Microflow liquid chromatography coupled to multinozzle electrospray ionization for improved lipidomics coverage of 3D clear cell renal cell carcinoma

In most bioanalytical laboratories, high resolution mass spectrometry (HRMS) systems with electrospray ionization (ESI) are hyphenated to liquid chromatography platforms. The latter typically operate under analytical flow (AF) regimes, with flow rates in the range of 0.2 to 1 mL/min. Hence, AF/ESI-HRMS methods prioritize detection of analytes of higher abundances or ionizability and tend to suffer from detrimental phenomena like matrix effects and/or ion suppression. A far higher sensitivity can be obtained with nanoelectrospray thanks a better ionization efficiency including a significant decrease of matrix effects. Both advantages are crucial to reliably access low-abundant compounds or weakly ionizable analytes. This work presents an original microflow (µF) chromatographic set-up coupled to a novel microfabricated multinozzle electrospray (mnESI) emitter with 5 nozzles (600 nL/min per nozzle) for untargeted HRMS lipidomic profiling. With a runtime of 19 minutes, similar to our established analytical flow (AF/ESI) lipidomics platform, µF/mnESI produced a 28-fold median increase across 69 deuterated lipid standards. The performance of this new configuration was also evaluated in the context of the profiling of 3D clear cell renal cell carcinoma (ccRCC) model exposed to a multidrug combination therapy. The processing of the acquired data resulted in 1270 (µF/mnESI) vs. 752 (AF/ESI) MS2-annotated lipids. Among those, 762 achieved <10% variation on pooled QC samples for µF/mnESI compared to only 361 for the AF method. In addition, the microflow ccRCC sequence confirmed improvements in ionization efficiency and adduct patterns observed in standards. These advantages enabled to annotate 79 oxidized triglycerides, 38 cholesterol esters (only 5 and 4 detected in AF/ESI, respectively), as well as 12 sitosterol esters, not yet detected in cell cultures.