I an running glucoraphanin (GR) and sulforaphane(SFN) standard. I prepared 0.5ppm in MeOH from 1000ppm in DMSO stock solution. I have tried several columns (C18, K5, hypercarb and suplco LC columns), after comparisons, we chose suplco columns.
we also tried several mobile phases:
mobile phase: A: 13 mM ammonium acetate, acetic acid, pH4
or water+0.1% formic acid and acetic acid
B: Acetonitrile +0.1% formic acid or acetic acid
or MeOH+0.1% formic acid or acetic acid
we found SFN always have very nice performance(stable rentation time and lots of signal), but GR sometimes did not show up, sometimes the rentation time shifted and signal decreased for the same samples when we do repeations.
So is GR stable? and how to get a stable signal in MS?
Looking at the respective structures, glucoraphanin contains an oxime derivative, which are known to be hydrolytically unstable which is further catalyzed by acidity, either from additives (eg, 0.1% FA) or the inherent acidity of a column, but since you're using a reverse-phase column (C18), the latter is not an issue for you. The oxime derivative can be thought of as a sulfated (NB not sulfonated) oxime, making the hydroxy even more hydrolytically unstable and susceptible to degradation in water, especially in acidic aqueous media. So, oximes are unstable and glucoraphanin is an especially unstable oxime. In contrast, sulforaphane only contains a sulfoxide functionality (stable) and an isothiocyanate, which is reactive (especially with nucleophiles) but is relatively stable.
As a side note, this chemical reactivity of glucoraphanin is exploited biochemically in cells to form the isothiocyanate of sulforaphane. This reaction is initiated by the breakage of the glycosidic bond. This is just to say that this functionality is very reactive and this might also be mechanistically what's driving the instability as well.
So I suspect you're getting degradation, which is why your getting lower signal, and the degradation products. The differences in retention time is strange, but I think this is actually just degradation products and not a change in retention time of the analyte. One option to get a signal on MS is to do direct-inject (avoiding the column entirely) or try some basic additives or buffered solutions, such as acetonitrile and phosphate buffer pH ~7.2 supplemented with triethylamine 0.1%. There are a few other possible reasons and related solutions, but the above is what I think is the most likely issue and solution.
Grant Simpson thank you so much for your so detailed answer.
yes, GR is really unstable. Currently we are trying to set up a method that can separate and have good signal of GR and SFN as well as other SFN derivates. but we repeat the same samples just in 2 hours (the sample kept in multisampler which is 8 degree), the signal and rentation time of GR changed. We tried, many times the GR did not show up at all in MS. i will tried what you suggest.
Next, we also need to do Caco-2 monolayer absorption and transport, and i will have a lot of samples, so if GR is so unstable for testing, it is impossible to do bulk samples testing.
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838987/pdf/nihms171315.pdf
https://pubs.acs.org/doi/pdf/10.1021/acs.jafc.9b07920
https://onlinelibrary.wiley.com/doi/pdf/10.1002/mnfr.201700911
Thanks
- Badges
- Science topic
- Similar topics
- Citations