It is not easy.. can you help me?

[Cl.Pharm]

I am trying to assay a mixture of Fexofenadine HCl and Pseudoephedrine HCl by HPLC. The ingredient exhibit too different characterestics to be determined in a single RP run.
What method can I follow to separate and quantitate such a pharmaceutical mixture?

[MG]

If you have only tried isocratic conditions and the retention times are too different, you could instead try gradient conditions. Or use a steeper gradient.

[SIELC_Tech]

Your compounds are drastically different in hydrophobicity and have some difference in pKa value. The development of the method, even gradient, might be quite challenging, because pseudoephedrine is not retained well on RP column. We recently develop a method for a different mixture which contains compounds with similar properties-NyQuil (Tylenol) caught medication. These compositions contain ephedrine, chlorpheniramine and dextromethorphan. Dextromethorphan is much more hydrophobic then ephedrine. Check the following link:

http://allsep.com/makeCmp.php?cmp=Cmp_118

As you can see from the link all compounds are eluted in reasonable time on Primesep column, while traditional RP gives a big gap between ephedrine (C10H15NO) and dextromethorphan (C18H25NO). You might need to go to higher organic concentration (70%) to elute fexofenadine. On Primesep C column you ephedrine will retain by ion-exchange mechanism while fexofenadine will retain mostly by reverse phase mechanism By paying with pH, buffer concentration and amount of the acetonitrile you have a good chance to elute these compounds in a reasonable time. You can also effectively play with pH of the mobile phase to gradually shut down ion-exchange properties of Primesep C column thus reducing ion-exchange interaction between stationary phase and fexofenadine.
What are you objectives for this separation (detection technique, run time, solvent and buffers of choice)?
If you can provide us with the sample of fexofenadine we will develop a method for you.

Contact us if you have questions.

Regards,

[Uwe Neue]

The retention of fexofenadine can be changed significantly by maipulating the pH. You get more retention at low and high pH, and less retention around pH 7. This manipulation might bring the two compounds into the same retention window. Or, as MG has pointed out, a gradient might be just fine as well.

[SIELC_Tech]

What retention time you expect for pseudoephedrine at pH=7? It has no hydrophobicity and you need 0% organic to have any RP retention, which I expect will be a notch of void (if any), then you need to rump your gradient to elute fexofenadine which has formula C32H39NO4, no matter how you change pH you are not going to change retention drastically. The gap between two compounds is very broad ion terms of hydrophobicity, pKa value is in the same range (around 9.5-10.2). In my opinion you either have to do a fast gradient from 0% organic to 100% organic or use mixed mode approach with high organic concentration (70-80%) and 10-30 mmol of buffer (various pH).
Maybe ion exchange chromatography will work too.
Also HILIC experts may suggest something different.

Regards,

Vlad

[Uwe Neue]

Pseudoephedrin is reasonably hydrophobic, and at neutral pH one could expect increased retention in the presence of an organic solvent. But afterall, one can always use a gradient...

[Dirk Hansen]

If you are able to use any RP-phase for this assay, a phenyl phase could also help to increase the retention of pseudoephedrine. In this case you may have to use Methanol in the mobile phase to get the maximum effect.

[Cl.Pharm]

Thank you all for your valuable participations. Some good approaches have been suggested.
A pH change to the higher RP column limit did not make any difference.
Using Ion Exchange Columns may be promising but I've never used them before, so I don't consider them as the first method of choice.
SIELC_Tech mentioned "mixed mode approach" and some thing about buffer concentration, can I have more details about this approach. If there is any published papers about this topic, I 'll be gratful if you inform me about it.

Thanks alot

H. ElSikhry

[MG]

Cl.Pharm, I'm not sure exactly what your problem is. Is it lack of retention for pseudoephedrine? I would be surprised if you didn't get some retention for it on C18. You could try an initial hold at 5% organic to retain pseudoephedrine, followed by a gradient to elute fexofenadine.

[SIELC_Tech]

Mixed mode columns usually combine several mechanisms of interaction. You can control both mechanisms independently, supress or enhance each of the mechanisms, use synergy of changes

http://allsep.com/Technology_NovelStationaryPhases.php

In your case it is reverse phase and ion exchange. Both of your molecules have a different hydrophobicity and slight difference in pKa. In order to have the eluted in a reasonable time you need to suppress hydrophobicity of fexofenadine, this will bring two compounds closer together. My guess that with 70-80% of CAN and ammonium acetate or formate in the mobile phase you can elute both of the within 5-10 minutes. You just need a proper choice of buffer and proper mixed mode column. If you can send us fexofenadine sample we will do free screening for you with no obligation to buy our column (we benefit from this too as we can have another neat application added to our website.

Download some brochures, posters and articles from our website. Also see some scientific background for Primesep columns:

http://allsep.com/Brochures_Home.php

Also check application page for similar compounds-you can sort it by compound name, date or type of column:

http://allsep.com/Applications_By_Publish_Date.php

Like I said NyQuil application is a good analogy to your separation.

Regards,

[Cl.Pharm]

Cyano columns gave good results, I think they are as promising as mixed mode columns of short carbon chain.. but I still need to understand more about the mechanism of retention of these columns. I searched the Internet to fine any specific paper focusing on this type of column but with no significant results. I'm looking for a help...

[SIELC_Tech]

Send us a sample of fexefenadine and we will show you how retention time changes with thr change of mobile phase and buffer. You can see from chromatograms how mechanisms of interaction, buffer concentration, pH, concentration of ACN affect retention time of your compounds in mixed mode chromatography.
I can send you our master presentation on Primesep columns (over 100 slides).

Regards,

Vlad