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Atmospheric pressure sampling mass spectrometric methods are ideal platforms for rapidly analyzing the metabolomes of biological specimens. Several liquid extraction-based techniques have been developed for increasing metabolome coverage in direct sampling workflows. Here, we report the construction of a dual-probe microsampling device (DPM), based on the design of the liquid microjunction surface sampling probe, for analyzing the metabolome of live microglial cells by drift-tube ion mobility spectrometry (IMS) quadrupole time-of-flight mass spectrometry. Utilizing two distinct solvent systems in parallel is demonstrated to extract a wide structural variety of metabolites and lipids, enabling a more comprehensive analysis of intracellular metabolism. Employing the DPM-IM-MS method to adherent cells yielded the detection of 73 unique lipids and 79 small molecule metabolites from each optimized solvent system probe, respectively. Integration of multiplexed ion mobility scans is also shown to increase extracted analyte signal intensities between 2- and 10-fold compared to traditional single-pulse IMS, enabling the detection of 38 low-intensity features not previously detected by single-pulse DPM-IM-MS. To examine the ability of the DPM system to differentiate between sample treatment groups, microglia were stimulated with the endotoxin lipopolysaccharide (LPS). Several metabolic alterations were detected between sample treatment groups by DPM-IM-MS, many of which were not previously detected with conventional single-probe liquid microjunction surface sampling.

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