title: Development and validation of a liquid chromatography-MS/MS method for simultaneous quantification of tenofovir and efavirenz in biological tissues and fluids.
abstract: Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV). Therefore, the aim of this work was to develop and validate a high performance liquid chromatography method coupled to triple quadrupole-tandem mass spectrometry (HPLC-MS/MS) for the quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood plasma). Chromatographic separation was achieved using a reversed phase C18 column (3μm, 100×2.1mm) at 45°C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at 0.35mLmin-1. Total run time was 9min, with retention time of 2.8 and 4.1min for TFV and EFV, respectively. The MS was operated in positive ionization mode (ESI+) for TFV and in negative ionization mode (ESI-) for EFV detection. Data were acquired in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500ngmL-1with LOD and LOQ for both analytes ≤0.4 and ≤0.7ngmL-1in sample extracts, respectively. The method was found to be specific, accurate (96.0-106.0% of nominal values) and precise (RSD<2.4%) in all matrices. Both TFV and EFV were found to be stable in all matrices after standing 24h at room temperature (20°C) or in the autosampler, and after three freeze-thawing cycles. Mean recovery values of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV determination in tissue and plasma extracts but compensated by the use of deuterated internal standards. The proposed methodology was successfully applied to a pharmacokinetic study following intravaginal administration of both ARVs.
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: lbarreiros@ff.up.pt.
authors.forename: Luisa
authors.initials: L
authors.lastname: Barreiros
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal.
authors.forename: Cassilda
authors.initials: C
authors.lastname: Cunha-Reis
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
authors.forename: Eduarda M P
authors.initials: EMP
authors.lastname: Silva
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
authors.forename: Joana R B
authors.initials: JRB
authors.lastname: Carvalho
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal; i3S - Instituto de Investigação e Inovação em Saúde & INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal.
authors.forename: José
authors.initials: J
authors.lastname: das Neves
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal; i3S - Instituto de Investigação e Inovação em Saúde & INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal.
authors.forename: Bruno
authors.initials: B
authors.lastname: Sarmento
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
authors.forename: Marcela A
authors.initials: MA
authors.lastname: Segundo
mesh_terms: D000282:Administration, Intravaginal; D000480:Alkynes; D000818:Animals; D019380:Anti-HIV Agents / Q000008:administration & dosage / Q000032:analysis* / Q000097:blood; D048588:Benzoxazines / Q000008:administration & dosage / Q000032:analysis* / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003521:Cyclopropanes; D004338:Drug Combinations; D005260:Female; D057230:Limit of Detection; D051379:Mice; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000068698:Tenofovir / Q000008:administration & dosage / Q000032:analysis* / Q000097:blood; D014621:Vagina / Q000737:chemistry; D044364:Vaginal Douching
chemical_list: D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; D004338:Drug Combinations; D000068698:Tenofovir; C098320:efavirenz
keywords: Antiretrovirals; Efavirenz; Intravaginal administration; Pre-exposure prophylaxis; Tenofovir