Hi Md. Mamunur Rashid 

The best option you have is a semi-quatntitation this is where you use a compound of similar molecular mass and structure to estimate a concentration assuming the intensity is comparable. This approach is highly questionable as even compounds with the same structure such as PCB congeners give differing intensities. The best that this will achieve is a ballpark estimate, and you should be careful in using the results to always remember that you are only estimating values and not giving exact figures. But if this is all you have available it at least gives you something to work off

If your work is more concerned with comparing something then you can simply use the intensity as a marker and not semi-quantify.

G

Hi, I think for doing quantification using chromatography (also LC MS MS), we need to have authentic standards, without  any standards,  it is impossible to do quantification. What did you mean by "Q3"? 

No standards, no quantification. This is because the ionization efficiency, matrix effect, and ion transport is compound specific in MS analysis.

One possibility is to go for semi-quantification, that is enough to compare your samples each other.

Otherwise, you can check in literature if there are indirect methods for the quantification of your metabolites of interest (for example through equivalents).

Again, what did you mean by "Q3"?

You can not ID the compounds with certainty unless you have the stds to compare them with. For a qualitative ID, you could collect enough pure sample material, then submit them to NMR, FTIR or other orthogonal techniques which looks at other chemical properties. This would provide you with additional data which might help.

BTW: I think by "Q3" he referred to the results obtained from a triple quad MS. Mass spec does not detect everything (only compounds which were ionized under the method conditions) so should not be relied on for identification or for quantitative results by itself.

Thanks a lot for all of your replies.

Yes, Q3 referred to the daughter ion generated from the parent ion (Q1) through triple quad MS.

Actually, I've fragmentation patterns of the metabolites (though the actual structure of the metabolites are not confirmed). Hence, I thought, if I use the high intensity MS fragment of metabolites as a Q3 it could work (Though, I've never done the quantification in this way. In addition, it's not possible for us to synthesize all those metabolites).

So, if you guys have this sort of experience, idea or references, please share.

'Luca Narduzzi' could you please share the information of semi-quantification?

Thank you all.

Hi Md. Mamunur Rashid 

The best option you have is a semi-quatntitation this is where you use a compound of similar molecular mass and structure to estimate a concentration assuming the intensity is comparable. This approach is highly questionable as even compounds with the same structure such as PCB congeners give differing intensities. The best that this will achieve is a ballpark estimate, and you should be careful in using the results to always remember that you are only estimating values and not giving exact figures. But if this is all you have available it at least gives you something to work off

If your work is more concerned with comparing something then you can simply use the intensity as a marker and not semi-quantify.

G

Dear Garry Codling,

Thank you for your kind reply. Actually, I've the standard of the parent compound and after the animal administration I've identified 7 plausible metabolites from that parent compound. I've already quantified the parent compound and now I wanted to quantified these new metabolites based on the peak area ratio. Its difficult to find out some compounds with similar mass and structure, but I think its not possible without standard. Therefore, I'm asking the expert scientist/ researchers, If they have any experience/idea/any paper reference/comments, please send me.

Thank you.

Hi Md. Mamunur Rashid,

in drug discovery various approaches have been described including the use of UV, NMR, ICP and CLND to provide correction factors for ms response. Metabolite enrichment might be needed to be able to use the less sensitive detectors (from in vivo studies or after in vitro incubation of parent providing metabolites of interest are present). 

UV approach is described in e.g. this BMS paper by Y. Yang: Quantitative estimation of circulating metabolites without synthetic standards by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction. Rapid Commun. Mass Spectrom. 2011, 25, 3245–3251 

You can only perform a Relative Quantifcation when you are comparing two or more groups. Just measuring 1 group of samples without standard it is not possible. You will need a standard curve with known concentrations to perform a real quantification of that metabolite.

Thank you Peter L Jacobs, for the material. I don't have access of that article. If it's possible for you, could you please send me that article ( [email protected] or [email protected] ).

Thanks to Albert Rivas-Ubach for sharing your helpful experiences.

It may be Possible.

It is quite related what we doing in unknown Impurity identification and its quantification.

Here, I suggest some steps which might be useful to you.

Firstly, whatever metabolite you have identified, Isolate the metabolite in its pure form by use of preparative chromatography or other techniques.

Considering his proper purity and develop the method by taking unknown metabolite as standard.

generate the calibration  curve and quantify unknown metabolites from samples.

It will give approximate quantification of metabolite.

study the fragmentation pathway  with the compound which is having similar fragmentation. In ms/ms MRM mode is only used for trace analysis of target analytes.

after studying the fragmentation of both try to perform confidence/selectivity method, to make sure the exact analyte is present.

Thank you all of you.