USP Monographs: Adenine

[tien]

Hi all,
May anyone explain this mobile phase composition for HPLC analysis of adenine?


Time (min) Buffer solution(%) Acetonitrile (%) Water (%)

0 5 5 90
20 5 5 90
20.1 10 10 80
30 10 10 80
30.1 5 5 90
40 5 5 90
Thank all in advance

[Gerhard Kratz]

An USP method: always a challenge
Gradient run: You start with high amount of water to get higher retention times.
And what is your question?

[Blazer]

Based on the USP monograph, you'll need three lines for your three mobile phases. Line A is the buffer (ammonium phosphate, pH 6.2), Line B is acetonitrile and Line C is water.

Initial conditions are 5% A, 5% B, 90% C. This stays isocratic for 20 minutes. At 20.1 minutes the proportion shifts to 10% A, 10% B and 80% C. This stays isocractic until 30 minutes. At 30.1 minutes you revert back to 5% A, 5% B, 90% C and stays isocratic until 40 minutes; this is a re-equilibration step.

[tien]

does it mean the run time for 1 sample analysis is 40 min?
Thank you all.

[itspip]

Yes.

Gradient methods often look something like what you posted in your question.

[hossam kamal]

is that method for assay or determination of the related substances because if it is for the related substance i think that increase in pH and % of the organic solvent after 30 min was for resolution of the impurities from the principle molecules