In most bioanalytical laboratories, high-resolution mass spectrometry (HRMS) systems with electrospray ionization (ESI) are hyphenated to liquid chromatography platforms. The latter typically operate ...

In most bioanalytical laboratories, high resolution mass spectrometry (HRMS) systems with electrospray ionization (ESI) are hyphenated to liquid chromatography platforms. The latter typically operate ...

One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0–12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8–26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.

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 the 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 in matrix effects. Both advantages are crucial to reliably accessing low-abundance compounds or weakly ionizable analytes. This work presents a microflow (μF) chromatographic setup coupled to a novel microfabricated multinozzle electrospray (mnESI) emitter with five nozzles spraying at 600 nL/min per nozzle for untargeted HRMS lipidomic profiling. With a runtime of 19 min, 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 a 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 five and four detected in AF/ESI, respectively), and 12 sitosterol esters, not yet reported in mammalian cell cultures.

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 the 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 in matrix effects. Both advantages are crucial to reliably accessing low-abundance compounds or weakly ionizable analytes. This work presents a microflow (μF) chromatographic setup coupled to a novel microfabricated multinozzle electrospray (mnESI) emitter with five nozzles spraying at 600 nL/min per nozzle for untargeted HRMS lipidomic profiling. With a runtime of 19 min, 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 a 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 five and four detected in AF/ESI, respectively), and 12 sitosterol esters, not yet reported in mammalian cell cultures.