As Andre Khoury already pointed out it is - especially in the polyphenol industry - quiet common to use another reference material e.g. gallic acid, but catechin is another example, than the component(s) one wants to quantify. the reason for this is usually dual: a pure reference material is lacking altogether or is prohibitive expensive, or the product that needs quantification is a complex mixture of polyphenols. In both cases it can be sensible to use another reference material that preferentially is structural related to the target compound, but even that is often not the case in industrial practice. For instance when quantifying cocoa polyphenols the standard most often used in the industry is gallic acid even though cocoa polyphenols are primarily oligomers of catechin and epicatechin, and catechin would be a more rational choice.

However in your case your reference "B" is structural related so this is good.

Keep in mind when using this approach that the assay values you obtain regardless of the assay method you apply (Folin, UV, permanganometry, others...) should not be interpretted as mass percentages and can in some instances be in excess of 100 %. It simply means that for example your component A is acting as if there is 110 % of reference "B" present. For certain purposes this can be sufficient e.g. to determine if one purification protocol is more efficient than another. However if you really need to quantify your component you will have to go for the elaborate approach already pointed out by some of the others: isolation, purification and identification of your own standard material.

As far as I know you cannot use B as a standard. As u clearly mentioned, on fragmentation it (A) does not cleave to produce B and added it also has an another combined molecule which may interfer in the reaction. There is no relationship with the absorbance hence most compounds are detected at this range and thats its the commanly preferred nm. U can try to cleave B molecule exactly from A using any other chemical reactions and then compare.

Dear Abirami,

Thanks for your reply. Really appreciate your answer. Let say in other situation, if fragmentation of phenolic acid A produces compound B as one of its daughter ion, can I use a standard of compound B to quantify phenolic acid A? Thanks :-)

in either case u cant.... unless otherwise the reaction used for quantification specifically reacts with the 'common structure' in both compound A and B.

Dear Chandra,

have you considered the possibility of employing B as internal standard?

Greetings Mr. Kathiravel,

May I know what reaction you meant when you said 'unless otherwise the reaction used for quantification specifically reacts with the 'common structure' in both compound A and B.'? Thanks

Greetings Mr. Fabrizio,

Can you help me to explain more details on employing B as internal standard as I am not sure how it can help in quantifying phenolic acid A? At first I thought of generating a standard curve using standard (B) via LCMS, then quantify phenolic acid A since both have a bit similarity in structure. But to think again, fragmentation of phenolic acid A doesn't give me (B) as one of the daughter ion.. I guess my methodology was wrong :-P I agree with what Ms. Abirami mentioned earlier as phenolic acid (A) has an extra bonding with other structure as well. Thus the absorbance at 280nm for both standard (B) and phenolic acid (A) will be different in terms of intensity and if I use standard (B) to quatify phenolic acid (A), the quantification will be inaccurate. Do you have any suggestions Mr. Fabrizio? Thanks in advance :-)

Hello dear!

As far as I know we can't use a different standard to quantify a particular compound because different compounds have different absorbitivity for particular wave length.. Absorbance is proportional to concentration, absorbitivity and path length or a cuvet diameter so the quantity you determine may not be due to concentration but also the nature of the compound. But you can use any standard to estimate the total phenolic content in any solution or extract.

Greetings Mr. Adam,

Thanks for sharing your experiences. Your statement does make sense since different compounds do have different absorbance ability.

Hi chandra,

Happy to hear from u. U can use B as a standard only if u exactly cleave A to produce B. Commonly, in polysaccaride estimation, we estimate the amount seperately after cleaving the sugars.

In this as i dont know the structure.. general assay to quantify the phenolics wuld be appropriate. On the other hand u can try to quantify using MS - by area under the cure.

This wuld be more of relative quantification.

If you have some colorimetric method for quantification for B you can do with A also by UV methods. You have to run also control.

Second you can isolate pure phenolic acid A from plant material and do quantitatively then by any method. Hope you can understand the way.

Hi guys,

Thanks so much for the fruitful information given. I found it is interesting to be able to get so much opinions and methodology for my problem :-) Really appreciate your help. I am sure will be able to quantify the phenolic acid as suggested by you guys. Thanks again :D

If you use GC-MS for the quantification, it would be possible to use B for the quantification of A provided that you use a similar fragment of the two compounds in their MS spectra using the SIC mode for the chromatogram.

I guess the easiest approach for this will be to make a standard of your own; what we call as a "test standard". I assume you are able to purify this compound of yours to a relatively good purity say about 90% or more. More the better. You can then use this standard to analyze all your other test samples after applying the purity factor. This is what I guess all the innovator companies worldwide are doing.

Greetings Mr. Mukul,

Thanks for your suggestion :-) I can isolate and purify this phenolic acid A using fraction collector and repurify again until I get a single chromatogram. However, to determine its purity, I think I have to use NMR to confirm 100% its structure and to make sure there are no other compounds in the isolate. Unfortunately, we can afford to purchase an NMR system at the moment :-P but I can always outsource it.

Dear Sir,

Usually, the quantification of many phenolic compounds extracted from a plant species is based on the standard curve made by Galic acid (a phenolic compounds too) and this is according to the Folin- Ciaucalteu method, so in my opinion if the quantification of many molecules presenting a phenolic rings are based on a standard curve made by one compound you can quantify ur extracted phenolic acid while you use a similar standard as you asked since these 2 present many similar characteristics.

Regards

Dear Chandra,

NMR can be used for structural identification. Even if there are other compounds there it does not matter as long as in your chromatography your compound peak is a "pure peak". I assume you have developed an HPLC method for the separation of related compounds for your target. You can calculate the purity on area percent basis; which is the most commonly followed practice. However, when you do that do not forget to confirm if all the impurities have a similar absorbance at your working wavelength.

Greetings Mr. Andre Khoury

Thanks for your suggestion :-) I think it does make sense and mentioned by fellow researchers in the previous comment as well. I shall give a try. Thanks yeah

I think Andre Khoury hit the nail on the head. If you can also isolate your compound, it is a solid as I would suspect, has a very narrow melting point (good simple evidence of resonably high purity), and you have a measure of the molecular ion mass from the mass spectra you obtain, then you can also determine the compounds molar absorptivity or extinction coefficient oand use its own UV absorbance to quantify based on the Beers-Lambert Law A=ebc

Greetings Mr. John,

Thanks for your reply :-) Yes, I am currently isolating the compounds and double confirm its molecular mass via LCMSMS. Planning to purify the compound in house and use it as a reference standard for further studies. Thanks also for the suggestion regarding the Beers-Lambert's Law. I could use it for future studies. Thanks alot :-D

Good Luck

I would say possible. but you need calibrate the relationship of A and B.

It is possible to quantify B using A as internal standard if this compound is absent from your matrix, or else you might have some bias if you don't take some extra precautions. You can use SIM mode to monitor both ions and use the ratio between them to obtain a relative quantification. If you can manage to build a calibration curve, then you can get absolute quantitative results.

As Andre Khoury already pointed out it is - especially in the polyphenol industry - quiet common to use another reference material e.g. gallic acid, but catechin is another example, than the component(s) one wants to quantify. the reason for this is usually dual: a pure reference material is lacking altogether or is prohibitive expensive, or the product that needs quantification is a complex mixture of polyphenols. In both cases it can be sensible to use another reference material that preferentially is structural related to the target compound, but even that is often not the case in industrial practice. For instance when quantifying cocoa polyphenols the standard most often used in the industry is gallic acid even though cocoa polyphenols are primarily oligomers of catechin and epicatechin, and catechin would be a more rational choice.

However in your case your reference "B" is structural related so this is good.

Keep in mind when using this approach that the assay values you obtain regardless of the assay method you apply (Folin, UV, permanganometry, others...) should not be interpretted as mass percentages and can in some instances be in excess of 100 %. It simply means that for example your component A is acting as if there is 110 % of reference "B" present. For certain purposes this can be sufficient e.g. to determine if one purification protocol is more efficient than another. However if you really need to quantify your component you will have to go for the elaborate approach already pointed out by some of the others: isolation, purification and identification of your own standard material.

Greetings Mr. Patrick,

You provided me with good informations on the above matter :-) Great points and explanation. Thanks again

Dear Chandra,

It is always a pleasure to assist. You might also like the below company brochure I wrote regarding this topic.

Best regards.

I used gallic acid as standard using the Folin method for measuring the phenolic content of my wood and bark extracts.