Bioequivalence and Bioavailability Forum

Dear sera,

❝ In BE studies, what are the acceptance criteria for the resolution value for analytical procedure of analyte of interest and internal standard? A resolution value >2 between the peak of interest and internal standard is desirable in analytical method validation but there are no acceptance criteria about resolution value in EMA and FDA bioanalytical method validation guidelines.

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❝ In a completed HPLC study, the resolution value was about 1.2 during the study. Analyte and internal standard peaks were free of distortions and splitting.

❝ How can the resolution value between analyte of interest and internal standard in the chromatograms for the above study be justified to be appropriate? What is your opinion for the baseline resolution lower limit?

It really depends on the method used.

If you are using UV or fluorimetric detection: resolution is crucial and the two peaks should be clearly separated.

If you are using LC-MS/MS, a perfect resolution is not required thanks to the selectivity of the detection. Co-elution of the analyte and of the IS can actually help to compensate for matrix effects, if both are affected similarly (that's one of the reasons why stable-isotope labelled internal standards usually perform better than structural analogues or unrelated molecules, though the couple of seconds of difference in retention times often seen with deuterium labelling has been described to create problems). However, during method development you should ensure that there is no ion suppression of the analyte by the IS and vice-versa.