An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleoside analog MP-, DP-, and TP- determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.50 to 2000 fmol/sample, and that for 3TC/FTC was 0.10 to 200 pmol/sample. Nucleoside analog MP-, DP-, and TP- determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.10 to 100 pmol/sample, and 5.00 to 2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research.
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 10μL to 50μL 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% to 50%). Numerous stability tests were completed showing that whole blood in an EDTA vacutainer could sit for 24 hours at room temperature prior to spotting, and DBS could remain at room temperature for up to five days including shipment at ambient using 2-day 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.
The pharmacokinetics (PK) of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP), the active anabolites of tenofovir disoproxil fumarate (TDF), and emtricitabine (FTC) in blood, genital, and rectal compartments was determined in HIV-positive and seronegative adults who undertook a 60-day intensive PK study of daily TDF/FTC (plus efavirenz in HIV positives). Lymphocyte cell sorting, genital, and rectal sampling occurred once per subject, at staggered visits. Among 19 HIV-positive (3 female) and 21 seronegative (10 female) adults, TFV-DP in peripheral blood mononuclear cells (PBMC) accumulated 8.6-fold [95% confidence interval (CI): 7.2-10] from first-dose to steady-state concentration (Css) versus 1.7-fold (95% CI: 1.5-1.9) for FTC-TP. Css was reached in ∼11 and 3 days, respectively. Css values were similar between HIV-negative and HIV-positive individuals. Css TFV-DP in rectal mononuclear cells (1,450 fmol/10 cells, 898-2,340) was achieved in 5 days and was >10 times higher than PBMC (95 fmol/10 cells, 85-106), seminal cells (22 fmol/10 cells, 6-79), and cervical cells (111 fmol/10 cells, 64-194). FTC-TP Css was highest in PBMC (5.7 pmol/10 cells, 5.2-6.1) and cervical cells (7 pmol/10 cells, 2-19) versus rectal (0.8 pmol/10 cells, 0.6-1.1) and seminal cells (0.3 pmol/10 cells, 0.2-0.5). Genital drug concentrations on days 1-7 overlapped with estimated Css, but accumulation characteristics were based on limited data. TFV-DP and FTC-TP in cell sorted samples were highest and achieved most rapidly in CD14 compared with CD4, CD8, and CD19 cells. Together, these findings demonstrate cell-type and tissue-dependent cellular pharmacology, preferential accumulation of TFV-DP in rectal mononuclear cells, and rapid distribution into rectal and genital compartments.
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