Quantifying Antiretroviral Pharmacokinetics

Ethylenediaminetetraacetic acid blood samples were collected at weeks 12, 24, and 48; and 50 microlitres of whole blood was pipetted onto Whatman 903 Proteinsaver cards which were dried overnight and then stored in airtight freezer-safe bags at -80°C. An indirect method for quantifying TFV-DP was used, which has been validated at the Division of Clinical Pharmacology, University of Cape Town.^{18 (link)} It consists of solid phase separation of tenofovir and TFV-DP, enzyme dephosphorylation of TFV-DP to tenofovir, and then high-performance liquid chromatography with tandem mass spectrometry to detect tenofovir.^{18 (link)} Dolutegravir trough concentrations were sampled 12±2 hours after the last dose for participants on supplemental dolutegravir, and 24±4 hours for participants receiving TLD only.^{17 (link)} Dolutegravir concentrations in plasma were analysed by liquid-liquid extraction in a method validated by the Division of Clinical Pharmacology, University of Cape Town.^{19 (link)} For TFV-DP concentrations, the lower limit of quantification (LLQ) is 16.6 fmol/punch and for dolutegravir concentrations the LLQ is 0.03 μg/mL. Viral loads were measured using Abbott Realtime® HIV-1 polymerase chain reaction assay (Abbott Molecular, Des Plaines, IL), which is able to quantify virus ribonucleic acid over a range of 20 to 10⁷ copies/mL.