Abstract

For the quantification of the novel non-nucleoside reverse transcriptase inhibitor etravirine in human plasma, dried blood spots and peripheral blood mononuclear cell (PBMC) lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Etravirine was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50 μL plasma. Extraction from dried blood spots was performed with a one-step extraction with a mixture of methanol, acetonitrile and 0.2 M zinc sulphate in water (1:1:2, v/v/v) and extraction from cell lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150 mm × 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. 13C 6-efavirenz was used as an internal standard. The analytical run time was only 10 min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 25–5000 ng/mL in plasma, 50–10,000 ng/mL in dried blood spots and a range of 5–2500 ng/mL in PBMC lysate. Accuracies ranged from 89% to 106% in plasma, from 94% to 109% in dried blood spots and from 91% to 105% in PBMC lysate. Precisions at the all concentration levels ranged from 1.9% to 14% in plasma, 4.7% to 20% in dried blood spots and from 3.1% to 11% in PBMC lysate. The bioanalytical assay was successfully incorporated with previously developed assays for the determination of all currently approved PIs and NNRTIs in plasma and dried blood spots and it is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with etravirine.

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