Peptide Stability Evaluation in Human Serum

Human serum was diluted to 25% (v/v) in sterile DPBS and equilibrated at 37°C for 15 min. Peptides were added to the diluted serum to a final concentration of 20 μM and incubated at 37°C with gentle mixing. At designated time points, 30 μL aliquots were withdrawn and mixed with 30 μL of 15% trichloroacetic acid in MeOH (w/v) and 30 μL of MeCN and stored at 4°C overnight to completely precipitate the serum proteins. Next, each aliquot was centrifuged (15,000g, 5 min, 4°C) and 14 μL of the supernatant was injected in an UPLC equipped with an analytical Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 mm I.D., 100 mm length) and an inline UV-Vis detector and a single quadrupole mass detector. The column was eluted with a linear gradient of 20–50% MeCN in ddH₂O containing 0.1% TFA. A UV/Vis detector at 214 nm and the mass spec quantifying the ion counts corresponding to the [M + 3H]³⁺ ion of the analytes were used to analyze the samples. The area under the peak of interest was obtained by integration, normalized to that of the peptide at t = 0, and plotted as a function of the incubation time to determine the serum half-life of the peptide.