Chromatography Forum

Hi Friends,

I have a fraction collected form sephadex LH-20, Analysis indicates that the fraction contains flavonoids in it. I ran a HPLC once for that fraction and it appears like the fraction has more than 7 compounds. I am wondering if would be able to find the name of individual compounds present in the fraction using LCMS. Waiting for your reply.

Flavonoids aren't too difficult by LC-MS; there are very large numbers of flavonoids in nature (the classic references are several books by Harborne). You can probably find out what flavonoids you'd expect in your sample, and then look with LC-MS to see which are actually present. They tend to be glycosylated, so you'll probably see the same fundamental flavonoid building-blocks (things like quercetin and kaempferol) with a range of different glycosylations and other additions. They separate quite well in straightforward reverse phase chromatography; there are huge numbers of references out there, but your favourite C18 column will probably do the job. If you only have LC-MS, you will see spectra containing source fragmentation, so you'll have to look for the flavonoid backbone as a (probably minor) fragment, and then look for additions of sugars; in this case it's important your flavonoids are well-resolved, or it can be easy to mistake which fragments belong to which precursor ions. If you have LC-MS-MS, life is simpler. Be aware that in either case, you will probably struggle to get details of sugar linkage. The glycosidic bonds will break most easily, and you won't have much clue as to which sugars are attached to which hydroxyl groups. Harborne's books give the old-fashioned ways to sort out the detail if necessary. Good luck.

Thank you very much for you reply.

So, Can we analyze fraction even if it has more than seven or eight flavonoids ?. I have the chance to use LC/MS/MS in a research institute. Should I run HPLC again to get good separation before I send the fraction to them?. They are asking few info for the analysis. I could not understand that. It will be very useful If you could give me some idea on this. I have pasted the info they asked me.

1. What does "matching" mean in "Charge per sample is Rs. 1000/- If you wanted to scan library there is
extra charge of Rs. 100 /- for 5 matchings" ,



2. These infos.
ESI MS Expected Mass
ESI MS/MS (list ions for which MSn or Zoom scan is required)


Actually I do know the major flavonoids present in my plant. And I need to find what are the major flavonoids present in the fraction that I have got as it has a bioactivity. One suggestion I got was to get the standards commercially and match it with compounds in fraction using HPLC, but those standard compounds are not affordable. So I am planning for LCMS.


Please give me some suggestions. Thank You.

(1) If they are doing LC-MS-MS, that means they have an LC system attached to their MS, and they can do chromatography just like you, but with the sample going directly into the MS. With such a system it would be no problem to analyse a fraction containing 7 or 8 flavonoids, as the LC will separate them on the way in.

(2) On the other hand, some labs talk about LC-MS-MS when they are actually using the LC system merely as an automated system for introducing the sample. If they do not intend to carry out any chromatography, you need to know! In this case, they probably can't safely identify 7 or 8 flavonoids in one go. For example, were you to inject kaempferol-glucose at the same time as kaempferol-glucose-rhamnose, you wouldn't know whether the kaempferol-glucose peak was a precursor ion of a chemical in its own right, or a fragment of the other ion!

Summary: clarify with them what they are doing, hplc-ms-ms or just using an lc-ms-ms to do ms.

(3) I would assume they are charging you Rs 1000 for actually analysing the sample, and are charging a further Rs100 for each peak that they identify and for which they return data. But again, you need to clarify this with them.

(4) Obviously if their typical clients have particular masses they are interested in, it helps them to know (some of my clients are synthetic chemists and can tell me exactly what to expect in their sample, and which ions I can ignore as salts and buffers). From the description, are they using a Thermo ion-trap? Again, I think you need to tell them the flavonoids you expect to be present, and explain that you'd like to know which subsets of this overall set of flavonoids are present in the samples. It will help them to report on the correct peaks, and not every other peak that is irrelevant to you.

Good luck!

Thank you so much lmh.

The answer was very helpful.

, Sham