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; 0.2-1 mL/min) regimes. Hence, AF/ESI-HRMS methods prioritize detection of analytes of higher abundances or ionizability and tend to suffer from matrix effects or ion suppression. A far higher sensitivity can be obtained with electrospray at nanoflow (10- 1000 nL/min) thanks to a better ionization efficiency and significant decrease of matrix effects. Both advantages are crucial to reliably access low-abundant compounds or weakly ionizable analytes. This work presents a microflow (μF) chromatographic set-up coupled to a novel microfabricated multinozzle electrospray (mnESI) emitter with 5 nozzles spraying at 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 16-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 measurements of ccRCC samples confirmed the improvements in ionization efficiency and adduct patterns observed with standards, enabling 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 reported in mammalian cell cultures.