I've been having a tough time finding it...suggestions?http://www.hmdb.ca/metabolites/HMDB00925
TMAO has a singlet peak in a 1H-NMR spectrum, at about 3.253 ppm. If you see a peak in this region I suggest that you spike in some pure TMAO solution and look for an increase in the peak intensity. Only a small amount is required, just enough to see an increase in peak height of your possible peak. This doesnt confirm TMAO but it is consistent with it being TMAO. I suggest that if you do see your peak increase in height, then you change the pH of your solution as well. Many biological molecules are pH sensitive and their chemical shifts will change as a result of a pH adjustment. If both peaks move in exactly the same way with changing pH this is very good indication that your peak and TMAO are one and the same. Good luck.
Thanks for the advice. I did try titrating the pH of my sample with a tiny amount of HCL and see if the peak at the 3.25 ppm part of the spectrum would shift because a part of the glucose signal seems to be so close (pratically on top), giving me a very hard time telling if TMAO was there or not. The fact that it is a singlet peak makes it hard to know if I'm actually seeing it or not. I have just ordered some TMAO to do what you advised but I like what you said about changing the pH after the addition of pure TMAO. Would you also suggest changing the pH some other way? Also, could 2D acquisition help here?
Probably better to use dilute DCl rather than dilute HCl as you will be adding less H2O to your sample. Since you have a singlet a 2D COSY is not going to help, but you could try a 2D HSQC to look for the H-C cross peak resulting from the 3 TMAO methyl groups.
HSQC is essential, as you have many compounds with singlet around that region - betaines etc.
This is always a difficult task...if you have a physical method for compound separation prior to NMR analysis it would be the best way to prove the existence of TMAO/Betaines...
Along with spiking experiments you can try 1D TOCSY which will give you information about the overlapping peaks also.
What Type of Sample? Urine? For mice normally TMAO is high for female animals and low/absent for males.
Do you want to distinguish TMAO from TMA, or from "any" other amine which carries a methyl? In any case ONLY the spiking proposed by Harold will clarify unequivocally! Low-sensitivity long-range 1H-15N HMBC can help - but again - at the end only spiking is a REAL proof!
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