To chemically define the structures of the plasma analytes selected for further investigation (i.e. analytes with m/z 76, 104 and 118 in positive MS1 mode), multiple approaches were used. Analytes of interest were isolated by HPLC, vacuum dried, re-dissolved in water and injected onto the same phenyl column with a distinct HPLC gradient (LC2, flow rate: 0.8 ml/min) starting from 0.2% formic acid over 2 min, then linearly to 18% acetonitrile containing 0.2 % formic acid over 18 min and further to 100% acetonitrile containing 0.2 % formic acid over 3 min. The targeted analytes were identified by their m/z and the appropriate fractions recovered. After removal of solvent, dry analytes were used for structural identification.