Plasma stability assays

The in vitro stability of Consomatin Ro1 was investigated in human plasma (mixed gender) and compared to human SS-14. Peptides were incubated in 500 μl of plasma at 37°C with a final concentration of 3 μM. Samples (40 μl) were taken at 0-, 5-, 15-, 30-, 60-, 120-, 240-, 480-, and 1440-min time points. A twofold volume of cold acetonitrile with 0.1% formic acid was added to terminate the incubation. Samples were applied on a C₁₈ column (Waters CSH; 2.1 mm × 50 mm, 1.7-μm particle size) with a flow rate of 0.650 ml/min and a gradient profile of 2 to 20% solvent B in 3.5 min and then 20 to 98% solvent B in 0.5 min and run on a Thermo Vanquish Horizon UHPLC. The HPLC solvents were 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). MS data were acquired using a Thermo Q-Exactive Focus Orbitrap mass spectrometer at 35,000 resolution (full width at half maximum at m/z 200) for full scan and 17,500 resolution for MS2 in data-dependent acquisition mode. Parent compound disappearance, based on relative LC-MS peak area (0 min = 100%), was used to calculate half-lives. Propanthelin bromide (1 μM) was used as a control, and two replicates for each compound were analyzed.