Bioequivalence and Bioavailability Forum

Dear Samar Sameh,

It is difficult to give precise comments without more knowledge of the chemistry of the metabolite and possible back-conversion. Even without disclosing the molecule, can you tell us what you are dealing with: acylglucuronide, N-oxide, lactone/acid...

❝ what we do is to spike the LLOQ samples of the drug with the maximum expected concentration of the metabolite

OK, worst case scenario.

❝ every time we have a failed accuracy, I don't know if there is really a back conversion or if our procedure is misleading.

My first question would be: how pure is your metabolite reference standard, and are you sure it does not contain even trace amounts of the parent ? Even 0.1 % would be enough in your experimental conditions. Try and inject a pure solution at the same level of concentration used in your experiment. If there is no parent peak: that's not the explanation. If there is one: it does not mean that your reference is contaminated, it could be in-source back-conversion (in-source collision-induced dissociation is well described in the literature for metabolites such as glucuronides and N-oxide). The next steps will then depend on the chemistry of the metabolite. If in-source back-conversion could be the explanation: changing your ionisation conditions may solve the issue (e.g. if glucuronide, try and decrease the cone voltage/declustering potential/whatever it is called on the instrument you use).

If you don't have any parent peak in a pure solution of the metabolite: then you may indeed have a back-conversion issue. You will have to run a series of experiments to find out at which step, and how to prevent it. I would suggest to start with the last steps of sample preparation, and then move backwards. For instance, extract blank plasma, spike the extract with a pure solution of the metabolite (minimum volume), reconstitute as usual, and inject at different times to see if a parent peak gradually appears.

If your metabolite reference standard contains trace amounts of the parent: you will have to take it into consideration to calculate the nominal concentration of the parent in your samples.