Journal of Chromatography B, 911 (2012) 76–83 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l ho me pag e: www.elsevier.com/l o cate/chromb Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments José das Neves a,b,∗ , Bruno Sarmento a,b,c , Mansoor Amiji d , Maria Fernanda Bahia a a Laboratory of Pharmaceutical Technology, LTF/CICF, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal b CICS – Centro de Investigac ¸ ão em Ciências da Saúde, Department of Pharmaceutical Sciences, Instituto Superior de Ciências da Saúde-Norte, CESPU, Rua Central de Gandra 1317, 4585-116, Gandra, Portugal c INEB – Instituto de Engenharia Biomédica, NEWTherapies Group, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal d Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, 110 Mugar Life Sciences Building, 360 Huntington Avenue, Boston, MA 02115, USA a r t i c l e i n f o Article history: Received 19 August 2012 Accepted 1 October 2012 Available online 2 November 2012 Keywords: Microbicides Liquid chromatography Box-Behnken design Method validation Polymeric nanoparticles Diphenylamine a b s t r a c t 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 50 L and the flow rate 1 mL/min. The total run time was 12 min and UV detection was performed at 290 nm 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 0.02–1.5 g/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02 g/mL), limit of detection (0.006 g/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. © 2012 Elsevier B.V. All rights reserved. 1. Introduction HIV/AIDS is one of the most important health, social and economic global burdens of our days. Prevention, particular to sexual transmission, is a keystone in the fight against infection and disease, but highly effective strategies are limited to condom use and sexual abstinence. Microbicides are products intended for vaginal or rectal administration around the time of sexual intercourse in order to avoid the transmission of HIV and poten- tially other pathogens [1]. Despite several initial disappointing results, recent success of a vaginal gel product containing 1% of ∗ Corresponding author at: Laboratory of Pharmaceutical Technology, LTF/CICF, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050- 313, Porto, Portugal. Tel.: +351 222 078 949; fax: +351 222 003 977. E-mail address: j.dasneves@gmail.com (J. das Neves). the antiretroviral drug tenofovir boosted interest in the field [2]. Alongside tenofovir and other promising compounds, dapivirine (TMC120; 4-[[4-[2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]- amino]-benzonitrile) (Fig. 1) is one of the leading antiretroviral agents currently being tested for the development of anti-HIV vaginal microbicides [1]. Preclinical testing of this potent non- nucleoside reverse transcriptase inhibitor provided encouraging results in terms of activity and toxicity for both vaginal and rectal applications [3–5]. Moreover, recent clinical studies indicate that dapivirine is safe and provides favorable pharmacokinetics when delivered either as a vaginal gel or ring [6–8]. Two phase III clinical trials testing dapivirine vaginal rings are currently underway [9,10]. Different dosage forms/delivery systems have been proposed for the vaginal administration of dapivirine. Alongside vaginal gels and rings, others such as films [11] and tablets [12] have also been suggested. Moreover, nanotechnology-based solutions have been 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2012.10.034