Chromatography Forum

I’ m routinely determining by HPLC the assay of a drug substance, which is an organic salt (basic + acid moiety)

I strictly reproduce the USP method under all aspects (mobile phase, stationary phase, concentrations, injection volumes etc etc etc) and I’m using official USP standards.

The method gives the assay of both the portions of the molecule.

The problem is, even if the results are in specification, both the portions of the molecule, which should give 100 (or less) as a sum, stay near the upper limit of the specification interval. When you sum them, you have more than 100% (101.5%, for example)

The USP standards were dried for the time and at the temperature prescribed, so I have no reason to suspect that standard areas should be higher than what I’ m finding.

Does anyone has a suggestion?

Thanks !

If you exactly follow USP prescribed procedure for RS drying and using, some other sourses can be considerd, like
1. Stability of standard and sample preparation
2. carry over phenomena
3. Problems coused by instrument/ column


what happens if you will invert the sequence ( first inject the sample and after the standard)?

Carryover does not seem to be a problem (standard deviation on standard injections is extremely low and the same results are obtained on two sample preparations, even on different days). Solutions injected hours after preparation does not show areas variation.

Sure I'm going to order a new amount of official standard and I'm also trying the same analysis on another instrument, inverting the sequence

Thanks for helping me!

Dear Elena,
could your own substance be better (higer HPLC assay value) than USP standard ? I thinh that it could be possible especially if the USP standard is an old bacth. So if a new analisys confirm higher HPLC value, obviously inside specifications limit, try to consider this ideas.

Sounds like it could be a standard problem. Can you pull some samples from the "morgue" that were run previously and see if the results can be duplicated?

Hi Elena,

Two other possible ways to find a solution:

First: If the product is a salt (basic + acid) moiety, could your product be performed a chemical assay (with perchloric acid in acetic or with NaOH in water) in order to know whereas the salt has been obtained correctly proportion.

Second: as GT comment, look catalog to know if USP reference STD is only to identify or to quantify... some standrads are only to identify (IR, Polimorphism...).

I hope it helps