3.8. Stability in Serum

The serum stability of peptides was determined in 25% human serum in PBS (pH 7.4) from healthy male donor as described previously [31]. Briefly, 20 mL of an aqueous peptide stock solution (3 mg/mL) was added to 480 µL of serum solution and incubated for: 2, 8 and 24 h at 37 °C. After incubation, serum proteins were selectively precipitated from the mixture by adding 10% TFA in the presence of 3 M urea. In the control samples, the precipitation was carried out immediately after the addition of serum. Next, the samples were stored at 0 °C for 30 min and centrifuged at 14,000× g for 20 min. The obtained supernatants were analyzed by analytical RP-HPLC (Symmetry 300 C18 column) and MALDI-TOF MS. The amount of the intact peptide was estimated from the height of the corresponding peak on the RP-HPLC chromatogram. Two independent experiments in duplicate were performed for each peptide.