Chromatography Forum

MPA: 0.1% TFA in water
MPB: 0.1% TFA in acetonitrile
Detection: 375 nm
Column temp:30oC
Column: Zorbax Extend C-18 (150mm x2.1 mm, 5u)
Product: dantrolene sodium

The reference standard peak has a right shoulder. The tailing is about 1. Peak purity is greater than 999.9
Could anyone please let me know why the RS peak has a shoulder and how to get rid of the shoulder.
I would really appreciate any help or suggestion,

First of all, I think your TFA should be precipitating in pure ACN...

This shoulder seems to be ionic dissociation. Look after the pKa of your analyte and adjust to a pH at leat -1.0 tham pKa.

Actually, trifluoroacetic acid is completely miscible with acetonitrile in all proportions. Since TFA is a strong acid, the pH of this mobile phase is about 2.

After looking up the structure of Dantrolene, I see that is is a hydrazone. As such it has cis and trans isomers. I have seen hydrazone isomers before, and often they are not fully resolved. Their UV spectra are going to be nearly the same, so peak purity won't distinguish them, nor will mass spec.

The pH of MP is about 2. Pka of the analyte is 7.5. It is a hydantoin derivative. The sample that I injected was prepared from the reference standard. It should be very pure.

Check the specification of the reference material. It may have a specification for cis/trans isomers. "Reference material" does not mean that it is pure; it means that its properties are well characterized. There are lots of drugs that exist as mixed isomers, and are characterized as such.

I’ve just called the vendor. They told me this RS is not mixed isomers.
Let me know if you have any more suggestions
Thanks,

What is your diluent? Injection volume?

Thanks,

Try diluting your standard in mobile phase (initial gradient composition). That often fixes ionization-related problems

My diluent is 50% acetonitrile in water. Injection volume = 10ul.

On Monday, I will try diluting my sample in the initial gradient composition. Hope this will fix my problem. I will let you guys know.

Thanks,

I believe it will fix your problem, as you do not have the acidic pH with just H2O + ACN, so it is possible you are already injecting the ionic form of your sample.

Maybe just adding TFA to your original diluent solve it as well.

By using the initial gradient composition or adding TFA to the original diluent, the peak no longer has the shoulder. Since the diluent is too acidic (pH ~2), the sample is degraded ~ 90% in a very short time.
Let me know if you have any more suggestions.

Thanks,

I would think that it is the acidic condition that degrades the sample. How about dissolving the sample in water or initial conditions without the acid?

Without acid, the peak has shoulder.

Chem1,
do I see this correctly? Your MP has a pH of ~2, yet your stuff degrades very fast at pH ~2? What´s very fast? Is it on the order of the retention time? It wouldn´t be surprising to get a shoulder or worse in that case.

What do you exactly mean by "sample degradation"?

Eventually migrating to another buffer might solve this problem, as TFA might be interacting with your sample...