Application of an intracellular assay for determination of tenofovir-diphosphate and emtricitabine-triphosphate from erythrocytes using dried blood spots

Abstract

This communication describes the application of an existing intracellular methodology to the quantitation of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) from erythrocytes using dried blood spots (DBS). Concentrations were determined from a 3mm DBS punch extracted into a 70:30 methanol:water solution (lysed cellular matrix). This extraction solution was then subjected to a previously validated analytical procedure for lysed cellular matrix. Experiments for DBS validation used replicate samples from study participants to demonstrate acceptable reproducibility with spot volumes ranging from 10-50 μL and punch location either from the edge or center of the spot. Analysis of paired DBS with purified red blood cells showed that a 3mm DBS punch contained an average of 11.9 million cells for the observed hematocrit range of the participants (35-50%). Numerous stability tests were completed showing that whole blood in an EDTA vacutainer could sit for 24h at room temperature prior to spotting, and DBS could remain at room temperature for up to five days including shipment at ambient using 2-days delivery. DBS stability in storage was acceptable up to 18 months at -20°C or -80°C and DBS could undergo 4 Freeze/Thaw cycles. The described method was applied to HIV prophylaxis studies, demonstrating powerful associations with HIV acquisition through its ability to discriminate gradients of adherence.

Keywords: Adherence; Analytical validation; Antiretroviral therapy; Dried blood spot; HIV-prevention; Intracellular nucleoside analog.

Copyright © 2016 Elsevier B.V. All rights reserved.

Figures

Figure 1

Blood was allowed to sit in EDTA whole blood for 24 hours prior to spotting versus immediate spotting (within 1 hour) for DBS. The difference in FTC-TP in DBS from 24 hour spotting versus control (y-axis) was associated with the FTC in plasma (x-axis), Y=−13.1+0.07*X (P=0.0001).

Figure 2

TFV-DP stability when duplicate DBS were stored at room temperature (RT), 4°C, and −20°C versus a DBS replicate stored at −80°C (n=60 pairs of −80°C with other conditions). The thin dashed line represents no difference and the thick dashed lines represent 15% above and 15% below the −80°C value.