Chromatography Forum

Hi,

I am developing an assay and impurity method for soft gel capsules that contains API in oleic acid (Mail component of oil). I use ACN/H2O 85/15 as mobile phase. Sample preparation is : add 1 capsule into a 25ml volumetric flaks, extracte with 20% of 1%TFA in H2O to break the capsule shell, then dilute to volume with Ethanol and filter.

However the oleic acid gives me a lots of peaks which interfere with my API and known impurity peaks. I am already using super-refined oleic acid. I can't really play with the mobile phase retio because the peaks are all over the place, What can I do during sample preparation to somehow "purify my sample"?

Thanks a lot.

After you have diluted with Ethanol please freeze your sample for 15 to 20 minutes and filter or centrifuge your sample. Do the same procedure with your standard solution. Hope that will help.

We've used a mixture of barium chloride and calcium chloride to precipitate fatty acids as insoluble metallic soaps, then filtered, and assyed the filtrate. I might even try some Ba+2 ion-exchange clean-up cartridges for your aqueous extract, depending upon what the API is.

After you have diluted with Ethanol please freeze your sample for 15 to 20 minutes and filter or centrifuge your sample. Do the same procedure with your standard solution. Hope that will help.

Freezing the sample seems to be a quick way so I'll try that first. Here are two question regarding this:

1. So I assume as long as I have enough sample left unfreezed for filtering and centrifuging I should be flexable about the freezing time, right?
2. Why do I do the the same thing with std? Is it to make sure the std go through the same temperature change so it won't affect my peak area?


Thanks a lot.

We've used a mixture of barium chloride and calcium chloride to precipitate fatty acids as insoluble metallic soaps, then filtered, and assyed the filtrate. I might even try some Ba+2 ion-exchange clean-up cartridges for your aqueous extract, depending upon what the API is.

I am working on a soft gel capsule, so could you provide me more detailed guidance on how to apply a mixture of barium chloride and calcium chloride to precipitate fatty acids in my case?

Right now my sample preparation is:

add 1 capsule to a volumetric flask, add 5ml of 1% TFA, sonicate for 20min so the cap shell breaks and dissolves, then QS with ethanol, the solution should be clear at this point.

So shall I add mixture of barium chloride and calcium chloride to the 1% TFA, if so how much shall I add?

regarding the Ba+2 ion-exchange clean-up cartridges, how do I use it? do I pour sample through it before analysis?

Thanks.

I am working on a soft gel capsule, so could you provide me more detailed guidance on how to apply a mixture of barium chloride and calcium chloride to precipitate fatty acids in my case?

Right now my sample preparation is:

add 1 capsule to a volumetric flask, add 5ml of 1% TFA, sonicate for 20min so the cap shell breaks and dissolves, then QS with ethanol, the solution should be clear at this point.

So shall I add mixture of barium chloride and calcium chloride to the 1% TFA, if so how much shall I add?

regarding the Ba+2 ion-exchange clean-up cartridges, how do I use it? do I pour sample through it before analysis?

Thanks.

Here's what I'd TRY:
add 1 capsule to a flask or beaker, add 1% TFA, sonicate for 20min so the cap shell breaks and dissolves. Add 10% BaCl2/CaCl2 solution to precipitate any fatty acids, QS with ethanol, then filter. How much BaCl2/CaCl2 solution to add? Take some of the filtrate and add a few drops of BaCl2/CaCl2 solution; if nothing precipitates, then you have excess BaCl2/CaCl2 solution, so experiment by cutting back to see.

If your API is soluble in your 1% aqueous TFA solution, that would be the poont if you wanted to try Ba extraction cartridges, as I don't think they work that well if the alcohol level is too high. Again: experiment.

Hi,

So can I add BaCl2/CaCl2 solution after adding some amount of Ethanol because my drug seems dissolve in ethanol and I am afraid precipitating oleic acid right after adding 1% TFA will also precipitate my API?

Thanks.

Here's what I'd TRY:
add 1 capsule to a flask or beaker, add 1% TFA, sonicate for 20min so the cap shell breaks and dissolves. Add 10% BaCl2/CaCl2 solution to precipitate any fatty acids, QS with ethanol, then filter. How much BaCl2/CaCl2 solution to add? Take some of the filtrate and add a few drops of BaCl2/CaCl2 solution; if nothing precipitates, then you have excess BaCl2/CaCl2 solution, so experiment by cutting back to see.

If your API is soluble in your 1% aqueous TFA solution, that would be the poont if you wanted to try Ba extraction cartridges, as I don't think they work that well if the alcohol level is too high. Again: experiment.

You can consider using normal phase condition such as heptane/methylene chloride s mobile phase. The oil (oleic acid) will elute close to the void and the API will elute later assuming it is more hydrophilic than oleic acid. The sample prep. only involves grinding, dilution and filtration. The normal phase condition takes care of matirx elimination. Attached is an application we did along the same rationale. Hope it will help.

http://www.dionex.com/en-us/images/pdf- ... -phase.pdf

Hi,

Using normal phase seems reasonable. I've never worked normal phase before so I have the following questions:

1. My API is vitamin D5, and I think it's more polar that oleic acid, could you confirm?

2. Now with the reverse phase, peaks due to oleic acid are all over the place. Do you think with normal phase, all the oleic acid related peaks will elute close to the viod?

3. The API will be dissolved in oleic acid containing 0.1%BHT and then filled into a sogt gel capsule.
For my current smp preparation, I dossolve 1 capsule into 5ml of 1%TFA, then add 20mL ethanol. Do I need to change sample prep diluent due to the normal phase study? If so what diluent do you suggest?

Thanks a lot.

You can consider using normal phase condition such as heptane/methylene chloride s mobile phase. The oil (oleic acid) will elute close to the void and the API will elute later assuming it is more hydrophilic than oleic acid. The sample prep. only involves grinding, dilution and filtration. The normal phase condition takes care of matirx elimination. Attached is an application we did along the same rationale. Hope it will help.

http://www.dionex.com/en-us/images/pdf- ... -phase.pdf

Hi,

D5 seems to be quite hyhdrophobic, too. I don't have firsthand experience with these two compounds, thus can't provide a definite answer to first two questions. You will need to try it. If you use base silica column, the electrostatic repulsion between oleic acid and the stationary phase will help oleic acid elute early.

Regarding the sample preparation, you want to dissovle the sample in a weaker diluent comapred to the mobile phase. D5 should be very soluble in heptane (or hexane) thus seems to be a reasonable solvent for sample prep.

Hello,

If you tell me the name of commercial product that containing this formula, I might be able to try it in the lab and get back to you with the result.

Dear XL:

Sorry that I was out of the loop for a while. The client changed their plan so we are not going for this formulation route. Thanks a lot for your great help.

due to the fact that the acids are acids, the following two ways should work:

1) use anion exchange cartridge to retain the acids;
2) dissolve the formualtion in diethyl ether, wash with NaOH solution, remove the solvent, redissolve in the mobile phase