Chromatography Forum

hi there,
I am trying to develop the method for analysis of cidofovir API. I got good retention of cidofovir using ion pair(tetrabutyl ammonium dihydrogen phosphate), but it is quite unstable as the method is gradient. The product is not getting retained without the use of ion pair. Does anyone have the method without the use of ion pair.

Thanks in advance

There are now many columns from multiple manufacturers that provide retention of very polar organics without using ion pairs. Look for a good polar embedded phase as one option. There are some "aqueous stable" phases that also could be evaluated. And SIELC offers a mixed mode phase that has embedded ions in the ligand, and Imtackt has a multi-mode phase as well.

I'm sure if you wait a bit longer the manufacturers will probably post their suggestions. Otherwise, check with your favorite column manufacturer for phases that retain very polar compounds. You should be able to do thiw without ion pairing.

here are methods for cidofovir without ion-pairing reagent, using Primesep D and Primesep 100 columns:

http://www.sielc.com/compound_153.html

Also here is article on tenofovir (no IP reagent, Primesep B4 mixed-mode column):
Evaluation of Hexadecyloxypropyl-9-R-[2-(Phosphonomethoxy)Propyl]-
Adenine, CMX157, as a Potential Treatment for Human
Immunodeficiency Virus Type 1 and
Hepatitis B Virus Infections, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2007, p. 3505–3509

Contact me if you have questions.

thanks vlad. But i already got this information. I am not having primsep B column but I tried YMC bASIC column instead but got the peak eluted at 2 min. Can u suggest any equivalent column for that.

thanks in advance.

here are methods for cidofovir without ion-pairing reagent, using Primesep D and Primesep 100 columns:

http://www.sielc.com/compound_153.html

Also here is article on tenofovir (no IP reagent, Primesep B4 mixed-mode column):
Evaluation of Hexadecyloxypropyl-9-R-[2-(Phosphonomethoxy)Propyl]-
Adenine, CMX157, as a Potential Treatment for Human
Immunodeficiency Virus Type 1 and
Hepatitis B Virus Infections, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2007, p. 3505–3509

Contact me if you have questions.

This compound is ideal for HILIC. Use an Atlantis HILIC silica column for this assay.

You can try ANY bare silica column in HILIC mode with 85% ACN and 5-10 mmol ammonium formate pH 3. Play with pH to adjust retention and peak shape. Play with ACN to adjust retention. Watch for solubility of buffer and your compound at high organic concentrations. This is most "economical" approach.

To add to Vlad's very useful comments: for HILIC, you need a high acetonitrile concentration, probably around 75%. Contrary to reversed-phase, retention increases as you increase the acetonitrile concentration. At such a high acetonitrile concetration, phosphate buffers are problematic due to solubility issues. You are better off using organic buffers such as formic acid/ammonium formate or simply just straight formic acid. Acidic acid, acetate is also an option.

thanks vlad. i will try this in our lab.

You can try ANY bare silica column in HILIC mode with 85% ACN and 5-10 mmol ammonium formate pH 3. Play with pH to adjust retention and peak shape. Play with ACN to adjust retention. Watch for solubility of buffer and your compound at high organic concentrations. This is most "economical" approach.