Bioequivalence and Bioavailability Forum

Dear ryraman2661,

❝ I have a query with reference to the anchoring point(s), (LLOQ and ULOQ) in method validation of normal, molecules.

Please check the definition of anchor points in section 9, glossary: Spiked samples set at concentrations below the LLOQ or above the ULOQ of the calibration curve and analysed to improve curve fitting in LBAs.

So by definition: LLOQ and ULOQ samples are not anchor points, and anchor points are only to be used for ligand-binding assays, not chromatographic methods.

❝ As per the recent M-10 guideline, the criteria for accepting calibration curve is minimum 75 % of non-zero standards should pass. There is no mentioning of anchoring points.

There is, for LBA, in section 4.2.3: At least 75% of the calibration standards excluding anchor points, and a minimum of 6 concentration levels of calibration standards, including the LLOQ and ULOQ, should meet the above criteria. The anchor points do not require acceptance criteria since they are beyond the quantifiable range of the curve.

❝ In Precision and Accuracy (P & A) experiments.

❝

❝ We do 8 points calibrators including LLOQ and ULOQ and 6 replicates of each level QC. If one anchor point, ULOQ calibrator not meeting, and other 4 QCs are within the accepted calibrators, can we accept?.

If you're using a chromatographic method, no, according to section 3.2.4: For accuracy and precision runs, if all replicates of the LLOQ or the ULOQ calibration standard in a run are rejected, then the run should be rejected, the possible source of the failure should be determined and the method revised, if necessary. If the next validation run also fails, then the method should be revised before restarting validation.

In ligand-binding assays, the LLOQ and ULOQ samples can't be excluded from the curve according to section 4.2.3, as mentioned previously.