Chromatography Forum

Hi friends,


Very tired of biphosphonates chemist looks for a hplc method for sodium ibandronate! Please help!
Tried RID but got poor results. Also added Copper(II) to make it UV detectable..no way! ELSD turned out fine but the substrate is volatile and can't detect it.
This is the hardest one among the biphosphonates I' ve worked with.
Any help is greatly appreciated.

You are dealing with an amphiphilic compound, i.e., it has both a non-polar and a polar end.

I suggest that you try ZIC®-HILIC that is available in PEEK columns and works well for both phosphates and amphiphilic compounds.

http://www.sequant.com/products/zicphil ... 00-03A.pdf

Hi Zompa

Here is a description of the HPLC for Ibandronate.
The Method is developed and validated!!! by the original Manufactor of the Substance (Boehringer Mannheim, now Roche Diagnostics).

HPLC-Column: BDS Hypersil C18 250x4.6mm

Mobile Phase contains:
99.5% Water pH 2.0 (adjusted with 85% Phosphoric Acid)
0.5% Acetonitrile
5mM Pentansulfonic Acid Sodium Salt per Litre
0.01% Titriplex III (Na-EDTA) per Litre

Detection at 195nm, Flow 1.0 ml/min

Greets
Ferris

Zompa,

In the past few days I tested the chromatographic properties of a couple of bisphosphonates (Alendronate and Clodronate). Previous attempts to separate the first of these two compounds proved to be problematic using SAX but my recent efforts were much more successful. The problem is that these compounds are chelating agents and traces of metals compromise the chromatography. This leaves you is only three prime options: work with a system free from metal contamination, add a chelating agent with a higher stability constant to the mobile phase or add a metal to the mobile phase (as you describe in your post).

The tactic I chose was to use a SAX column designed for Ion Chromatography (the exact column I used was a prototype column I developed for another purpose), together with a hydroxide eluent system, an eluent suppressor and conductivity detection. This combination provided completely Gaussian peaks with good sensitivity (signal-to-noise ratio for a 20 microliter injection of 30mg/L is at least 5000:1) for both of the above bisphosphonates.

If you are interested in more details, I'll rerun the chromatography on a commercially available column next week and supply the details.

Thanks a lot my friends.

Very good advice.

Chris, unfortunately I don't have that kind of detector.Your advice is very useful, though.

Ferris,

Don't know how to thank you.. I'll go to the lab to try it.
If I can ever help you both..

Regards.

Zompa,

I I would be very supriced if the ibandronate is volatile so I am supriced you are saying that you can not detect by ELSD. What is the temperature of the evaporative tube that you selected?

And in order to back up what I just said I went on and found a reference for you that describes the analysis of ibandronate by ELSD... The method, being isocratic, looks pretty straightforward...


Title: Determination of ibandronate and its degradation products by ion-pair RP LC with evaporative light-scattering detection
Author(s): Jiang Y, Xie Z
Source: CHROMATOGRAPHIA 62 (5-6): 257-261 SEP 2005
Document Type: Article
Language: English
Cited References: 19 Times Cited: 0
Abstract: A simple method has been developed for analysis of ibandronate and related substances by ion-pair reversed-phase high-performance liquid chromatography (RPIC) with evaporative light-scattering detection (ELSD). After optimization of the chromatographic conditions satisfactory separation of the compounds was achieved on an Intersil C-8 column with an isocratic mobile phase-8:4:88 (v/v) acetonitrile-methanol-12 mM ammonium acetate buffer containing 35 mM n-amylamine (pH 7.0). The mobile phase flow rate was 1.0 mL min(-1). The calibration plot was linear in the range 352 to 1760 mu g mL(-1) for ibandronate. The precision and reproducibility were 0.3% and 0.5%, respectively. The average recovery of ibandronate was 100.4% and RSD was 0.6%. The method was validated and shown to be precise, accurate, and specific far assay of ibandronate in bulk material and dosage forms. The proposed liquid chromatographic method can be satisfactorily used for quality control of ibandronate.

Hi Kostas,

I can detect Ibandronate on the ELSD but can't detect one of the possible impurities. Moreover,I was asked not to use the ELSD and to develop a method with UV detection.

Thanks for answering.

Kostas,

I just saw your second message.. Thanx a lot. Unfortunately, as I said to you before, I am not allowed to use RID, but only UV detection.
I thanks a lot for your research. Really kind of you.

If UV is the detector of choice, you can either try to derivatize you compound (and I have not idea how you can do that -maybe it is not even possible-) or you can try indirect detection. You said that you did try CuII and did not worked...

I remember some publications that are using a UV detectable cationic ion pairing reagent to do that and maybe it is what you can do. Indirect UV detection though is not very easy to achieve and you have to understand it's mechanism before you try it...

Hi Kostas,

I'm starting put into practice your suggestions!
Cu(II) works for ibandronate but not much with one of the likely impurities. There are system peaks and the detector goes over very near the Rt of this impurity.
I'll let you know what the results are. I also want to try the method suggested by Ferris. It's something I haven't tried so far.

Thanx, Regards.

Zompa.

Dear Friends,

Thank you all for the helpful hand. It's been a daunting task and I really appreciated your advice.
Hope I can help you back at some stage.
I tried the method suggested by Ferris. It's great, indeed! Thank you very much.
Unfortunately I don't have a hypersil BDS and used an Agilent Zorbax XDB.
The peak shape is excellent although there are some system peaks and the response of the Api is quite low. Tried to add Cu++ to this system but as expected it hasn't worked out fine. If you have any ideas...it's more than welcome and appreciated.
I also worked on ion-exchange at the same time and found out that there is a very good separation if you use a Allsep anion column with formic acid 98% (0.2 mL /L) and Cu(NO3)2 0.5 mM. The peak shape is not the best but acceptable.
I hope to receive other suggestions from you as far as the peak response and the system peaks are concerned.
Thanks especially to Ferris. It was very kind of you, and you all.

Zompa.