Laboratory of Pharmaceutical Technology, LTF/CICF, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Dec 12;911:76-83. doi: 10.1016/j.jchromb.2012.10.034. Epub 2012 Nov 2.
Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50μL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02-1.5μg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02μg/mL), limit of detection (0.006μg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti-HIV microbicide drug.
地匹福林，一种非核苷类逆转录酶抑制剂，目前正被用于开发潜在的抗 HIV 杀微生物剂制剂和传递系统。建立了一种新的高效液相色谱（HPLC）-紫外检测法，用于测定不同生物基质中的地匹福林，包括细胞裂解物、通透性实验的受体介质和黏膜组织匀浆。该方法采用反相 C18 柱，以三氟乙酸溶液（0.1%，v/v）和乙腈的梯度模式作为流动相。进样量为 50μL，流速为 1mL/min。总运行时间为 12min，紫外检测波长为 290nm，用于检测地匹福林和内标二苯胺。采用 Box-Behnken 实验设计研究了方法的不同实验变量，即流动相组成和梯度时间的比例，以及它们对保留因子、拖尾因子和地匹福林和内标理论板数等响应的影响，以及两种化合物之间的峰分辨率。优化后的方法进一步进行了验证，并评估了其在使用地匹福林或载有地匹福林的纳米粒子的体外和体内实验中的有用性。该方法表现出选择性、线性、准确性和精密度，在 0.02-1.5μg/mL 范围内。其他色谱参数，如拖尾、定量下限（0.02μg/mL）、检测限（0.006μg/mL）、回收率（等于或高于 90.7%）以及不同储存条件下的样品稳定性，也进行了测定，结果表明该方法适用于预期目的。该方法成功地用于细胞摄取试验和跨细胞单层和猪生殖黏膜组织的通透性研究。总之，该方法为研究地匹福林在不同生物基质中的行为和评估其作为抗 HIV 杀微生物药物的潜力提供了一种简单、灵活和可靠的方法。
