Bioequivalence and Bioavailability Forum

Dear Sagar!

❝ […] in the example you mentioned, can we say that the method was not properly developed. Ideally one would have expected a method which separates the RT of the two enantiomers by aconsideration time to minimise overlapping.

Especially for a chiral method I would say that peaks were sufficiently separated. Pharmacopoeias (like USP) call for a chromatographic resolution R_s of ≥1.5, whilst FDA (Reviewer Guidance - Validation of Chromatographic Methods, Nov 1994) recommends an R_s >2 (see also this post). At the right is the chromatogram of a high aqueous standard, showing a resolution of ≈2.5.

❝ You said the first peak was not integrated at all.

Yes; IMHO that’s a flaw in the integration method.

❝ Does that mean the separation was achieved but not quantification of the first peak isomer was achieved?

Exactly. He excluded the first peak. Therefore even for the aqueous standard with a perfect baseline the area of the second enantiomer is systematically too low. That’s why I stated in my previous post:

❝ ❝ • Even if the second peak would have been correctly

❝ ❝   integrated by his method (▬▬▬), the area would be underestimated.

No big deal for this particular method (BE of one the second enantiomer to the racemate; no in vivo interconversion expected), since all chromatograms are evaluated in the same way. But think about another drug where enantiomers are interconverted. The ratio of the enantiomers will not be the same in subject’s samples compared to CCs and QCs. Furthermore the ratio will change within the time profile… Only the perpendicular drop integration (▬▬) in my first example will give correct results.