM, a V(max) of 0.0249 nmol min cm(-2) and an intrinsic clearance (V(max)/K(m)) of 0.19 microL min cm(-2). No saturation could be observed for the efflux transport of EGC, ECG and EGCG even at concentrations up to about 200 microM, which may be due to their low affinity towards the transporters at the concentration range studied. In conclusion, the extent of efflux transport of GTCs in Caco-2 cells was, in descending order, EC > EGC > ECG approximately EGCG, which may reflect the order of elimination occurring in the intestine. The kinetic studies showed the importance of efflux transporters in basal-to-apical transport of EC and suggests their role in the limited oral bioavailability of EC.     

Effect of benzo[a]pyrene on P-glycoprotein-mediated transport in Caco-2 cell monolayer

The main exposure pathway of benzo[a]pyrene (Bap) for humans is considered to be via the daily diet. The purpose of this study was to investigate the effect of BaP on the intestinal transport of chemicals mediated by P-glycoprotein (P-gp). The intestinal epithelial membrane transport of rhodamine-123 (Rho-123), a substrate of P-gp, was examined using a monolayer of the human Caco-2 cell line grown in transwells. In the monolayer exposed to Bap for 72 h before transport experiments, the ratio of the apparent permeability coefficients (P(app)) of Rho-123 efflux increased compared to that of the control. The permeability of rhodamine-B (Rho-B), not a substrate of P-gp, showed no difference between the monolayers. Treatment with quinidine or cyclosporine A, which are P-gp inhibitors, decreased the P(app) of Rho-123 to the same degree in both monolayers. The transport of Rho-123 was not influenced by the presence of Bap. Thus, Bap seemed not to act directly on the efflux activity of P-gp and be a binding site competitor of Rho-123. In the Caco-2 cells that enhanced the efflux of Rho-123 by the treatment with Bap, an increase in mRNA expression of MDR 1 (P-gp) was confirmed compared to that of control by RT-PCR. Furthermore, Western blot analysis using a monoclonal antibody, C219, demonstrated the increase of P-gp in Caco-2 cells exposed to Bap, compared with controls. It was inferred that Bap exposure induced the expression of P-gp, which led to the observed increase in efflux transport of Rho-123. The possibility was suggested that Bap might affect the disposition of medicines by increasing P-gp expression.     

Intestinal transport of pure diester-type alkaloids from an aconite extract across the Caco-2 cell monolayer model

Aconitine (AC), mesaconitine (MA), and hypaconitine (HA) are the active alkaloids identified in aconite tuber, an important traditional Chinese medicine. The study is aimed to investigate their intestinal transport profiles and potential interaction during the intestinal absorption using the Caco-2 cell monolayer model. All three alkaloids had good permeability with P(app) values greater than 1 × 10 (-6) cm · s (-1). However, AC, MA, and HA in a mixture and as an extract, in both cases with the same content of alkaloids, showed higher transport efficiency in the apical to basolateral, and lower transport efficiency in the basolateral to apical directions. Digoxin, as a P-glycoprotein (P-gp) substrate, was substantially effluxed in the basolateral to apical direction but inhibited by the three alkaloids. Furthermore, the backwards transport of MA and HA was inhibited by the P-gp inhibitor verapamil. These observations indicated that the three alkaloids may not only be P-gp inhibitors but also its substrates; they interact with each other and can potentially enhance their own bioavailability when taken concomitantly.     

盐酸赛庚啶在Caco-2细胞中吸收转运的研究

目的 研究盐酸赛庚啶在Caco-2细胞模型中的吸收机制。方法 建立Caco-2细胞体外吸收模型,通过测定荧光黄透过率、安替比林和普萘洛尔的渗透系数、地高辛的外排率验证Caco-2细胞吸收模型的成功建立。采用LC-MS/MS法测定盐酸赛庚啶在膜两侧的浓度,计算渗透系数和外排率。结果 Caco-2模型建立成功,采用LC-MS/MS法可以准确测定缓冲体系中盐酸赛庚啶的浓度。结论 盐酸赛庚啶的渗透系数随药物浓度的变化而变化,且与转运方向有关,可能为P-gp和BCRP的底物。     

鲑鱼降钙素在Caco-2细胞模型中的吸收机制

目的 研究鲑鱼降钙素(SCT)在Caco-2细胞模型的吸收机制.方法 采用人源结肠癌细胞系Caco-2细胞模型研究不同浓度SCT由绒毛面(AP侧)到基底面(BL侧)的转运过程,用HPLC法测定SCT在转运液中的浓度.结果 SCT 0.5～ 1.5 mg·mL-1对其表观渗透系数(Papp)无显著影响.结论 SCT在小肠上皮细胞主要以被动转运的方式吸收,有望将其开发成生物利用度较高的口服给药系统.