Chromatography Forum

Hi Everyone

Currently working with a USP method where we are testing for ethanol and formamide in an API.

Our results for ethanol is comparable to that on the CofA. Our result for formamide is not. We do not detect formamide at all.

We see formamide in the standard, we have performed recovery (spiking formamide directly on the API and by adding formamide to water solution then adding that to the sample). We get ~105% recovery with excellent precision <2.0%. Thus we suspect that we are having an extraction issue.

The procedure for extraction is as follows:
120 mg of API, accurately weighed, to a 15-mL centrifuge tube, and dissolve in 2.0 mL of m-xylene, heating in a water bath at 45 ± 5°, if necessary. Add 2.0 mL of water, mix, and centrifuge. Transfer the m-xylene layer to a 15-mL centrifuge tube, and extract with 2.0 mL of water. Discard the upper m-xylene layer, combine the two retained lower aqueous layers, add 1.0 mL of Internal standard solution, and mix.

Can anyone offer some advice on where we might be going wrong in the sample preparation stage?

Thank you,

Hello.
Does internal standard is a related substance to formamide? Anyway imho it should be added with initial xylene and standard solution should be prepared in the same manner.

The internal standard is IPA. It is added to the standard curve solutions post treatment as per the test preparation. The only difference is that we don't heat the standards.
We have prepared a test preparation without heating to see if we then see Formamide but this makes no difference.
Vice Versa, we have tried heating the standard solution to see if this removes formamide, it made no difference to the standard response.

Try to add IS to the substance via xylene solution and try to make the standard solution in the same manner.

You've certainly done what I would have done. Spiking directly onto the solid and getting recovery certainly seems to validate your method. Are you sure the CoA is correct? Did they use the same method for analysis?

The internal standard is IPA.

Really IPA (2-propanol) as internal standard? Most GC procedures for ethanol use 1-propanol as internal standard, and some use ACN.

The internal standard ... is added to the standard curve solutions post treatment as per the test preparation.

Usually best if internal standard is added early, if it's an appropriate internal standard.

Thanks for all the response.

In answer to a few questions:

The method states to use IPA and this is why we are using it. It doesn't seem to have caused any problems and is very consistent in PA.

We are now starting to questions there CofA results. All the experiments we have conducted lead to Formamide just not being present, at least not at detectable levels (approximately 0.8%).

We have used two methods where the instrument parameters are slightly different but not significantly different. We have only attempted the one extraction process. We have conducted experiments introducing a number of controls and variations on how the extraction is performed. We have even analysed various fractions collected to see if we were loosing it at one particular step. Independent analysts have all obtained the same results where no formamide is present.

Nothing seems to make much sense.