title: 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.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Jia-Hua
authors.initials: JH
authors.lastname: Zheng
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Caitlin
authors.initials: C
authors.lastname: Rower
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: McAllister
authors.affiliation: School of Medicine, Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Jose
authors.initials: J
authors.lastname: Castillo-Mancilla
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Brandon
authors.initials: B
authors.lastname: Klein
authors.affiliation: School of Medicine, Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Amie
authors.initials: A
authors.lastname: Meditz
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: L Anthony
authors.initials: LA
authors.lastname: Guida
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kiser
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Lane R
authors.initials: LR
authors.lastname: Bushman
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. Electronic address: Peter.Anderson@ucdenver.edu.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Anderson
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D059788:Dried Blood Spot Testing / Q000379:methods*; D000068679:Emtricitabine / Q000737:chemistry*; D004912:Erythrocytes / Q000737:chemistry*; D015658:HIV Infections / Q000097:blood; D006400:Hematocrit / Q000379:methods; D006801:Humans; D055118:Medication Adherence; D000432:Methanol / Q000737:chemistry; D010755:Organophosphates / Q000737:chemistry*; D011122:Polyphosphates / Q000737:chemistry*; D015203:Reproducibility of Results; D012996:Solutions / Q000737:chemistry; D013696:Temperature; D014867:Water / Q000737:chemistry
chemical_list: D010755:Organophosphates; D011122:Polyphosphates; D012996:Solutions; C583447:tenofovir diphosphate; D014867:Water; D000068679:Emtricitabine; D000225:Adenine; C005692:triphosphoric acid; D000432:Methanol
keywords: Adherence; Analytical validation; Antiretroviral therapy; Dried blood spot; HIV-prevention; Intracellular nucleoside analog
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