only observed three geometrical isomers (instead of four) for the UV-irradiated sample of 3,4-diCQA using a phenyl-hexyl column .
When the UV-generated geometrical isomers of 3,5-diCQA were analyzed on the phenyl-containing column matrices (at a column temperature of 30[degrees]C), only three peaks (instead of four peaks) were observed and resulted with the elution order T[M.sup.*][M.sup.#] (Figure 2(d), Table 2), as observed by Clifford et al.
Thus, to achieve the separation of 3,5-diCQA geometrical isomers on the phenyl-containing column matrices, a column temperature of 50[degrees]C was introduced and resulted in the elution order TC[M.sup.*][M.sup.#] (Figure 4) instead of the elution order T[M.sup.*][M.sup.#] seen in Figure 2(d).
Finally, the elution order for the geometrical isomers of 4,5-diCQA was seen to be [M.sup.*]TC[M.sup.#], only on three bi-phenyl columns and two C18 columns as summarized in Table 2 and demonstrated in Figure 2(e).
The Effect of Column Temperature on the Separation of diCQA Geometrical Isomers. From the above results, it is apparent that determining the elution order of diCQA geometrical isomers on different reverse-phase column matrices shows inconsistencies (Table 2).
From the above results, it is apparent that determining the elution profile/order of diCQA geometrical isomers on different reverse-phase column matrices (phenyl versus alkyl) shows inconsistencies.
For instance, using the Ultra C18 column, the separation of the 3,4-diCQA geometrical isomers was enhanced by the introduction of column temperature; however, the di-cis-isomer was not observed on the chromatogram, suggesting coelution of the di-cis-isomer.
Figure S1: Elution profile of 3,4-diCQA geometrical isomers obtained using Phenomenex phenyl-hexyl and acetonitrile as the mobile phase composition.
As previously stated, in an attempt to distinguish the geometrical isomers of CGA, Clifford et al.
Here, we suggest thus an MS-based method that differentiates CGA geometrical isomers (cis and trans) (Figure 4).
Despite such a limitation, the current study develops and suggests a Q-TOF MS approach that could accurately annotate CGA molecules (both regional and geometrical isomers), providing also a particular interpretation of the spectrum from MS fragmentation of these compounds.
Thus, this study clearly shows the ability of Q-TOF-MS as a technique for metabolite annotation in a mixture of compounds, with a unique promise to distinguish between geometrical isomers (particularly in the case of CGA molecules, as indicated in this study).