Chromatography Forum

Hi ,

Anybody carried out force degradation (acid ,base ,peroxide) when the sample prep is made in internal std for HPLC analysis (note old methods)

ie
Accurately weigh x mg of the y standard into an amber zml volumetric flask. Dissolve the solid in xml of internal standard, mix well and make up to volume with internal standard

would you spike in internal std to acid base ,peroxide (though seems strange to me to force deg internal std ?)
Exclude internal std - but how would you quantify
Prepare samples slightly different to fit in with same internal concentration for force deg samples?

ie

using xml acid - yml base to neutralise => Zml internal std = vol - (y-x) ?

Much appreciated

I would 'force degrade' the sample then add the internal standard in preparation for the method assay.

Hi ,

But then you will not be at the correct area ratio for internal standard

ie 20mg sample - add 5ml acid - 24 hours - neutralise with 5ml naoh - make to 100ml with internal standard .. therefore 10% less internal std

Sample prep - 20mg sample - dissolve in internal std - make to 100ml in internal std

Unless control and stds are prepared taking this 10% 'correction' factor into account I guess ?

Sample prep - 20mg sample - dissolve in internal std - make to 100ml in internal std

Or, if you want to avoid the calculation just add 10 mL of salt solution to match the acid + base.

The calculation is easier, but unless you *know* that your internal standard is stable under your forced degradation conditions, adding the IS *before* degradation defeats the purpose.

Hi Tom,

Thanks for advice
so your recommendation is :
Exclude internal standard from stress testing altogether

This would require control sample including and excluding the Istd for specificity

Would quantification therefore be assessed by % area (versus control samples) or by external standards?

Thanks

We may be talking at cross purposes here. What I'm suggesting is that the internal standard be added immediately after the degradation of the sample but before it is diluted (i.e., you add some amount of IS and then do your dilution).

The calculations involved with external vs internal standards are virtually the same; the only difference is that with external standards you work with areas whereas with internal standards you work with area ratios. You don't actually have to know what the concentration of the internal standard actually is, you merely have to ensure that the concentration is the same in both calibrator and sample solutions.

The "elephant in the room" if you are measuring degradants using area ratios is the assumption that the response factors of all degradants are the same as the parent compount.

Hi Tom ,

Thanks for you input.
Sorry I think I am over complicating this but think I get it now
My concern is that the internal standard would behave differently degrade even after neutralisation after say 24 hours
I am thinking for quantification and specificity stress samples, control samples and blank solutions should be prepared with and without internal standard
Covers all bases I guess
ie ID of acid/base /peroxide unknowns - blank soln
ID of possible degs peaks at RT of Istd - confirmed in control/stress no ISTD
ID of stress peaks and quantified as per method - stress/control containing ISTD

overkill or acceptable approach ?

Thanks