Fused silica capillaries with internal diameter of 150-200 µm were considered as optimal columns for the current experiments due to their operation range of 2 to 5 μL/min flow rates. With a 5-or 8-nozzle emitter, the resulting flow per nozzle was reduced to sub-µL range, enabling a multiplexed nanoflow electrospray. Next, the 150 mm length was chosen to minimize the ratio of the injection-to-column volumes for good chromatographic performance, while achieving tolerable system backpressure. To our knowledge, the only commercially available column with these specifications was the Bruker PepSep C18-AQ 0.15 x 150 mm with a pressure tolerance up to 1'000 bar. We calculated the flow rate for this stationary phase to be 3 µL/min to achieve the targeted method runtime. The recommended nozzle design was the 8-nozzle emitter (10 µm ID), resulting in 0.375 µL/min/nozzle. To reduce the probability of clogging over a sequence with biological matrix, the 5-nozzle design (20 µm ID) providing 0.6 µL/min/nozzle was also tested. Mobile phase composition was adapted from the commonly used 60% acetonitrile in water (A) and 90% isopropanol in acetonitrile (B) to 50% acetonitrile (A) and 80% isopropanol (B), respectively, to decrease viscosity and to limit the maximal working system pressure to below 800 bar (tubing contribution ~200 bar). Gradient started with an isocratic step of 1 min at 3% B to allow sufficient time to separate early eluting lipids from the initial flow-through eluate which displays increased nanospray instability. Gradient program was adapted to obtain an elution pattern of major lipid classes and a run time similar to our in-house conventional lipidomics method (i.e. 19 min). The optimization of the M3 emitter placement was achieved together with a careful voltage ramping at initial gradient conditions for maximizing spray stability and ion sampling while avoiding discharges. Total current was kept below 10 µA (4.5-6 µA observed across the gradient). Sheath gas flow was adjusted to avoid accumulation of droplets at the nozzle tips. Finally, the lower mass limit of the survey scans was raised from m/z 220 to 400 to avoid the environmental contaminant ions of m/z 327.008 and 371.316 to affect the available C-trap capacity.