UPLC–MS/MS method for the simultaneous quantification of anti-HBV nucleos(t)ides analogs: Entecavir, lamivudine, telbivudine and tenofovir in plasma of HBV infected patients

• First UPLC–MS/MS method to simultaneously quantify anti-HBV nucleoside analogs.
• Fully validated method following FDA guidelines.
• Fast and cheap protein precipitation protocol, suitable for clinical routine.
• The method has been tested on real-life specimens with good results.

Hepatitis B infection affects two billion people worldwide and 350 million of these are chronically infected. Chronic hepatitis B virus is one of the most important cause of mortality and morbidity worldwide. If it is left untreated, about one-third of affected people will develop progressive and possibly fatal liver disease, like hepatic cirrhosis and primary hepatocellular carcinoma. Currently, five nucleos(t)ide analogs are approved for the treatment of chronic HBV infection. They are: lamivudine, adefovir dipivoxil, telbivudine, entecavir and tenofovir disoproxil fumarate.In this work, we developed and validated an UPLC–Tandem mass spectrometry assay method capable of monitoring lamivudine, telbivudine, tenofovir and entecavir plasma concentrations.Both standards and quality controls (high, medium and low) were prepared in human plasma.Each sample was added with internal standard (5′amino-5′deoxy-thymidine) and then drugs were extracted through a protein precipitation protocol with acetonitrile + 0.1% formic acid and then dried. The extracts were resuspended in water and then injected into the chromatographic system. The chromatographic separation was performed on an Acquity UPLC HSS T3 1.8 μm 2.1 × 150 mm column, with a gradient of water and acetonitrile, both added with formic acid (0.05%). Accuracy, intra-day and inter-day precision at quality controls levels fitted all FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 30 HBV+ patients with good results. This simple analytical method could represent a useful tool for the management of anti-HBV therapy.

Overlaid chromatograms of tenofovir (A), lamivudine (B), telbivudine (C) and entecavir (D) at the level of lower limit of quantification and in blank plasma samples.Figure optionsDownload as PowerPoint slide