Chromatography Forum

hi ltnguy3,

I started this morning by using a primesep column, one of those suggested by sielc-tech. I'm trying a Primesep B and have attempted H2O/CH3CN/TFA as a mobile phase. So far I have got poor results as I want the main peak to elute at about 15 mins.
If you have any news let me know.
I'll stay in touch if I see improvements.

TFA might be to strong for this application. Try to use 0.1% formic acid instead of TFA or 5-10 mmol ammonium formate at pH=3-3.5. We will run one of *dronic acids today or tomorrow.
Make sure that what you see is zoledronic acid and not some other impurity. Try to inject your sample without column and measure peak area, then do the same with column attached.
What detection technique you are using UV or ELSD? A lot of *dronic acid show very low (or none) UV activity, although zoledronic should be UV active due to the heterocyclic ring.
I will update you shortly on the development.

Regards,

Vlad

Hello Itnguy3,

I think the basic idea of a Primesep B2 is not that bad, but the polar group is close to the silica and your very analytes have somehow to get their (pass the alkyl chaines). Maybe the solution for your problem lies in more classical phases. I would use either an amino phase, which has weak anion exchange capabilities or a SAX phase or a C18 phase with tetrabutylammonium salt as ion-pair reagent in the mobile phase.

I don't know if you have already tried this approaches, or if anybody else knows that they are not working.

Dirk

Aminopropyl phase might be too strong for this compound and elution and peak shape might be a problem. Aminopropyl is stronger the Primesep B and much more stronger than Primesep B2. If you inject for example maleic acid you aminopropyl phase will give you twice more retention compare to Primesep B and 5 times more retention then Primesep B2. The shileding effect of alkyl chain in Primesep columns helps to achieve better peak shape. There is no need for zoledronic acid to fully penetrate the layer to have ion-exchange interaction with the phase.

I forgot to add in my previous message that in case of zoledronic acid and UV detection you can use phosphoric acid (or phosphate buffers). This is not suitable when ELSD or LC/MS is used as detection technique.

Zompa,
Since API does not have sodium citrate and mannitol, you can use the method that I posted on 04/05/05 but go with isocratic elution.

Dirk Hansen,
I have tried C18 phase with tetrabutylammonium bromide as on the published application:â€

hi Itnguy3, Dirk and Vlad,


I wanted to try CH3CN/H2O/AmAc 20 mM pH 4.0 but had problem with the instrument pump. I'm gonna do it right now and let you know.

Thanx Vlad for trying and for the information. I'll try to follow your suggestions as soon as I finished this attempt.

Itnguy3,
I would appreciate if you send me your article, just to have a look at it.
my email address is paologas@mailcity.com

The method you started has zoledronic acid elute at low rt, if I am not mistaken.

TFA is doubtless too strong. I quitted it no problem.

Thanx to all of you.

Itnguy3,

Have you tried tertbutylammonium hydroxide (as ion pair reagent) (2mM), pH=2.3 with H3PO4 and 5% MeOH?
It might work for your purpose.
Let me know.

Vlad,
I have made many attempts with the Primesep B column but got poor results both in shape and rt. If you manage to obtain a better result let me know. I'm trying IPC with YMC C18 now.

zompa.

Thanks Zompa. I will update you on the result.

Hi itnguy3,

I'm gonna read your email at soon. Know you sent it but had problems logging in. Thanx a lot. The method I suggested to you might work. I'm getting the best results I've had so far by using it.
Talk to you.

zompa

Sorry itnguy 3,

In the method I mentioned and tried the ion-pair reagent is tetrabutylammonium hydroxide (sorry for the mistake). I got a bit better result with tetrabutylammonium bromide. Zoledronic acid elutes at about 10 mins. Reducing the % of MeOH I moved it to about 15 mins.
Let me know how it goes.

Z.

Zompa,
Which column are you using? Do you have any problem with baseline noise?
I’m using Hypersil GOLD (C18) 4.6 X 150 5u.
MPA: 98% (0.002M tetrabutylammonium bromide; pH=2 with H3PO4)
MPB: 2% MeOH
Flow rate: 1ml/min
Detection: 217 nm
Zoledronic acid elutes @ 4 min; mannito@ 1.3 min and sodium citrate @ 4.5 min.

itnguy 3,

While trying out this method I used a YMC ODS-AL C18, non endcapped, 5 uM, 250 mm x 4.6mm, 120 A. I've left this method aside for a while cuz I wanted to try the one you sent me. The baseline wasn't too bad. The main peak shape had to be perfected, though, as it is a bit tailed. With this column zoledronic acid elutes at about 11 mins, imidazolylacetic acid at 3.2 mins and imidazole at 2.4 mins.
I'll go back to this method soon . Unfortunately I've problems with the instrument right now.

let me know...

Z.

itn,

flow was 0.8 mL/min and detectoe wavewlength 210 nm.

z.

Zompa,
let me know how things go with the method that I sent to you.
Thanks,

Hi itnguy3,


I wish I had a working HPLC to try your method on!
The article sounds great and promising.
How re you getting on with your excipients?
I wish the primesep column had worked on this product!
Have you ever come across Sodium Risedronate, by the way?
I will update you on my results.
Thanx a lot.

z.