11
orientin [8]. Subsequently, using UPLC- IM-MS negative mode ESI, characteristic 6-C and 8-C glycoside fragment ions were identified (see Figure 6) [25]. Comparing Figure 5 and 6, informatics evolution is also illustrated, where detected analyte structure and fragmentation dissociation pathways are incorporated into the targeted data processing results. Despite the improved peak capacity brought on by an evolution of chromatographic and mass resolution, coelution resulting from sample complexity can prevent the acquisition of non-targeted single component fragment ion data. However, ion mobility facilitates an additional dimension of separation. Co-eluting isobaric species, metabolites or isomers (that may produce the same product ions) would produce a mixed fragment ion spectrum (MSE
), but when they are
additionally separated using ion mobility (HDMSE
spectra occur.
Investigations into non-targeted authentication profiling using the combined specificity of UPLC-IM-MS and CID have been carried out to provide unambiguous identification of flavonoid analytes TW
CCSN2
Figure 5. I) 8-C glycoside (orientin) and II) 6-C glycoside (isoorientin) positive ion characteristic fragment ion ratios (highlighted) and elemental compositions.
(CCS values generated using travelling wave ion mobility and a nitrogen buffer gas) values were used to produce unequivocal identification of marker flavonoid isomers (6-C and 8-C-glycosylflavone isomer pairs orientin (187.7 Å2
and vitexin (195.5 Å2
)/isoorientin (198.1 Å2 )/isovitexin (188.8 Å2
) ))
[25]. Coeluting with structurally related analytes in the complex leaf extracts of Passiflora, the retention times determined for isoorientin and orientin were different by 0.05 min and for isovitexin/vitexin by 0.12 mins. In the Passiflora extracts analysed, the concentrations of isoorientin and orientin are such that they merge to form one chromatographic peak. Using accurate mass measurements alone, these coeluting marker isomers would be indistinguishable, however using both IMS and optimised UPLC conditions, both critical isomer pairs are resolved (see Figure 7) [25]. Not achievable with mass resolution alone, the isomeric product ion spectra shown in Figure 6 are produced from IM resolved chromatographically coeluting orientin and isoorientin isomers shown in Figure 7. Utilising HDMSE
, ‘fragment ions’ are
Figure 6. IMS resolved coeluting I) 8-C glycoside (orientin) and II) 6-C glycoside (isoorientin) negative ion characteristic product ion ratios (highlighted) and annotated structures.
generated from a specific retention time/ drift time aligned precursor ion, hence are referred to as product ions [26,27]. Detailed profiling of Passiflora species using ion mobility mass spectrometry have previously been described [25].
), single component fragment ion
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