Simultaneous measurement of etravirine, maraviroc and raltegravir in pigtail macaque plasma, vaginal secretions and vaginal tissue using a LC–MS/MS assay

• Simultaneous LC–MS/MS assay for etravirine, maraviroc, and raltegravir.
• Drugs can be quantified in pigtail macaque plasma, vaginal tissue and secretions.
• A simple precipitation method allows for >94% overall recovery of each drug.
• Low limit of quantification enables detection after vaginal or oral delivery.

Etravirine (ETR), maraviroc (MVC) and raltegravir (RAL) are promising antiretroviral drugs being used in HIV treatment and may be interesting for prevention applications such as oral or topical pre-exposure prophylaxis. Here we describe a sensitive and accurate method for the simultaneous detection of ETR, MVC and RAL from pigtail macaque plasma, vaginal secretions, and vaginal tissue. This method is characterized by a straightforward precipitation extraction method, a limit of quantification <0.5 ng mL−1 for all three antiretrovirals bolstered by a corresponding internal standard for each drug analyte, and short run time. Quantification is performed using positive ion electrospray triple quadrupole mass spectrometry. This method was validated over clinically relevant ranges for the three ARV drugs in all three matrices: 0.1–100 ng mL−1 for ETR, 0.05–100 ng mL−1 for MVC and 1–100 ng mL−1 for RAL. Our method is accurate and precise, with measured mean inter-assay precision (%CV) and accuracy (% bias) of 5.08% and 1.96%, respectively, while the mean intra-assay precision and accuracy were 3.44% and 1.08%. The overall post-extraction recovery for ETR, MVC and RAL was >94% in all cases. We also show that extracted biological samples are stable after storage at room temperature or 4 °C and after three freeze/thaw cycles. This is the first analytical method capable of quantifying ETR, MVC and RAL in biological matrices relevant for pre-clinical testing of oral or topical HIV prevention methods in pigtailed macaques.