title: A UPLC-MS/MS method for the simultaneous plasma quantification of all isomeric forms of the new anti-HCV protease inhibitors boceprevir and telaprevir.
abstract: HCV infection affects over 170 milions people in the world. Up to now standard-of-care therapy consisted in ribavirin plus interferon-α 2a or 2b. Recently, two new Direct Acting Antivirals (DAA), inhibitors of NS3 HCV protease, have been developed and approved for the routine use on HCV genotype 1 infected patients: boceprevir and telaprevir. Protease inhibitors show complex pharmacokinetics (strong metabolism of both drugs by CYP3A and drug interactions), they require a TID dosage and, furthermore, they are present in plasma patients in two different isomeric forms. In this work, we developed and validated an UPLC-tandem mass spectrometry assay method capable of monitoring the telaprevir and boceprevir concentrations in plasma, discriminating each isomer of both drugs. Blank plasma used for Standards and QCs preparation, was obtained from blood of healthy donors. The calibration curves for each isomer of telaprevir and boceprevir were linear in a range from 46.87 ng/mL to 6000 ng/mL and from 23.43 to 3000 ng/mL, respectively (mean r(2)>0.998 for all analytes). QCs at three different concentration were prepared: High, Medium and Low. Each Standard, QC and patient sample was treated with a protein precipitation protocol with a solution containing acidified acetonitrile and Internal Standard (6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline). An aliquot of supernatant was diluted and then directly analyzed by reverse-phase UPLC-MS/MS. Accuracy (mean 98.47%), intra-day (mean <5.21%) and inter-day (mean <7.57%) precision for telaprevir and boceprevir quality controls fitted all FDA guidelines. All compounds resulted stable in our method conditions and the extraction procedure showed a recovery near to 100%. Finally, we tested this method by monitoring plasma concentrations in 9 HCV+ patients, for each triple combined therapy, with good results. The observed median ratio between S and R isomers for boceprevir and telaprevir were 1.22 (IQR 1.10-1.33) and 1.52 (IQR 1.21-1.67), respectively. The use of this simple assay method could be an important tool for management of HCV-1 DAAs treated patients.
authors.affiliation: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy. antonio.davolio@unito.it
authors.forename: Antonio
authors.initials: A
authors.lastname: D'Avolio
authors.forename: Amedeo
authors.initials: A
authors.lastname: De Nicolò
authors.forename: Danilo
authors.initials: D
authors.lastname: Agnesod
authors.forename: Marco
authors.initials: M
authors.lastname: Simiele
authors.forename: Adnan
authors.initials: A
authors.lastname: Mohamed Abdi
authors.forename: Sujan
authors.initials: S
authors.lastname: Dilly Penchala
authors.forename: Lucio
authors.initials: L
authors.lastname: Boglione
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cariti
authors.forename: Giovanni
authors.initials: G
authors.lastname: Di Perri
mesh_terms: D002853:Chromatography, Liquid; D006526:Hepatitis C / Q000097:blood / Q000188:drug therapy / Q000201:enzymology*; D006801:Humans; D007536:Isomerism; D057230:Limit of Detection; D009842:Oligopeptides / Q000097:blood* / Q000627:therapeutic use; D011392:Proline / Q000031:analogs & derivatives* / Q000097:blood / Q000627:therapeutic use; D011480:Protease Inhibitors / Q000097:blood* / Q000627:therapeutic use; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D009842:Oligopeptides; D011480:Protease Inhibitors; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide; D011392:Proline