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Back to the forum  Query: 2017-07-23 20:44 CEST (UTC+2h)
 
samar sameh
Junior

Jordan,
2017-07-16 07:47

Posting: # 17554
Views: 340
 

 Metabolite back conversion [Bioanalytics]

Hello Every body:
can any one please share with me the procedure conducted for the back conversion test during bioanalytical method validation. what we do is to spike the LLOQ samples of the drug with the maximum expected concentration of the metabolite, and the calculating the accuracy of the LLOQ through a calibration curve of the drug the covers a range from LLOQ to ULOQ. every time we have a failed accuracy, i dont know if there is really a back conversion or if our procedure is misleading.

BR
S.S
Ohlbe
Hero

France,
2017-07-17 10:24

@ samar sameh
Posting: # 17557
Views: 261
 

 Metabolite back conversion

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.

Regards
Ohlbe
samar sameh
Junior

Jordan,
2017-07-18 13:29

@ Ohlbe
Posting: # 17558
Views: 192
 

 Metabolite back conversion

Dear ohlbe

Thank you very much for the valid and effective reply, it is very helpful. below the answeres too your question:
We are dealing with 2 metabolites both on the +ve ionization mode; the N-oxide (potency=96.8%) and sulfoxide (potency=99.9%)
Ohlbe
Hero

France,
2017-07-18 16:29

@ samar sameh
Posting: # 17559
Views: 172
 

 Metabolite back conversion

Dear Samar Sameh,

» We are dealing with 2 metabolites both on the +ve ionization mode; the N-oxide (potency=96.8%) and sulfoxide (potency=99.9%)

I have no experience with sulfoxide. N-oxides are known for in-source dissociation. My understanding is that source temperature is an important factor. However if the metabolite is chromatographically separated from the parent, in-source dissociation should not create any problem.

If you are dealing with 2 metabolites and are finding potential issues, I would suggest to study the two separately in order to facilitate troubleshooting.

Regards
Ohlbe
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