title: UHPLC-MS/MS method with automated on-line solid phase extraction for the quantification of entecavir in peripheral blood mononuclear cells of HBV+ patients.
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. 20 μL 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 0.039 ng to 5 ng (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.
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy. Electronic address: amedeo.denicolo@unito.it.
authors.forename: Amedeo
authors.initials: A
authors.lastname: De Nicolò
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Bonifacio
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy.
authors.forename: Lucio
authors.initials: L
authors.lastname: Boglione
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy.
authors.forename: Jessica
authors.initials: J
authors.lastname: Cusato
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy.
authors.forename: Debora
authors.initials: D
authors.lastname: Pensi
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy; S.C Pharmacy of "Maria Vittoria" Hospital, ASL TO2, Turin, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Tomasello
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Di Perri
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, "Amedeo di Savoia" Hospital, Turin, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: D'Avolio
mesh_terms: D057205:Automation, Laboratory / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D006515:Hepatitis B virus / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry* / Q000821:virology*; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C413685:entecavir; D006147:Guanine
keywords: Entecavir; HBV; On-line SPE; PBMC; Tandem mass spectrometry; UHPLC