Acquity lcms system

Five mg of lyophilized PEP04314 (0.71 µmol, Affibody AB, Solna, Sweden) was added to 0.5 mL of 0.2 M ammonium acetate buffer (pH 7.5), and the solution was added to a reaction vial containing 0.4 mg (0.72 µmol) of (±)-H₃RESCA-maleimide or maleimide-mono-amide-NOTA (Macrocyclics, Plano, Tx, USA). The reaction mixture was kept at room temperature for 90 min before being transferred to an ultracel 3K centrifugal filter (Merck) containing 3 mL of 0.1 M ammonium acetate buffer (pH 4) and centrifuged at 4000 rpm for 90 min. The flow-through was discarded, and 4 mL of fresh 0.1 M ammonium acetate buffer (pH 4) was added. The filter was then centrifuged again for 90 min, and the flow-through was discarded. Purified (±)-H₃RESCA-PEP04314 or H₂NOTA-PEP04314 was collected via reverse spin in 1 mL of 0.1 M ammonium acetate buffer (pH 4.5), and stored at -70 °C in 100 µL aliquots prior to use. Purity of the final product was determined via a Waters Acquity LC/MS system equipped with a Waters Xselect CSH C18 column (250 mm ×10 mm, 130 Å) at a flow rate of 5 mL/min using a gradient of EtOH (10-40% over 15 min) and 0.1% formic acid.