Abstract
RationaleFor the characterization of the chemical composition of complex matrices such as tobacco smoke, containing more than 6000 constituents, several analytical approaches have to be combined to increase compound coverage across the chemical space. Furthermore, the identification of unknown molecules requiring the implementation of additional confirmatory tools in the absence of reference standards, such as tandem mass spectrometry spectra comparisons and in silico prediction of mass spectra, is a major bottleneck.MethodsWe applied a combination of four chromatographic/ionization techniques (reversed‐phase (RP) – heated electrospray ionization (HESI) in both positive (+) and negative (−) modes, RP – atmospheric pressure chemical ionization (APCI) in positive mode, and hydrophilic interaction liquid chromatography (HILIC) – HESI positive) using a Thermo Q Exactive™ liquid chromatography/high‐resolution accurate mass spectrometry (LC/HRAM‐MS) platform for the analysis of 3R4F‐derived smoke. Compound identification was performed by using mass spectral libraries and in silico predicted fragments from multiple integrated databases.ResultsA total of 331 compounds with semi‐quantitative estimates ≥100 ng per cigarette were identified, which were distributed within the known chemical space of tobacco smoke. The integration of multiple LC/HRAM‐MS‐based chromatographic/ionization approaches combined with complementary compound identification strategies was key for maximizing the number of amenable compounds and for strengthening the level of identification confidence. A total of 50 novel compounds were identified as being present in tobacco smoke. In the absence of reference MS2 spectra, in silico MS2 spectra prediction gave a good indication for compound class and was used as an additional confirmatory tool for our integrated non‐targeted screening (NTS) approach.ConclusionsThis study presents a powerful chemical characterization approach that has been successfully applied for the identification of novel compounds in cigarette smoke. We believe that this innovative approach has general applicability and a huge potential benefit for the analysis of any complex matrices.
Highlights
High‐resolution accurate mass spectrometry (HRAM‐MS)‐based non‐ targeted screening (NTS) is a key methodology for characterizing the chemical composition of complex matrices.[1]
Ammonium fluoride was added to the mobile phase to enhance ionization efficiency in the negative ionization mode, a recommendation that has been reported in the literature for global metabolite profiling.[28]
Given that the same analytical column and solvents were used for RP methods with positive ionization, these numbers indicate both a high degree of complementarity and many differences due to the varying susceptibility for matrix effects using either heated electrospray ionization (HESI)(+) or atmospheric pressure chemical ionization (APCI)(+) ionization mechanisms, as has been reported previously.[29,30]
Summary
High‐resolution accurate mass spectrometry (HRAM‐MS)‐based non‐ targeted screening (NTS) is a key methodology for characterizing the chemical composition of complex matrices.[1] One major part within such a workflow is compound identification that can be achieved by either matching compound features against spectral databases (suspect screening analysis [SSA]) or, without any prior knowledge, by comparing first‐order fragmentation (MS/MS) derived information with in silico predicted fragments from compound databases (non‐ targeted analysis [NTA]).[2] NTS enables the simultaneous identification and semi‐quantification of a large number of compounds using an unbiased approach. In addition to these separation modes, heated electrospray ionization (HESI) and atmospheric pressure chemical ionization (APCI), using both positively (+) and negatively (−) charged ionization, have provided complementary information[12,13] depending upon analyte polarity, size, and the presence or absence of heteroatoms and functional groups
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