Barreiros Luisa, Cunha-Reis Cassilda, Silva Eduarda M P, Carvalho Joana R B, das Neves José, Sarmento Bruno, Segundo Marcela A
UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal.
J Pharm Biomed Anal. 2017 Mar 20;136:120-125. doi: 10.1016/j.jpba.2016.12.028. Epub 2016 Dec 27.
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. 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 with LOD and LOQ for both analytes ≤0.4 and ≤0.7ngmL in 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.
全球数以百万计的人感染了人类免疫缺陷病毒（HIV），因此有必要持续探索新的预防和治疗策略，包括将抗逆转录病毒药物（ARV）如替诺福韦（TFV）和依非韦伦（EFV）联合使用的局部杀菌剂产品。因此，本研究的目的是开发并验证一种高效液相色谱法与三重四极杆串联质谱联用（HPLC-MS/MS）方法，用于定量生物基质（小鼠阴道组织、阴道灌洗液和血浆）中的TFV和EFV。采用反相C18柱（3μm，100×2.1mm）在45°C下进行色谱分离，以0.1%（v/v）甲酸水溶液和0.1%（v/v）甲酸乙腈溶液按0.35mL/min的流速进行梯度洗脱。总运行时间为9分钟，TFV和EFV的保留时间分别为2.8分钟和4.1分钟。质谱以正离子模式（ESI+）检测TFV，以负离子模式（ESI-）检测EFV。数据在选择反应监测（SRM）模式下采集，氘代ARV用作内标。ARV浓度在4至500ng/mL范围内时校准曲线呈线性，样品提取物中两种分析物的检测限（LOD）和定量限（LOQ）分别≤0.4和≤0.7ng/mL。该方法在所有基质中均具有特异性、准确性（名义值的96.0 - 106.0%）和精密度（相对标准偏差<2.4%）。在室温（20°C）下放置24小时、在自动进样器中放置24小时以及经过三次冻融循环后，TFV和EFV在所有基质中均保持稳定。小鼠组织或体液中加标的ARV平均回收率≥88.4%。在组织和血浆提取物中测定EFV时观察到基质效应，但通过使用氘代内标进行了补偿。所提出的方法成功应用于两种ARV阴道给药后的药代动力学研究。
