differences in response

[usatinsky]

I have two fully qualified reference standards for a drug substance – first is a freebase and second is a TFA salt. The test method is a regular HPLC on C18 column with 0.1% TFA/Water/Acetonitrile mobile phase, gradient program up to 75% organic, and detection at 215 nm. The response of freebase standard (area/concentration) is about 104% of the response of TFA salt standard (converted on freebase for calculations of concentration). I have no reason to suspect an overestimation of purity value of the TFA standard because it was investigated and corrected for all organic and inorganic impurities, moisture content and residual solvents. I may suspect that there is an impurity (with higher UV absorption) hiding under the parent peak in freebase standard although it is highly unlikely. What else could cause this phenomenon?

[DR]

Any differences in peak shape and/or retention time can exacerbate the random variation expected between different preparations of the same standard material.
Using different salts will often result in larger differences in peak shape and retention time than what you get from random variation, so I would think that 104% is not bad (since, to a small extent, you are comparing apples to oranges).

If you were to work the free base up in a solution of TFA so that your final dilution of it chemically matched the composition of the tfa salt preparation, I'd expect a smaller residual difference between the 2.