Quercetin and morin have the same molecular formula (C15H10O7) and molar mass (302.2 g/mol). Quercetin is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one while morin is 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one, so they differ only in the positions of the two hydroxy groups of the B ring of flavonoids (quercetin is 3,4 while morin is 2,4). I want to know how we can differentiate between them (identification) with LC/MS.
Dear Fuad Al-Rimawi
Recently I had a problem with the isomers orientin and isoorientin: RT and LC-MS+ was the same and I solved this with LC-MS-.
Do you mean without reference standards?
If you have reference standards, then just develop a LC method that adequately separates the compounds and compare the retention times to the reference standards. Here is a reference that separates the two compounds using HPLC: DOI: 10.1016/j.jpba.2005.03.016
Without standards is less straight forward. You could use a similiar HPLC method to a literature source and assume that the order of elution for the two compounds would be the same. This is less rigorous for sure, so I would highly advise purchasing standards if possible.
John T Barr's answer is likely best if measuring aglycones but in addition their ms/ms fragmentation likely also differs and would give more confidence in the separation. if only measuring aglycones and standards are available then fairly straightforward. if occur as glycosides and you want to confirm those then in-source fragmentation to liberate aglycone and perform ms/ms on it or use an ion trap to do MSn. various glycosides of same aglycone should yield similar aglycone in the MS and aglycone will fragment similarly.
I know you asked for Mass Spectrometry, but do you have access to an NMR? You should be able to differentiate them that way.
Before mass spectrometry and NMR became widely available there were techniques to characterise flavonoids using UV/visible spectrometry in combination with chemical derivitising agents. For example, treating a flavonoid with boric acid and sodium acetate will identify orthodihydroxyl groups (i.e. the 3,4 OHs in quercetin) by the formation of a borate complex and a shift in the UV absorption spectrum.
It might be possible to use these techniques with mass spectrometry to distinguish your two structures. I'm not sure how you would work this in with an LC/MS method. Perhaps you could collect a sample of the relevant peak from you LC, treat it with the derivitising agent and examine it by MS for the formation of the borate complex.
An old reference for the UV/visible technique is:
Mabry, T. J.; Markham, K. R.; Thomas, M. B., The systematic identification of flavonoids. Springer-Verlag: New York, 1970.
If you are interested in these procedures let me know and I can provide a pdf of a thesis where I summarised the techniques and applied them to the identification of flavonoids isolated from honey by HPLC.
by fragmentation patterns, both the compounds can be distinguished. I hope the following article will help you- Anal. Chem. 2011, 83, 409–416.
http://www.academia.edu/4104820/Polyphenol_Identification_Based_on_Systematic_and_Robust_High-Resolution_Accurate_Mass_Spectroscopy_Fragmentation
Indeed, Manoj, the fragmentation method is explained in there, and both quercetin and morin were fragmented in both negative and positive mode displaying differential fragmentation. Also, they do separate in a rather straightforward LC gradient using 5 - 35 % ACN in H2O with 0.1% FA.
I agree with Nagaraju. I think EI-MS is the easiest way to identify flavonoids. It is not difficult to find fragmentation pattern from library.
One other thing to try- quercetin should react with acetone to form an acetonide, while morin won't. Google acetonide, but the reaction is dissolving your compound in dry acetone with a small amount of acid ( sulfuric acid works for me). Neutralize with TEA and dry off the excess acetone. you should be able to see the different masses using ESI.
Visit ESI-MS/MS library www.tmsdatabase.org to see the difference in fragmentation patterns of quercetin and morin.
Quercetin and morin have different fragmentation pattern (RDA fragmnents) and from my own experience morin is eluting before quercetin on C18 RP column. I am using LTQ Orbitrap XL with heated electrospray ionization, and I have reference standards of quercetin and morin. My MS/MS fragments (% MS/MS base peak) for quercetin: 273(20), 257(20), 179(100), 151(70), while for morin: 273(50), 257(50), 229(80), 151(100), 125(70), 107(10).
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