U.S. Department of Energy

Pacific Northwest National Laboratory

Building a Library with >1000 Ion Mobility Collision Cross Sections for Ultrafast Small Molecule Analyses


Metabolomics and exposomics studies have been growing rapidly during the past decade and providing insights into cellular metabolism and how a system responds to stimuli. Ion mobility spectrometry-mass spectrometry (IMS-MS) has become an appealing tool for small molecule studies as it enables the analysis and separation of the many isomeric species. However, to facilitate the identification of endogenous metabolites and xenobiotics from complex samples, a library with accurate collision cross sections (CCS) is needed. In this work, we generated a CCS library for >1000 small molecule standards using triplicate analyses in positive and negative polarity. This library was then coupled with ultrafast small molecule analyses using a sub-minute online SPE-IMS-MS method for characterization of different metabolic and xenobiotic conditions.


IMS-MS is capable of separating molecules that have the same mass-to-charge (m/z) ratios, but different sizes, shapes or conformations. In this work an Agilent drift tube-IMS-QTOF MS platform was used to characterize over one thousand endogenous metabolites and xenobiotics. The CCS values were measured using seven stepped fields in both positive and negative polarity. For small molecule studies, the samples were analyzed by coupling Rapidfire, an online SPE system to the IMS-MS platform which enables sub-minute ultrafast analyses. Different SPE cartridges such as C18, graphitic carbon, HILIC and phenyl cartridges were explored to effectively extract different classes of molecules. In addition, different ionization sources including ESI, APCI and APPI were evaluated for efficient small molecule studies.

Preliminary Data 

A large scale ion mobility CCS library including over 800 endogenous metabolites and over 200 xenobiotics was generated using drift tube IMS-MS. Different classes of metabolites displayed different trend lines for their CCS values. For instance, fatty acids show larger CCS values than carbohydrates or amino acids. We were also able to map the CCS values for key metabolites and intermediates in important metabolite pathways such as glycolysis/gluconeogenesis, pentose phosphate pathway and citrate cycle, etc. These results are essential for future studies aimed at identification of metabolites within different structure classes and pathways. To evaluate the use of this CCS library for metabolite identification, we analyzed how small molecules change in patients with and without exercises, as well as those with and without ethanol use, and compared the results from the SPE-IMS-MS method with those from LC-MS method.

Xenobiotics that are important for the evaluation of environmental exposure such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) exposures were also studied. Different ionization sources including ESI, APCI and APPI, were evaluated for efficient characterization and detection of the PAHs and PCBs and their metabolites. Preliminary data showed that we were able to detect PAHs in [M∙]+ and [M + H]+ forms and PCBs in the [M-Cl+O]- form using APCI/APPI methods. We also showed that the pre-separation, clean-up and enrichment steps by Rapidfire SPE greatly improved the sensitivity, enabling ultrafast and simultaneous analyses of PAHs and PCBs and their metabolites in complex biological and environmental samples.


Novel Aspect 

Development of a large small molecule CCS library and a SPE-IMS-MS platform for ultrafast metabolomics studies

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