The instrumentation was a Thermo Scientific TSQ Vantage В® triple quadrupole mass spectrometer coupled with a Thermo Scientific Accela В® UHP pump (Thermo Scientific, San Jose, USA) and CTC Analytics HTC PAL В® auto sampler. Quantification of TFV and FTC and their respective internal standards was performed with high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) with an approach similar to previously published methods. All solutions were stored at 4вЊ in glass vials. Purity and salt content were taken into consideration when preparing prep stocks. The combined working internal standard (IS) solution of TFV-iso and FTC-iso were made in UPH 2O yielding a final concentration of approximately 16.7 ng/mL. These were combined to prepare QC working stocks and the validation QC samples in the same way as standard calibrators. Separate quality control (QC) prep stocks (1 mg/mL) of TFV and FTC were prepared in UPH 2O. These were used to make the combined standard working stocks of TFV/FTC in UPH 2O. Individual standard prep stocks (1mg/mL) of TFV and FTC were prepared in ultra-pure water (UPH 2O).

Preparation of stocks and standard calibrators, quality control and internal standard samples This communication provides the methodology and supporting analytical validation results for the measurement of TFV and FTC in DBS.Дў.2. We previously described preliminary analyses and the potential clinical application of DBS for adherence monitoring in subjects receiving tenofovir and emtricitabine, but we did not describe the method validation. An important consideration for tenofovir and emtricitabine in DBS will be the high accumulation of phosphorylated tenofovir in red blood cells. Because of these advantages, many DBS methods have been developed for a variety of drugs, including antiretrovirals, but a DBS method for tenofovir and emtricitabine has not been developed, to our knowledge. The dried blood spot (DBS) blood sampling strategy offers advantages to plasma drawbacks: blood for DBS can be collected and processed at any clinic or research facility quickly and easily, and DBS requires only ~25 ВµL of whole blood, making it suitable for pharmacokinetic studies in special patient populations. Blood collection for harvesting plasma typically requires в‰Ґ 4mL of blood, which can add up to large volumes when conducting pharmacokinetic studies among infants or pediatric patients. Plasma processing is time consuming, and it requires technical personnel and onsite or nearby centrifugation. Thus far, most assays have utilized plasma or serum matrices for these studies. Measurement of TFV and FTC in plasma has been used to monitor adherence to therapy, and to evaluate pharmacokinetics in special populations such as infants, children, pregnancy, and for drug-drug interaction studies. Both agents are also active against Hepatitis B virus (HBV) infection, although only tenofovir has an indication for HBV treatment. These agents are co-formulated as Truvada, among other co-formulation products.

Tenofovir (TFV) and emtricitabine (FTC) are widely-prescribed antiretroviral drugs used for the treatment and prophylaxis of HIV infection. Comparison of TFV and FTC in DBS versus plasma yielded r 2 в‰Ґ 0.96, indicating that DBS can be used as a plasma alternative for pharmacokinetic analyses in vivo. Analytes were stable for five freeze/thaw cycles and up to 6 days at room temperature, whereas long-term storage required в€’20вЊ or в€’80вЊ. The method was accurate (within В± 15% of control) and precise (coefficient of variation в‰¤ 15%) for hematocrit concentrations ranging from 25% to 76% using edge punches versus center punches and spot volumes of 10ВµL to 50ВµL. The assay was validated over the range of 2.5ng/mL to 1,000ng/mL for TFV and 2.5ng/mL to 5,000ng/mL for FTC. 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. 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.

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