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

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.