A PCA iKey Separation Device, coupled with IM-MS was successfully used to profile ginsenosides Rb1, (Rb2, Rc), (Rd, Re), (Rf, Rg1), and Rg2. IPA at 1 μL/min flowed into the PCA channel and a MS ES voltage of 2.6 kV was applied. IM-MS was used to generate TWCCSN2 values, precursor ion accurate mass, accurate mass mobility product ions, and retention times. Using TWCCSN2 measurements can increase targeted screening specificity.
A ginsenoside CCS scientific library within UNIFI was previously generated.2 This allows the expected and previously determined CCS values to be utilized to screen and confirmthe presence of isomeric flavonoid markers. Three extracts: gingko biloba+red panax, red panax, and Korean ginseng were screened against the library in order to determine their presence, and unequivocally identify isomeric ginsenosides. CCSvalues (derived from ion mobility drift times) are used as an identification parameter that can distinguish ginsenoside isomers, as well as to profile unknowns.
In Figure 3, the ionKey/MS System IM negative mode base peak ion chromatogram obtained for analysis of 10:1 diluted Korean ginseng tea extract is shown. Figure 3 depicts a conventional view of the complex sample profiled. However, in Figure 4 the ionKey/MS System IM negative mode plot of the drift time (ion mobility resolution), versus the retention time for 10:1 diluted Korean ginseng extract is presented. Leveraging the unique software functionality of UNIFI.
Figure 4 visually illustrates how an orthogonal separation to the chromatographic separation is achieved with ion mobility, and the increased peak capacity that is possible. The retention time region between 5 and 14 minutes in Figure 4 shows there are a large number of compounds that are now resolved, compared to the same region on the conventional base peak ion extracted mass chromatogram shown im Figure 3.
The ion mobility data viewer in UNIFI enables investigative interaction with acquired ion mobility data. UNIFI incorporates many easy-to-use features, such as Zoom to Component and Bookmark, that enable the same investigative interrogation of data to be applied across many acquisitions. It is possible to select any one of these components and generate the drift plot, mass spectrum, and extracted mass chromatogram.
The true complexity of the profiled sample is illustrated when both ion mobility and UPLC chromatographic resolution are combined. Ginsenoside isomers Rg1 m/z 845.4897 (green) and Re m/z 991.5484 (brown) in Figure 5 are chromatographically coeluting at 8.058 minutes. Figure 5 shows the combined precursor and fragmentation spectra data of the two coeluting ginsenoside components.
In Figure 6 the single component retention time aligned and drift time aligned ion mobility product ion spectrum for the ginsenoside isomer Rg1 is presented, further illustrating the utility of ion mobility separations.
In Figure 7 the ion mobility separated ginsenoside Re:A and ginsenoside Rg1:B mobility peaks are presented. Here, the single component precursor/ion mobility product ions are obtained, even thoughthey chromatographically coelute with other compounds present in the complex ginseng extract.TWCCSN2 measurements can increase confidence in identification. The results are summarized in the UNIFI Component Summary shown in Figure 8, where the values obtained for the profiling of Korean ginseng are presented. For the marker ginsenoside isomer pairs (Rb2, Rc), TWCCSN2 measurements of 355.24 Å2/344.50 Å2, (Rd, Re), 328.31 Å2/323.46 Å2, and 301.60 Å2/292.03 Å2 (Rf, Rg1) were obtained. The TWCCSN2 measurement errors were typically <2%, when compared to the study performed using UPLC-IM-MS in 2013.2 This further confirms that it is possible to confidently distinguish, the marker isomer pairs of ginsenosides from the extracts of the specified products analyzed, using TWCCSN2 measurements.
This approach offers unique selectivity for profiling complex mixtures. The results obtained clearly show the benefits of using CCS measurements and the combined peak capacity of ionKey/MS System with IM. Coeluting analytes and isomers have been resolved, as well as unequivocally identified in the three extracts profiled. In addition, it is possible to acquire the cleaned up mobility specific product ion spectra, that are mobility resolved from coeluting components. This approach has the potential to change the scope of authentication profiling.
The added confidence in making identifications using TWCCSN2 measurements has the potential to reducethe need to use expensive high purity standards, where assay confirmation relies on retention time and accurate mass measurement. The cost of purchasing 10 mg of each standard for the assay performed totalled £2483.00, a significant cost undertaking. Cost savings across many application areas can be made.
