Investigation of isomeric structures in a commercial mixture of naphthenic acids using ultrahigh pressure liquid chromatography coupled to hybrid traveling wave ion mobility-time of flight mass spectrometry

The separation of isomeric naphthenic acids (NAs) is of high importance to obtain more detailed and therefore useful information to understand their fate, transport, toxicity and potential removal treatment methodologies. In the current study, the capabilities of the ultrahigh pressure liquid chromatography/traveling wave ion mobility-time of flight mass spectrometry (UPLC/TWIM-TOF-MS) to separate and study different isomeric structures of NAs were investigated. Fifty seven standard compounds belonging to different chemical families of classical NAs were analyzed to obtain their experimental drift times in addition to chromatographic retention time and mass-to-charge information (m/z). These acyclic and cyclic molecules yielded ions with collision cross section (CCS) values ranging from 110 to 210 Å2. The feasibility of the UPLC/TWIM-TOF-MS method to provide a higher degree of confidence in the identification of isomeric structures was evaluated by analyzing the commercial (Sigma-Aldrich) NAs mixture. Identification and structure confirmation of several alicyclic compounds of similar m/z in the NAs mixture were possible by this method. For instance, the presence of previously tentatively identified compounds by the ultrahigh pressure liquid chromatography/quadrupole time of flight mass spectrometry (UPLC/QTOF-MS), such as 4-tert-butylcyclohexanoic acid, 4-dicyclohexylacetic acid or 3,5-dimethyladamantane-1-carboxylic acid, was confirmed. Combining ion mobility separation with UPLC/TOF-MS gives a higher level of selectivity to the overall method by selective interrogation of specific retention time, mass-to-charge and mobility regions. However, there are cases where it is not possible to resolve many similar molecules such as acyclic isomeric compounds in commercial NAs mixture by this technique. This was likely due to the very small CCS area differences among the structural isomeric species. Considerable improvements in the ion mobility resolution and separation will be required for this technique to be able to resolve isomeric species with slight differences in physicochemical properties (e.g., size and structure).