Quantitation of tenofovir and emtricitabine in dried blood spots (DBS) with LC-MS/MS

Abstract

A reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of tenofovir (TFV) and emtricitabine (FTC) in dried blood spots (DBS) from human whole blood was developed and validated. Whole blood samples were spotted, dried, and a 3mm punch was extracted with methanol for analysis by LC-MS/MS utilizing stable isotope labeled internal standards. The assay was validated over the range of 2.5-1000ng/mL for TFV and 2.5-5000ng/mL for FTC. The method was accurate (within ±15% of control) and precise (coefficient of variation ≤15%) for hematocrit concentrations ranging from 25% to 76%; using edge punches vs. center punches; and spot volumes of 10-50μL. Analytes were stable for five freeze/thaw cycles and up to 6 days at room temperature, whereas long-term storage required -20°C or -80°C. Comparison of TFV and FTC in DBS vs. plasma yielded r(2)≥0.96, indicating that DBS can be used as a plasma alternative for pharmacokinetic analyses in vivo.

Keywords: Analytical method; Antiretroviral therapy; Dried blood spot; LC–MS/MS; Nucleoside analog.

Figure 1

Representative overlay of extracted DBS blank, DBS blank with internal standard and the lower limit of quantitation for TFV and FTC are in the upper left and right panels, respectively. The TFV and FTC retention times were approximately 2.2 and 5.0 minutes respectively. The y-axis was scaled to the lower limit of quantification response. The lower left and right panels represent the TFV-IS and FTC-IS signals, respectively.

Figure 2

Results from the different temperature storage stability of clinical samples. Four storage conditions were tested represented by a dashed line (RT) and solid lines (4°C, −20°C, −80°C). The Y axis represents the mean of measured concentrations at the different storage conditions of TFV (2a) and FTC (2b) from three subjects receiving 30 days of daily dosing followed by off-drug 30 days.

Figure 2

Results from the different temperature storage stability of clinical samples. Four storage conditions were tested represented by a dashed line (RT) and solid lines (4°C, −20°C, −80°C). The Y axis represents the mean of measured concentrations at the different storage conditions of TFV (2a) and FTC (2b) from three subjects receiving 30 days of daily dosing followed by off-drug 30 days.

Figure 3

Intensive pharmacokinetic results evaluating the long term storage stability of clinical samples at room temperature from two different dosing days (first dose, i.e. Day 1 and Day 30). The Y axis represents the mean of measured TFV (3a Day 1;3b Day 30) and FTC (3c Day 1;3d Day 30) of three subjects at the respective time point and dosing days from the initial run (solid line) and re-run (dashed line). The elapsed time between two runs was 404 days.

Figure 3

Intensive pharmacokinetic results evaluating the long term storage stability of clinical samples at room temperature from two different dosing days (first dose, i.e. Day 1 and Day 30). The Y axis represents the mean of measured TFV (3a Day 1;3b Day 30) and FTC (3c Day 1;3d Day 30) of three subjects at the respective time point and dosing days from the initial run (solid line) and re-run (dashed line). The elapsed time between two runs was 404 days.

Figure 4

Results from hematocrit experiments for TFV (4a) and FTC (4b). The Y axis represents the deviation from the control condition (original hematocrit) for the QC Level 4 (15/15 ng/mL) represented by open circles and the Level 6 (800/4000 ng/mL) represented by open squares.

Figure 4

Results from hematocrit experiments for TFV (4a) and FTC (4b). The Y axis represents the deviation from the control condition (original hematocrit) for the QC Level 4 (15/15 ng/mL) represented by open circles and the Level 6 (800/4000 ng/mL) represented by open squares.

Figure 5

DBS versus plasma from 2 intensive samplings (5 time points over 24 hours) in 3 subjects. One sampling was after the first dose and the other was after 30 days of daily dosing. TFV in DBS versus plasma (5a) and FTC in DBS versus plasma (5b) showed linear relationships defined by 1.4x, r²=0.96 for TFV and 0.8x, r²=0.99 for FTC. The regression did not account for repeated measures. Reprinted with permission from Castillo-Mancilla et al. AIDS Res Hum Retroviruses. 2013; 29(2):384–90.

Figure 5

DBS versus plasma from 2 intensive samplings (5 time points over 24 hours) in 3 subjects. One sampling was after the first dose and the other was after 30 days of daily dosing. TFV in DBS versus plasma (5a) and FTC in DBS versus plasma (5b) showed linear relationships defined by 1.4x, r²=0.96 for TFV and 0.8x, r²=0.99 for FTC. The regression did not account for repeated measures. Reprinted with permission from Castillo-Mancilla et al. AIDS Res Hum Retroviruses. 2013; 29(2):384–90.