To date five nucleoside analogs are used in the treatment of chronic hepatitis B: among these, entecavir is the most used. Nevertheless a few information about its distribution in tissues is currently known. Since the determination of entecavir disposition in the hepatocytes is impracticable because of its invasiveness, the quantification in an "easier-to-obtain" cellular model could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry assay based on an automated on-line SPE, to quantify entecavir concentrations in peripheral blood mononucleated cells (PBMCs), in both its phosphorylated and un-phosphorylated forms. To achieve this, each PBMC isolate was divided in two aliquots, one was treated with acid phosphatase to convert entecavir phosphorylated metabolites into free form, the other one was not-treated. Standards and quality controls were prepared in PBMCs, isolated from healthy donors, and underwent the same process. 20μL of the resulting solutions were injected in the on-line SPE system. Thymidine was used as internal standard. Calibration curves fitted a linear model for entecavir levels in a range from 0.039ng to 5ng (mean r(2)=0.998). Accuracy, intra-day and inter-day precision of the method fitted FDA guidelines recommendations. Moreover, recovery was consistent and matrix effect resulted low and reproducible. We tested this method by monitoring entecavir concentrations in PBMCs from 28HBV mono-infected patients, confirming its reliability and suitability for the evaluation of intracellular entecavir penetration.

UHPLC-MS/MS method with automated on-line solid phase extraction for the quantification of entecavir in peripheral blood mononuclear cells of HBV+ patients

DE NICOLO', AMEDEO
First
;
BOGLIONE, Lucio;CUSATO, JESSICA;PENSI, DEBORA;DI PERRI, Giovanni;D'AVOLIO, ANTONIO
Last
2016-01-01

Abstract

To date five nucleoside analogs are used in the treatment of chronic hepatitis B: among these, entecavir is the most used. Nevertheless a few information about its distribution in tissues is currently known. Since the determination of entecavir disposition in the hepatocytes is impracticable because of its invasiveness, the quantification in an "easier-to-obtain" cellular model could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry assay based on an automated on-line SPE, to quantify entecavir concentrations in peripheral blood mononucleated cells (PBMCs), in both its phosphorylated and un-phosphorylated forms. To achieve this, each PBMC isolate was divided in two aliquots, one was treated with acid phosphatase to convert entecavir phosphorylated metabolites into free form, the other one was not-treated. Standards and quality controls were prepared in PBMCs, isolated from healthy donors, and underwent the same process. 20μL of the resulting solutions were injected in the on-line SPE system. Thymidine was used as internal standard. Calibration curves fitted a linear model for entecavir levels in a range from 0.039ng to 5ng (mean r(2)=0.998). Accuracy, intra-day and inter-day precision of the method fitted FDA guidelines recommendations. Moreover, recovery was consistent and matrix effect resulted low and reproducible. We tested this method by monitoring entecavir concentrations in PBMCs from 28HBV mono-infected patients, confirming its reliability and suitability for the evaluation of intracellular entecavir penetration.
2016
118
64
69
Entecavir; HBV; On-line SPE; PBMC; Tandem mass spectrometry; UHPLC
DE NICOLO', Amedeo; Bonifacio, Gabriele; Boglione, Lucio; Cusato, Jessica; Pensi, Debora; Tomasello, Cristina; DI PERRI, Giovanni; D'Avolio, Antonio...espandi
File in questo prodotto:
File Dimensione Formato  
3 UHPLC-MS method with automated on-line solid phase extraction for the quantification of entecavir.pdf

Accesso riservato

Tipo di file: PDF EDITORIALE
Dimensione 597.6 kB
Formato Adobe PDF
597.6 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1563690
Citazioni
  • ???jsp.display-item.citation.pmc??? 4
  • Scopus 16
  • ???jsp.display-item.citation.isi??? 14
social impact