Chromatography Forum

Hello forum,

API is similar to Boceprevir. C36H61N5O7S (contains ((1R,2S,5S)-3{N[....sulfonyl....cyclohexyl}amino)carbonyl...(1S)-1-.....(oxo)acetyl]pentyl......azabicyclo[3.1.0]hexan-2-carboxamide)
MM=707.4 Da
3-4 chiral centers?!

One diastereomer is well resolved with reversed HPLC and UPLC. But the possible conformers? Are they impacting on peak purity?

First we started with HPLC. ACE C18 column, MPA/MPB both containing MeOH/ACN/Phosphate buffer pH7. Gradient from 33% aqueous to 10% aqueous. In peak purity, autothreshold approach failed numerically and graphically (purity plot) for even the reference standard.

Then, redevelopment to UPLC. BEH Phenyl column, MPA Phosphate buffer pH7, MPB MeOH/Phosphate buffer pH 7, gradient from 40% to 10% aqueous. In peak purity, autothreshold approach passed numerically but purity plot present a "impure" zone at the ascending part of main peak even for the reference standard.

All requirements for correct peak purity evaluation were satisfied.

In both method, diluent was 0.01% TFA in MeOH. This is critical to extract and dissolve API from tablets.

From some experiments, it is suspected to have conformers since when column temperature is increased peak purity passes. But higher temperature dramatically reduces the sensitivity for several other resolved impurities.

Different pHs (FA, NH4OH) in mobile phase decreases sensitivity.

Should we try chiral approach in reversed or normal phase? With which type of columns? Any other thoughts?

Many thanks,
Pedro

I would give HILIC a trial.

I would give HILIC a trial.

Thanks Kratz. But HILIC could be really efficient to separate isomers (conformers), even knowing that API and isomers are hydrophobic compounds?

Thanks again for your interest.

BEH Phenyl is being tentatively used. C18 seemed to be worst in terms of separation of diasterisomer. Are these ones the most appropriate for this case? Any other suggestions? Any specific chiral column? Other?

Have you considered using a cyclodextrin (either alpha, beta or gamma) at low level in the mobile phase with reverse phase ?

Have you considered using a cyclodextrin (either alpha, beta or gamma) at low level in the mobile phase with reverse phase ?

Thanks JMB for the suggestion. But are you indicating the use of cyclodextrin directly in mobile phase? At which concentration? We have ried Chirobiotic column but separation was not achieved. Is there any most promising chiral column that you can reccommend? Or any other specific column based on the API structure.

Thanks

If I understood correctly your basic problem is different conformers - which is a standard problem with proline-including peptides or peptide-like tructures. Trying to achieve baseline separation for these usually will not work - the interconversion is slow enough to skew your peak, but it's too fast to separate two distinct species. The best that you can (sometimes) get is two peaks with a interlying "saddle". So, usually the best option is to ramp up the temperature in order to speed up the conversion and merge the confomers into one peak - as you already have observed. I would focus on this approach and try to optimize the method for the other impurities at higher temperature.
Chiral chromatography won't help you in separating confomers.

Have you considered using a cyclodextrin (either alpha, beta or gamma) at low level in the mobile phase with reverse phase ?

I have a question about this approach. I have a compound that I wanted to try this technique on. Based on this https://books.google.com/books?id=3qv-o ... VE&f=false

I'm trying to to mimic it's prep 20mmol beta cyclodextrin/10 mmol potassium phosphate monobasic, but I've noticed that the cyclodextrin is not readily soluble at this concentration in water. How is the author achieving solubility at 20 mmol?

What is your recommended concentration to use this as an additive for RP? thx