title: Enzymatic digestion as a tool for the LC-MS/MS quantification of large peptides in biological matrices: measurement of chymotryptic fragments from the HIV-1 fusion inhibitor enfuvirtide and its metabolite M-20 in human plasma.
abstract: The use of enzymatic digests of the peptide HIV-1 fusion inhibitor enfuvirtide as a tool for the absolute quantification of this polypeptide (MW 4492 Da) in human plasma by LC-MS/MS has been evaluated. Two different methods applying digestion of enfuvirtide with chymotrypsin after solid phase extraction (SPE) of the plasma samples have therefore been developed and validated. One method used a stable isotopically labeled analog of the complete peptide (d60-enfuvirtide) as internal standard (IS) and could use as much as four different chymotryptic fragments for the quantification of enfuvirtide in a range of 100-10,000 ng/ml. Intra- and inter-assay precisions and deviations from the nominal concentrations varied for the different fragments, but were below 9% when the four results were averaged. The other method used a stable isotopically labeled chymotryptic fragment of the peptide (d10-ASLW) as IS. Although this IS does not correct for variations in digestion recovery, it allows the selective quantification of enfuvirtide (100-10,000 ng/ml), besides the quantification of the sum of enfuvirtide and its de-amidated metabolite M-20 (120-12,000 ng/ml). Both methods were suitable for the absolute quantification of enfuvirtide and M-20 in plasma, but proper selection of the fragment(s) used for the quantification appeared crucial when the deuterated fragment was used as IS.
authors.affiliation: Department of Pharmaceutical Sciences, Section of Biomedical Analysis, Division of Drug Toxicology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. I.vandenbroek@pharm.uu.nl
authors.forename: Irene
authors.initials: I
authors.lastname: van den Broek
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Sparidans
authors.forename: Jan H M
authors.initials: JH
authors.lastname: Schellens
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000077560:Enfuvirtide; D015700:HIV Envelope Protein gp41 / Q000097:blood*; D023581:HIV Fusion Inhibitors / Q000097:blood*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D010446:Peptide Fragments; D000077560:Enfuvirtide; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase