In the three-valve system, during this wash run, the phospholipid trap column remains configured off-line from the protease column and is re-equilibrated with solvent A (typically 0.23% formic acid), preparing it for the subsequent sample injection. The three-valve configuration also provides flexibility, as the phospholipid trap column can be positioned up-or downstream the protease column, allowing sample delipidation to be performed at protein or peptide level, respectively, without requiring further modifications on the LC methods.