title: Development and validation of a quantitative assay for the measurement of two HIV-fusion inhibitors, enfuvirtide and tifuvirtide, and one metabolite of enfuvirtide (M-20) in human plasma by liquid chromatography-tandem mass spectrometry.
abstract: A method for the quantification of two peptide HIV-1 fusion inhibitors (enfuvirtide, T-20 and tifuvirtide, T-1249) and one metabolite of enfuvirtide (M-20) in human plasma has been developed and validated, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). The analytes were extracted from plasma by solid-phase extraction (SPE) on vinyl-copolymer cartridges. Chromatographic separation of the peptides was performed on a Symmetry 300 C(18) column (50mmx2.1mm I.D., particle size 3.5 microm), using a water-acetonitrile gradient containing 0.25% (v/v) formic acid. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for peak detection. Deuterated (d60) enfuvirtide and (d50) tifuvirtide were used as internal standards. The assay was linear over a concentration range of 20-10,000 ng/ml for enfuvirtide and tifuvirtide and of 20-2000 ng/ml for M-20. Intra- and inter-assay precisions and deviations from the nominal concentrations were </=13%. Stability of the analytes was tested under all relevant conditions for sample handling. The method was capable to measure concentrations of enfuvirtide and its metabolite in plasma samples of human immunodeficiency virus type-1 (HIV-1) infected patients treated with the drug.
authors.affiliation: Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Section of Biomedical Analysis, Division of Drug Toxicology, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
authors.forename: I
authors.initials: I
authors.lastname: van den Broek
authors.forename: R W
authors.initials: RW
authors.lastname: Sparidans
authors.forename: A D R
authors.initials: AD
authors.lastname: Huitema
authors.forename: J H M
authors.initials: JH
authors.lastname: Schellens
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D000077560:Enfuvirtide; D015700:HIV Envelope Protein gp41 / Q000097:blood* / Q000737:chemistry; D023581:HIV Fusion Inhibitors / Q000097:blood* / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D010446:Peptide Fragments; C474741:peptide T1249; D000077560:Enfuvirtide