Bioequivalence and Bioavailability Forum

Hi Mutasim,

❝ Does it mean measuring each one alone? or the total as in FDA guidance?

The glucuronide is much more hydrophilic than the parent. No way to extract both together in sample preparation (not to speak about having them in the same chromatographic run – unless you apply an aggressive gradient elution). Hence, use glucuronidase. This gives you the total.

❝ CI be for each one alone or both unconjugated and total or only total?

Total.

Hi Mutasim,

reading the guidance again I understand your confusion.

On top of page 3 we have

Analyte: ☐ parent ☐ metabolite ☒ both
Background: Ezetimibe undergoes extensive pre-systemic metabolism; ezetimibe-glucuronide is the major active metabolite. Because of extensive hepatic recirculation, the exposure to ezetimibe is less representative to evaluate absorption.

And then

Bioequivalence assessment: Background/justification: On total (parent + glucuronide metabolite together)

(my emphases)

Analyte: ☒ both seemingly tells us to measure them separately but the remainder tells us that only the total should be assessed for BE. So why all that fuzz?

I still think that cleaving by glucuronidase and analyzing ezetimibe is the way to go. What I wrote above was based on extraction techniques (SPE, LLE). If you opt for protein precipation it could work but as a hydrophilic compound it might well be that some part of the glucuronide gets trapped in the precipitate. I also believe that you need two chromatographic conditions. Furthermore, glucuronides are known for back-conversion in the ion-source. Hence, what you might get are wrong values for both the parent and the metabolite but the sum will be correct (assuming that nothing of the glucuronide gets trapped – what I doubt). Hence, I would keep it simple and go for cleavage and have just one run.
If the EMA really want both separatelly IMHO, it would have been better to state in the GL:

Analyte: ☒ parent ☒ metabolite ☐ both

Out of curiosity: Since dealing with glucuronides was the topic of my first paper* – which stationary & mobile phase are you using?

---

• Mascher H, Nitsche V, Schütz H. Separation, isolation and identification of optical isomers of 1,4-benzodiazepine glucuronides from biological fluids by reversed-phase high-performance liquid chromatography. J Chr B. 1984; 231–9. doi:10.1016/S0378-4347(00)80885-3.

Hi Mutasim & all,

yesterday at BioBridges Paola Coppola (MHRA, PKWP observer) presented the state of affairs. The ezetimibe guidance is finalized and will be published soon. No changes to the draft. You are right and I was wrong. One should indeed measure both ezetimibe and its glucuronide. BE should be based on their sum* only.

I still think that’s stupid. Both methods have errors (inaccurate, imprecise). If you evaluate the sum of concentrations, errors will sum up as well. Given, the concentrations of the glucuronide are two orders of magnitude higher than the parent’s and hence, the impact of the latter is negligible.
At the BioInternational 1994 in Munich there was a nice distinction between “Need to know” and “Nice to know”. I don’t like the idea that something which is only nice to know (i.e., not used in the assessment for BE) is mandatory in a guidance. 

---

• Bioequivalence assessment: Background/justification: On total (parent + glucuronide metabolite together)
  (my emphases)
  Cave: Apples & oranges. Total should be expressed as ezetimibe after correction for the molecular masses (M_ezetimibe 409.4 g·mol^–1, M_{ezetimibe glucuronide} 585.5 g·mol^–1).

"The applicant should document that the enzymatic de-conjugation can be considered quantitative under assay conditions"