Development and validation of an assay for the simultaneous determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography-tandem mass spectrometry.

This paper describes the development and validation of an assay for the simultaneous quantification of the antiviral and antiretroviral drugs zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography coupled to tandem mass spectrometry. Sample pretreatment consisted of protein precipitation with 0.1% (v/v) formic acid in methanol, evaporation and reconstitution. Chromatographic separation was performed on a Synergy Polar reversed phase C18 column (150 mm × 2.0 mm ID, particle size 4 µm) using a stepwise gradient with 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in methanol at a flow rate of 300 µL/min. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for drug detection and quantification. Isotopically labeled zidovudine, lamivudine, tenofovir and ribavirin were used as internal standards. The method was validated over a clinical range of 20-2500 ng/mL for zidovudine, lamivudine and tenofovir, 4-500 ng/mL for abacavir and emtricitabine and 160-20,000 ng/mL for ribavirin. The inter and intra-assay accuracies and precisions were between -8.47% and 14.2% for zidovudine, emtricitabine and ribavirin. For abacavir, lamivudine and tenofovir, the inter and intra-assay accuracies and precisions at the lower limit of quantification were between -11.0% and 18.3%, whereas at all other levels these accuracies and precisions were between -11.7% and 12.0%. The described method is suitable for the determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma in clinical practice to monitor plasma concentrations in selected cases to optimize therapy.

Quantitative determination of oseltamivir and oseltamivir carboxylate in human fluoride EDTA plasma including the ex vivo stability using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Oseltamivir, the ethyl ester prodrug of the neuramidase inhibitor oseltamivir carboxylate, is licensed for the treatment of patients with influenza virus infection. Here we describe the development and validation of an assay for the simultaneous quantification of oseltamivir and oseltamivir carboxylate in human fluoride EDTA plasma including the ex vivo stability using liquid chromatography coupled to tandem mass spectrometry. Sample pretreatment consisted of protein precipitation with 8% (v/v) trichloroacetic acid in water using only 50 µL plasma. Chromatographic separation was performed on a reversed phase C18 column (150 mm × 2.0 mm ID, particle size 4 µm) with a stepwise gradient using 0.1% formic acid and methanol at a flow rate of 250 µL/min. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for detection and drug quantification. The method was validated over a range of 3-300 ng/mL for oseltamivir and 10-10,000 ng/mL for oseltamivir carboxylate. Deuterated oseltamivir and oseltamivir carboxylate were used as internal standards. The intra-assay accuracies and precisions for oseltamivir were between -8.8 and 16.3% at the LLOQ level, whereas for all other concentration levels this was -8.6 and 14.5%. For oseltamivir carboxylate the intra-assay accuracies and precisions were between -10.9 and 10.7% at all levels. Furthermore, oseltamivir was stable in plasma and whole blood ex vivo in commercially available fluoride EDTA tubes for at least 24h at 2-8 °C. This method is now applied for the determination of both compounds in specific patient populations to evaluate current dosing guidelines.