Chromatography Forum

We are trying to develop a method for measuring mercaptopurine in a suspension prepared with the USP Vehicle for Oral Suspension. This vehicle contains microcrystalline cellulose, xanthan gum, carrageenan, carboxymethylcellulose sodium, citric acid, sodium phosphate dibasic, simethicone, potassium sorbate, methylparaben, and water. We started out with the USP HPLC method which uses a C18 column with a ACN/ 0.1% AcOH in water gradient. We found that if we use any ACN, our compound co-elutes with the DMSO in the solvent front. Also we found that we needed to drop the pH to improve the mercaptopurine peak shape so now we are running just pH 2 phosphate buffer. (pKa ~ 3.7)

We've tried injecting mercaptopurine standards in DMSO and in the suspension vehicle. The mercaptopurine peak in just DMSO elutes around 4 minutes. The mercaptopurine peak in the suspension elutes at about 6 minutes. I am wondering if something in the vehicle is complexing the mercaptopurine, causing it to be less polar and elute later.

I have two questions. Is there a different column that I should be using so that this compound is better retained and I can add some organic to the mobile phase? How can I prepare my samples so that the suspension vehicle does not cause a peak shift.

Thanks very much for any suggestions.

KarenJ

You could try adding an ion pairing to the mobile phase.
Maybe something like TFA or HFBA to increase retention.

Do you have access to the individual compounds in the USP vehicle
for Oral solution? Maybe add these on at a time to the std solution
and compare the RT for solutions of a). std vs. b). std + excipient
to find out which excipient is contributing to the shift in RT.

Here is method for purine based compounds on mixed-mode Primesep column. Purines will retain based on combination of reverse phase and cation-exchange mechanisms. If you reduce amount of TFA to 0.1 you will probably get around 10 minutes retention. Weaker acids (formic) will give you even longer retention:
http://www.sielc.com/compound_345.html

Retention will significantly increase on Primesep 100 and Primesep A column. DMSO will elute in the same way as in other column (2-3 min). If you increase organic to 20-40% DMSO will elute even faster. Retention time of purine based molecules can be adjusted with the ion-strength of the mobile phase. All these columns have ion-pairing reagent attached to the surface.

Contact me if you have questions.

Vlad