Bioequivalence and Bioavailability Forum

Dear Mohamed,

❝ For bioequivalence/bioanalytical LC-MS/MS assays we used to calculate accuracy and precision for QC samples by carrying out three samples or five samples at three concentration levels...

I would even expect 4 levels: LLOQ, LQC, MQC, HQC.

❝ ...and back calculate concentrations by taking area ratios and applying regression equation.

Yes, that's the correct way to do it.

❝ Recently, a regulatory reviewer is insisting that we calculate precision from area ratios disregarding regression equation.

From which country, and based on which guideline ?

IMHO that's not the correct way to go. The aim of the validation is to ensure that the PK data are based on reliable information i.e. reliable concentrations. AUC is the area under the curve of concentrations versus time, not of peak area ratio versus time.

The EMA and FDA guidelines don't specify whether the CV should be calculated using the concentrations or the peak area ratio. But I've never heard so far of a lab calculating the CV of the ratio, or of an authority requesting this type of calculation.

❝ We examined several validated assays and found that using area ratios only resulted in huge CV% reaching up to 45% in same assays and within limits for some others.

There I'm extremely surprised. I would expect the difference to be really minimal, and I would certainly not expect the CV of the peak area ratio to reach 45 % if the CV of the concentrations was below 15 %. But there may indeed be a difference at low concentration levels, depending on the intercept.