Gemini nx c18 5 m column

Plasma-stability assays were performed by applying a modified procedure described by Seebach et al. (2011) and Roščić et al. (2008) . Each peptide solution was prepared by dissolving 1 mg of a peptide in 1 mL of PBS buffer (pH 7.42). The samples containing the peptide (200 µL) and a freshly refrozen plasma (50 µL) were incubated at 37 °C. They were analyzed after 0, 2, 24 h (sialorphin (peptide I) and peptide II) and 0, 2, 24 and 48 h (peptide XI). At each time interval 40 μL of the mixture was taken and deproteinised by addition of a 48% aqueous TFA (8 µL) and PBS buffer (112 µL). The aliquots were centrifuged at 14,500 rpm for 5 min. The supernatants were filtered over Millipore Millex-GV syringe filters with a 0.22 μm pore size PVDF membrane (Millipore) and analysed with RP-HPLC on a Phenomenex Gemini-NX C18 5 µm column (4.6 mm × 150 mm) using the solvent system of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B) and linear gradients of 1-30% (B) over 20 min (peptides I and XI) and 2-60% (B) over 20 min (peptide II). To assess the stability at each time interval, the area of the peptide peak from the chromatogram was calculated and expressed as the percentage of the area of the peak recorded just after mixing a peptide with serum (0 hour). The assay was performed at least in triplicate for each peptide.