Will O-methyl-chrysoeriol-7-O-rhamnosyl-glucosyl-glucoside elute before Chrysoeriol-7-O-coumaroyl-glucosyl-glucoside on a C18 reverse phase column analyzed with UPLC-MS/MS methodology?
Hello,
Trying to address it from tool-based approach, than chemistry.
Worth trying out, the two cool tools for this question:
[a] PredRet: http://predret.org/how-to-use-predret/ needs login etc. but super-fun! and
[b] Not sure how this works: http://www.retentionprediction.org/hplc/howitworks.php
But then, the important question would be : I hope you are seeing the MS/MS spectra as well? Without it, not sure with just MS1 it is anywhere close to the true compound identification/ annotation. if you have high res MS/MS spectra matching for the two compounds, then the elution order may not matter a lot. But without it, everything is doubtful, unless validated with true authentic chemical standards run the same way.
Hope it helps.
Thanks,
The answer depends on the strength of your mobile phases and the difference in solubility (and the selective retention) of the methyl and acyl molecules. It also depends on whether the column is 'normal phase' or 'reverse phase', temperature, protonation.... This is the rationale behind the use of LC-MS (differentiation)!
Biswapriya Biswavas Misra Thank you so much for your response.
Yes, I am doing MS/MS, however, I am confused on the identity of a certain mass.
For O-methyl-chrysoeriol-7-O-rhamnosyl-glucosyl-glucoside (m/z 787), I can see fragments corresponding to losses of a O-methyl group (m/z 15), chrysoeriol (301), rhamnosyl (146) and 2 glucosyl's (162).
For Chrysoeriol-7-O-coumaroyl-glucosyl-glucoside (m/z 771) I can see fragments 301 (chrysoeriol), 162's (corresponding to the two glucose moeities) and again m/z 146 (coumaroyl).
However, both rhamnose and coumaroyl moeities give a mass loss of 146. Generally in reverse phase the O-methyl counterpart of the same flavonoid elutes after. However, I am seeing the m/z 787 peak eluting before the m/z 771 peak. And hence I am unable to differentiate if the observed mass loss of 146 corresponds to a rhamnose or a courmaroyl unit. So, I thought may be a rhamnose moiety may be more polar than a coumaroyl moiety and hence is increasing the polarity of the peak with m/z 787 compared to m/z 771?
Any inputs on this?
There will present a daughter Ion of coumaroyl moiety (145) in the ms/ms of Chrysoeriol-7-O-coumaroyl-glucosyl-glucoside (m/z 771), perhaps no in O-methyl-chrysoeriol-7-O-rhamnosyl-glucosyl-glucoside .
I will look at the LogP for both compounds and see how close they are togethet and compare with something that you alteady know its retention time, and LogP; elution time have nothing to do with mass spec or MRM
Thank you for the responses everyone.
Jinle Xiang Yes, that is a very good suggestion.
I can now see minor fragments of 146 in the peak eluting earlier however no real presence in the later one.
So, surprisingly 1. O-methyl-Chrysoeriol-O-coumaroyl-hexosyl-hexoside is eluting before 2. Chrysoeriol-O-rhamnosyl-hexosyl-hexoside.
Is this possible on reverse phase UPLC C18 column? I would expect 1 to elute after 2 as it seems more non-polar overall. However, can this possibly happen in a complex natural extract?
Shreeya Ravisankar it's possible indeed! Sometimes, it seems that some compounds have minor different elution order in different C18 column.
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