Chromatography Forum

Hi,

Just can´t find exact description why is saponification needed for example for the determination of vitamin A and E with HPLC analysis. Can anyone explain it or point to a source where I can get the info?

looking around on the internet, I see some methods that include saponification and others that do not. This appears to be tied to the objective of the method and the matrix.

These vitamins may be present as esters. To obtain a total content, it may be approporatie to saponify the esters to have all of the vitamin present as the free alcohol.

Saponification can be used to degrade a fatty matrix.

So to answer the question regarding the the analysis you are using, it might be appropriate to know what matrix and if you have the possibility of esterified vitamins.

Don H has got this answered well.

looking around on the internet, I see some methods that include saponification and others that do not. This appears to be tied to the objective of the method and the matrix.

These vitamins may be present as esters. To obtain a total content, it may be approporatie to saponify the esters to have all of the vitamin present as the free alcohol.

Saponification can be used to degrade a fatty matrix.

So to answer the question regarding the the analysis you are using, it might be appropriate to know what matrix and if you have the possibility of esterified vitamins.

The method is for foodstuffs in general and same sample preparation is for vitamines D2 and D3, alpha-,beta-,gamma- and delta-tocopherols and for vitamin A. So it is meant for example to transfer Retinyl palmitate to retinol? Also does the the degradation of fatty matrix serve any purpose? Is it bad for RP-column?
Thanks alot.

So it is meant for example to transfer Retinyl palmitate to retinol?

It would appear that this is the case. However, the best way to find out the intent of the method is to find out what the intent of the method was when developed or selected. A properly documented method should state analytes of interest and matricies for which it is intended. If this is a method that was handed to you, you may have the procedural steps that were extracted from a larger document or set of documents. The answer to this means tracking back the source of your method.

Also does the the degradation of fatty matrix serve any purpose?

Yes. If the saponification was intended to degrade fatty matrix, it would be intended to improve the analyis in some way. This could be to remove interfering peaks in the chromatogram. It could be to improve peak shape. It could be to... Well, without details about the method, I can make a lot of guesses.

Is it bad for RP-column?

This may be better answered by a person who does LC, but I can say that this depends on what mixture goes on the column. Again details would be helpful.

If you can put the specific method steps here, someone running a similar method may be able to address specifics.

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Is it bad for RP-column?

This may be better answered by a person who does LC, but I can say that this depends on what mixture goes on the column. Again details would be helpful.

If you can put the specific method steps here, someone running a similar method may be able to address specifics.

Most triclycerides are not too soluble in solvents like methanol or ACN. That's why saponification of such samples is commonly done.

After saponification, samples would likely be brought back to neutrality, and typically a mobile phase with a low-pH buffer would be used.

It would appear that this is the case. However, the best way to find out the intent of the method is to find out what the intent of the method was when developed or selected. A properly documented method should state analytes of interest and matricies for which it is intended. If this is a method that was handed to you, you may have the procedural steps that were extracted from a larger document or set of documents. The answer to this means tracking back the source of your method.

I found them all but only managed to work through vitamin D. The method was validated for drinks and foods in general(does not mention any specific matrixes used(seems wrong to me, but I don´t have alot of experience so it could be ok)). The method was validated for sum of vitamin D(D2+D3). I also found the document which the method was based on and it was european standard EVS-EN 12821 Foodstuffs- Determination of vitamine D by HPLC- measurment of cholecalciferol(D3) and ergocalciferol(D2). This one said that it has been validated for margarine, milk, milk powder etc. Both of them were not specific about the saponification but said that D2 and D3 are saponified in foodstuffs using alcoholic potassium hydroxide and extracted by an appropriate solvent.
I also found a point which was disturbing to me. The original method said that it is suitable for determination of D2 or D3 not for both of them at the same time. The reason being that if one is determined the other is used as internal standard which seems reasonable as the extraction procedure can have some losses and also the analytes can degrade. The method validated here determines both of them at the same time and doesn´t use any internal standard.
But back to the saponification purposes. The procedure is following:
I add KOH in ethanol to the sample and heat the mixture ~80C for 30 minutes. If oili content is visible I add more KOH in ethanol and heat it more. After that I add water and hexane and shake it for 3 minutes. Then I take most of the hexane phase and centrifuge it after which I take certain amount from the top and dry it in rotary evaporator. Finally I dissolve it in certain amount of ethanol and inject it to HPLC.

The oil is glycerides that you do not want to put on the column as triglycerides. With your quantitative eyeball, you determine how much KOH is needed to degrade the triglycerides.

The KOH stays in the aqueous phase. Any quantity of potassium salts you can get into the hexane will be minuscule.