The Agilent 1290 Infinity 2D-LC solution was used to study the lipid composition of blood plasma and induced sputum samples. Applying multiple heart-cutting enabled a fully automated HILIC-based lipid (sub-)class type fractionation in the first dimension and online transfer of the fractions onto a high-resolution reversed‑phase UHPLC column in the second dimension for separation of group members according to hydrophobicity. Detection and identification of the lipids was carried out with the Agilent Accurate-Mass Q-TOF LC/MS System. The main advantage of this approach is a signifi cant reduction of the number of lipids that enter the mass spectrometer at a given time. This reduces the risk of misidentification and erratic quantification in complex lipid samples due to interfering isobaric lipids or isotopes of lipids.