How can i get rid off placebo?

[Mr.Buffer]

Dear members ;

I am working on a impurity method for a drug product which contains ibandronic acid as drug and natrium acetat , acedic acid ,natrium chlorur , water as placebo. Since ibandronic has no UV absorbtion so i use refractive index dedector to develop method.
My problem is placebo gives giant peak and changes ibandronic acid retention time due to placebo adsorbs by column stronger than my analyte and cause to retention time much ( at least i think this might be problem ) . it is about 1 min and results a splited peak ( normally it doesnt split )

I can not use gradient system nature of refractive index dedector .
So i thought if i can get rid of place i might be successful but i dont know how ...

Any suggestions ?

Thanks in advance.

[Tjalling]

- Which placebo component is causing the problem?
- What are your chromatographic conditions?

[Mr.Buffer]

I dont know which one(s) makes that problem. I had not have time to try each placebo components invidually. But as you can see there are natrium , acetate and chlorur ions in the matrix. Btw this is a intravenous drug product and i have also some response problems about known impurities cause of its concentration ( i need to increase sample concentration but simply i cant make it ) but it is another story.
Chromatograhic conditions are ;
-Mobil phase ; %0,1 formic acid pH 3.5 with 2N NaOH
-column ; ion-exchange column 150 mm x 4,6 mm x 7µm
- 35°C column and dedector temperature
-flow : 0,7 ml/min 100µl injection volume.

I heard same problems from my collages about similar products and same product with different methods , placebo interfere with known impurities and concentration , response problems with product(s).

[tom jupille]

To develop a successful separation, you will have to find conditions that separate your analyte peak from the placebo peaks.

Assuming you are getting a reasonable k' for the analyte, try changing the ionic strength and/or the pH of your buffer. At a high enough pH, ibandronic acid should be -2 charge, which means that an ionic strength change should move it with respect to chloride and acetate (which are both -1).

[Mr.Buffer]

I could not explain my problem exactly. When i injected ibandronic acid standard (1mg/ml)alone it has 9.6 min retention time and it changes dramaticly and splits when i inject sample.This is first problem.
Second one is more important ;
I can not dedect 0.001 mg/ml ibandronic acid which is my impurity level standard. So i have to find way to increase sample concentration.
(product is 1mg/mL i.v. solution so i need a way to incrase it )
I am thinking SPE but never had experince with it.

[tom jupille]

Diluting your sample 1:1 with mobile phase may help the peak splitting issue (by making the injection diluent "weaker"), but will obviously hurt the sensitivity. Another approach would be to decrease the injection volume, but that has the same drawback.

Another thought: Since ibandronate complexes to Ca++, do a Google search on "IMAC" ("Immobilized Metal Affinity Chromatography"). It's usually used for protein work, but an IMAC column (or cartridge) should selectively retain/concentrate ibandronate as well as any impurities that share the bisphosphonate moiety. You would not see other impurities that way, though.

[Alera]

MrBuffer,

The impurity method you are trying to develop is fairly advanced. I have developed methods for similar compounds in the past and can provide further guidance if you wish. You can contact me directly via email.

[HW Mueller]

Why take this underground? I think many of us, me inclued, can profit from problem solving in IC. I know that mobile ions (analyte) can "stick" to immobilized ions (stat. phase) very strongly if competing other mobile ions are at a low concentration. There should thus be a possibility to concentrate on column.

[Mr.Buffer]

I am all ears to hear some advices.
I mentioned my all problems in previous posts.

[Alera]

Dear HW, sure, no problem, we can keep it online. I don't think RI is a viable option for the impurity method for this particular separation, it just does not have enough sensitivity, plus no ability to concentrate the sample. You are right about possibility of concentrating the sample on the column, but it would require diluting it first substantially to decrease salt concentration in the sample. In my experience, such large injection volumes (several mLs) lead to extremely noisy baseline and do not produce an advantage at the end of the day.

I would probably try to use amino group or phosphate group for derivatization and use conventional IP-RP (ion pair) chromatography.

[HW Mueller]

Reading some of the contributions again I now think that Tom´s suggestions should lead to success.

[Mr.Buffer]

First of all thanks for concern and replies;

Now , I am thinking to prepare a tetrabutylammonium hyroxide buffer for pH 3 for suitable concentration ( e.g 0.1 mM since sample matrix has lots of ions , but I do not know if I think right about buffer concentration and as far as I know buffer concentration has critical role in IP-RP . , details of sample matrix mentioned on first posts)
But I still think to use RI since neither phosphoric acid nor phosphrous acid has chromoforb groups to give UV absorbance.
I know RI has its own problems ( e.g sensivity ) .
We have a ELSD in the lab. but I do not have any experince of it so still RI is better option for me.