Untargeted Exhaled Breath Condensate Metabolomics by ESI, ESCi, and DART Ion Mobility-Time-of-Flight Mass Spectrometry

JOSÉ J. PÉREZ; MARÍA EUGENIA MONGE; NAEL A. MCCARTY; FACUNDO M. FERNÁNDEZ

Baltimore

Conferencia; 62nd ASMS Conference on Mass Spectrometry and Allied Topics; 2014

American Society for Mass Spectrometry

Introduction It is widely established that the chemical composition of airway secretions is affected by pulmonary diseases. This can be probed non-invasively through sampling of exhaled breath condensate (EBC), allowing new insight into biochemical and inflammatory processes in the lung via untargeted metabolomics. Typically, chromatography-mass spectrometry methods are used in the metabolomics discovery phase. However, direct ionization or continuous infusion techniques coupled with ion mobility-time-of-flight mass spectrometry (IM-TOF MS) may offer alternative, higher-throughput approaches while still providing adequate metabolome coverage due to the relatively lower complexity of EBC. The present work investigates several direct ionization techiques for their feasibility in generating complimentary datasets to profile the EBC metabolome. Methods EBC samples collected with an R-TubeTM from healthy volunteers were pooled to make one homogenous EBC sample for method comparison. This pool was frozen at -80 ºC (2-h minimum); freeze-dried overnight; and reconstituted, without derivatization, in acetonitrile:water 80:20 v/v (concentration factor=20). Direct-infusion electrospray ionization (ESI), direct-infusion electrospray chemical ionization (ESCi), and transmission-mode direct analysis in real time (TM-DART) traveling wave ion mobility spectrometry (TWIMS) with TOF-MS detection were performed using a Synapt G2 High Definition Mass Spectrometry system (Waters Corporation). Positive and negative ion, high-resolution mass spectra were acquired in the 50-1200 m/z range. MZmine 2.10, MassLynx V4.1, and DriftscopeTM HDMS v2.1 were used for data analysis. Exact mass and drift time matching with standards was used for metabolite identification. Preliminary Data ESI, ESCi, and TM-DART TWIMS-TOF MS-based methods were developed and evaluated for comparability and feasibility in untargeted EBC metabolomics analyses. Positive and negative ionization conditions which yielded the most detected peaks were selected for each ionization mode. For direct infusion methods (i.e. ESI and ESCi), a 2-min acquisition time and 2-µL/min infusion flow rate (4-µL total sample volume) were determined to be optimal for acquiring sufficient spectra for comparison. For TM-DART, a 4-µL sample volume added to the mesh resulted in comparable, if not better, mass spectra than larger sample volumes (e.g. 15 µL). This volume resulted in a shorter drying time (5 min) prior to TM-DART analysis and allowed for more accurate comparisons between methods. Finally, the DART gas heater temperature was varied between 250-450 ºC to evaluate its influence on sensitivity and number of detected peaks. This temperature was optimized at 250 ºC and 300 ºC for positive and negative ion modes, respectively. TWIMS separations were achieved with the use of linear wave height and velocity ramps of 10-40 V and 200-800 m/s, respectively. Under the studied instrumental conditions, the ESI and ESCi methods resulted in ions generally within the range of m/z 100-800 in positive mode, and m/z 100-970 in negative mode; with negative mode exhibiting more distinct features. TM-DART spectra resulted in ions within a smaller m/z range in both ion polarities (150-750 in positive; 150-650 in negative) with clear differences in the ionic species generated and detected when compared to those in ESI and ESCi. It was concluded that direct infusion methods offer more rapid EBC analysis than traditional LC-MS methods, and that TM-DART possessed additional advantages with regards to even shorter analysis time, minimization of carryover, and ultimately, higher throughput. Novel Aspect Direct-infusion ESI, direct-infusion ESCi, and TM-DART TWIMS TOF-MS methods for EBC metabolomics studies are compared for the first time.