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Title:   Author  item
                                            Berhow, Markitem
                                            Bowman, Michaelitem
                                            GU, MING - Cerno Bioscience
  Submitted to: Meeting Abstract
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 5/31/2015
                                    Publication  Date: 6/4/2015
                                    Citation: Berhow, M.A., Bowman, M.J., Gu, M. 2015. Determining elemental composition of phytochemicals in camelina seed meal by high mass accuracy and spectral accuracy [abstract]. American Society for Mass Spectrometry Annual Conference.Interpretive Summary:
                                Technical Abstract: An optimized single run evaluation that would accurately determine the elemental composition of as many compounds present in an extract would greatly aid in the evaluation of plant tissues. For phytochemicals, we have used accurate mass analysis to quickly characterize the potential chemical formulas for both known and unknown compounds in the seed extracts. We were able to identify several phenolics in the methanol extracts of defatted camelina seeds including rutin and two derivatives of rutin. The identification of the later two compounds was accomplished by elemental composition determination through high spectral accuracy and confirmed with CID/HCD spectra. This result allowed for the focus of isolation and characterization of other unknown compounds in the camelina seed extract. Ground defatted seed samples were extracted with 50:50 DMSO:MeOH. The phytochemicals were separated by HPLC on a reverse phase C18 column with mobile phases of water vs. methanol with 0.15% formic acid, monitored with a photo-diode array detector set at key UV wavelengths, and evaluated on a Thermo Electron LTQ Orbitrap Discovery MS, with an electrospray ionization (ESI) source. The mass spectrometer was run in negative ionization mode, with a scan range from m/z 280 to 2000, and at resolving power of 7500. Mass-dependent MS/MS on the most abundant ion trapped by the Ion Trap MS scan was performed by higher energy C-trap dissociation (HCD) at 25% energy with the resulting fragmentation ions being detected by the Orbitrap MS. Spectral accuracy has been proved to be a powerful metric to eliminate false positive formulas during elemental component determination through peak shape calibration. In this preliminary study, we employed the spectral accuracy approach to determining elemental composition of three compounds from the extract through commercial available software. Under the search conditions with 5 ppm mass tolerance, possible elements of C H N O S, and some constraints of empirical rules, elemental composition determination of two high abundant ions of m/z 609 and 741 were performed to find their correct elemental composition as C27H29O16 and C32H37O20, respectively. Both C27H29O16 and C32H37O20 achieved very high spectral accuracy of 99.4% and ranked number one out of 47 and 102 possible candidates respectively, according their spectral accuracy. Under the same search conditions, elemental composition determination for a weak ion at m/z 941 with 10 times less intensities than m/z 609 was found to be C43H47O24 with spectral accuracy of 96.7%, which was ranked as number two out of 58 possible candidates, according to its spectral accuracy. In all three cases, spectral accuracy of correct elemental composition has at least 1.5% higher than the spectral accuracy of the compounds at rank four or below. This significant difference indeed removes at least 95% false positives, allowing confident determination of their elemental composition. These determinations were further confirmed by MS/MS of 941 which produced fragment ions at both 741 and 609 as structurally related species although they were eluted at different retention time as independent compounds. This method was used to identify 18 compounds in a methanol extract of defatted camelina seeds, seven of these were confirmed by comparison to standards or MS/MS fragmentation patterns. Elemental composition determination of unknowns in plant extracts based on both high mass accuracy and spectral accuracy.


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