A sensitive method for the simultaneous determination of five antiretroviral compounds and a pharmacoenhancer from human plasma has been developed using strong cation mixed-mode solid phase extraction (SPE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This assay represents three drug classes utilized in management of HIV, including nucleoside reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TNF), integrase inhibitors, raltegravir (RAL), dolutegravir (DTG), and elvitegravir (ETG), as well as the pharmacokinetic enhancer, cobicistat (COBI). The drugs were extracted from plasma using Phenomenex Strata X-C solid phase extraction cartridges (100mg), evaporated to dryness under a stream of nitrogen, and reconstituted in mobile phase. The chromatographic separation involved a Waters XSelect HSS T3 column (150 x 4.6 mm, 3.5 micron) and a gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) (5-100%). Mass spectrometric detection was achieved using direct MS/MS channels for each drug, specific to their [M+H]+ ion (RAL, DTG, ETG, TNF, FTC) or [M+2H]2+ ion (COBI). The instrumentation utilized was a Shimadzu LCMS-IT-TOF (ion trap-time of flight) system, which included two LC-AD20 pumps, a SIL-20ACHT autosampler, an in-line degasser (DGU-20A3), and a CTO-20A column oven. A positive electrospray (+ESI) source was used to facilitate ionization, with liquid nitrogen as the carrier gas, and a source temperature of 200oC. Verapamil was used as the internal standard. The method is capable of monitoring concentrations of these drugs in plasma in the range of 0.01 – 1.5 μg/ml, which is suitable for therapeutic drug monitoring (TDM) of these compounds in patients whose blood is sampled at trough. Furthermore, by utilizing the HSS T3 column, a wide range of lipophilicities is accommodated on a single column, thus facilitating TDM for patients on combination products, such as Stribild™, or other combinations of drugs for HIV treatment.

Simultaneous determination of five antiretroviral drugs plus cobicistat in human plasma using strong cation mixed-mode SPE and HPLC-MS/MS

254th National Meeting (2017)

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