Bioequivalence and Bioavailability Forum

Dear Ladi,

❝ However, after talking with sponsor, we decided to put everything on hold and investigate.

Great !

❝ I understand that the n-oxide is more polar and should come out first in reverse phase column...

Not necessarily. I worked on some compounds in the past where the N-oxide was eluted late.

❝ And what about third peak at 2.0 min? Not sure but we also learned during literature review that these n-oxide compounds can easily form dimers.

Could be. Glucuronides are also known to give extra peaks due to collision-induced in-source dissociation - but this time I would expect them to be eluted earlier.

❝ 1.Since we using MS/MS, will it be acceptable to see more than one peak in study sample?

Yes, sure, as long as the peaks are well separated and do not interfere with the analyte/IS peak.

❝ 2.If the inference peak is well separated, is it necessary to identify what those peaks are?

There we come to the difference between "nice to know" and "need to know". As an analyst I'd say it is very, very helpful to know what these additional peaks are: it helps to ensure whether there is a risk of back-conversion, potential stability issues etc.

❝ 3. I have adjusted my gradient table and flow rate so that the two interference peaks are farther away (real itopride 1.4 min, first interference at 2.0 min and second interference at 2.2 min). Will it be OK if I set up my method to stop acquiring itopride at RT 1.9 so that the two interference are no longer visible. What else we have to do to be able to use this new gradient method for study.

It is possible. Some people even use multiplexing i.e. two HPLC systems connected to a single MS/MS system. Two runs injected in parallel, one on each HPLC system. A sample is injected from HPLC 2 during the "non-interesting" part of the chromatogram from HPLC 1. This being said, you have to be sure that the retention times are stable enough. Personally, unless using multiplexing I would acquire the full chromatogram.

❝ 4. Once we get our hands on the n-oxide, we will add it to the QC to test for selectivity, back conversion during extraction and long term storage. Is that enough or should we do more.

It sounds good !