Document Detail


A HPLC-MS method for the simultaneous quantification of fourteen antiretroviral agents in peripheral blood mononuclear cell of HIV infected patients optimized using medium corpuscular volume evaluation.
MedLine Citation:
PMID:  21071165     Owner:  NLM     Status:  In-Process    
Abstract/OtherAbstract:
A sensitive and accurate high performance liquid chromatography-mass spectrometric (HPLC-MS) method for the intracellular determination of 14 antiretroviral drugs in peripheral blood mononuclear cells (PBMCs) for HIV+ patients was validated. PBMCs are isolated by Ficoll density gradient centrifugation and cells count and the relative mean volume is performed with a Coulter(®) instrument. Extraction of drugs from PBMCs pellets was obtained with methanol:water (70:30, v/v), with quinoxaline added as internal standard, after a sonication step. Supernatant was dried and then dissolved in water/acetonitrile (60/40, v/v), before injection into a 2.1 mm×150 mm Atlantis(®) T3 3μ column. Chromatographic separations were performed using a gradient program with a mixture of water (0.05% formic acid), as mobile phase A and acetonitrile (0.05% formic acid), as mobile phase B. Analytes quantification was performed by electro-spray ionisation-single quadrupole mass spectrometry using the selected ion recording (SIR) detection mode. The positive ionization was used for the HIV protease inhibitors (PIs) indinavir, saquinavir, nelfinavir, nelfinavir M8 metabolite, amprenavir, darunavir, atazanavir, ritonavir, lopinavir, tipranavir, the integrase inhibitor (II) raltegravir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and etravirine, while the negative ionization is applied for efavirenz. The calibration curves were built using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.1 to 32 ng/mL (1-320 ng/mL for tipranavir) and fitted to a quadratic regression model weighted by 1/X. The mean extraction recovery for all PIs, II and NNRTIs was always above 82%. The method was precise, with a range of intra/inter-day percent standard deviation within 2.6-14.8%, and accurate with mean of percent coefficient of variation (CV%) from nominal values -7.85 to +9.7%. Each drug concentration evaluated was expressed in ng/mL and optimized using each patient medium corpuscolar volume and cell number. This analytical method is routinely used in our clinical research center for the assessment of intracellular levels of all PIs, raltegravir and NNRTIs commercially available at present.
Authors:
Antonio D'Avolio; Marco Simiele; Marco Siccardi; Lorena Baietto; Mauro Sciandra; Valentina Oddone; Francesca Romana Stefani; Silvia Agati; Jessica Cusato; Stefano Bonora; Giovanni Di Perri
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Publication Detail:
Type:  Journal Article     Date:  2010-11-10
Journal Detail:
Title:  Journal of pharmaceutical and biomedical analysis     Volume:  54     ISSN:  1873-264X     ISO Abbreviation:  J Pharm Biomed Anal     Publication Date:  2011 Mar 
Date Detail:
Created Date:  2010-12-21     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8309336     Medline TA:  J Pharm Biomed Anal     Country:  England    
Other Details:
Languages:  eng     Pagination:  779-88     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier B.V. All rights reserved.
Affiliation:
Laboratory of Clinical Pharmacology and Pharmacogenetics, Department of Infectious Diseases, University of Torino, Amedeo di Savoia Hospital, Corso Svizzera 164, Turin, Italy. antonio.davolio@unito.it
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