Chromatography Forum

I work for a pharmaceutical company. We have an HPLC method with a validated relative response factor for an impurity. We are switching to a different HPLC method with the same wavelength on the UV detector. Unfortunately, there is not enough material to re validate the relative response factor of the impurity. It appears that the impurity has the same response by the new method. Is there a USP/EP approved way to prove the relative response factor equivalency between the methods?
I've thought about making injections of lots on both methods and compare their area%.

Thanks,
Denise

As long as the UV cutoff for each mobile phase used is similar and the wavelength(s) used in the methods are not close to the UV cutoff, the relative responses should remain constant as they are a function of each component's molar absorptivity.

I can't provide a USP citation for this argument but I think it is sound.

Hi Denise,

I've thought about making injections of lots on both methods and compare their area%.

That’s exactly the way to do it. You don’t need to rationalize as to why the values obtained with the “newâ€

DR, I don´t see how the cutoff has anything to do with the response of an analyte unless the detector is ~saturated by the mobile phase. Do I have a mental block somewhere?
If the poloarities of the mobile phases are quite different there could be a wavelength maximum shift in the analyte, and therefore a change in the absorptivity at a given wavelength.
(I have checked many books and other sources, but found very little and no really helpful information on influene of the solvent on absortion, directly, not over wavelength shift. That is, I don´t know of any rules, or whatever, about influence of light absorbance probability dependence on solvent. Anybody know something about this?)

The cutoff could be important in terms off LOQ/LOD. If the detection is at 210 nm or 220 nm and the mobile phase switches from ACN to MeOH or something else I would make sure that the LOQ is still sufficient. That could be done even by diluting sample solutions.
Aditionally I would run the methods with a PDA to see wether the absortioption maxima change or not (assuming that detection is at the maxima).
Alex

I still don´t follow.
Also, if the max shifts, everything else shifts more or less also.

"Just show/document the equivalency" - agreed

"We are switching to a different HPLC method" - any validation already performed?

The cutoff could be important in terms off LOQ/LOD. If the detection is at 210 nm or 220 nm and the mobile phase switches from ACN to MeOH or something else I would make sure that the LOQ is still sufficient. That could be done even by diluting sample solutions.
Aditionally I would run the methods with a PDA to see wether the absortioption maxima change or not (assuming that detection is at the maxima).
Alex

^^ pretty much what I was thinking - this is for impurities and their responses relative to main peak, so LOD/LOQ are going to be important. I agree that revisiting PDA and runnign some samples via both methods would be a good idea.

HW - I lack the benefit of any handy reference explaining this, but I am inclined to think that analyte maxima and minima shouldn't change as a function of solvent composition (unless you *really* alter solvent polarity and/or pH). If analyte spectra do change, I would hope that they would all shift by the same amount and that the change would be very modest. Again - a PDA will help determine this.

As the OP is in Pharma, the bottom line will be demonstrating equivalence with any compendial method that would be used to referee any impurity profile questions down the road.

Thanks for the comments thus far.

The wavelength for both methods is 280nm. Mobile phases are 0.1% TFA in water/methanol.
The new method was validated for all impurities except one impurity of interest. We only had enough material for specificity. We also used a lot with significant levels of the impurity to test for stability, ruggedness, reproducibility. There is not enough of the impurity standard to test for LOD, LOQ, linearity (and therefore, RRF), or accuracy.

To me it sounds like: â€

We also used a lot with significant levels of the impurity to test for stability, ruggedness, reproducibility.

If you get the same response ratio between the impurity and the API using each of the two methods, I think you're probably OK...

"The wavelength for both methods is 280nm. Mobile phases are 0.1% TFA in water/methanol. " - so what changed? cos if it was only column, or some minor changes these could be done in robustness

how much does the impurity cost by the way?