Bioequivalence and Bioavailability Forum

Dear Sri!

❝ The calibration (standard) curve should cover the expected unknown sample concentration range in addition to a calibrator sample at lower limit of quantification.

In method development you should target the calibration range based on the lowest and highest concentrations expected in the study.
The LLOQ should be chosen in such a way that you are able to reliably describe the plasma profile. Reliable in this sense means that you are able to estimate the apparent elimination and (in most regulations) AUC_t ≥80% AUC_∞.
The ULOQ should be chosen based on the expected C_max in the majority of subjects.
The guidance suggests six to eight calibrators for a linear function (probably more for nonlinear functions). Although linear regression theory calls for equidistant points, in bioanalytics most (all?) people opt for a geometric progression of calibrators in order to

• reduce the variability at the lower range and
  
• deal with the multiplicative error due to serial dilutions.

You may use following formula as a starting point:

Ci = Ci-1 × (Cn/C1)1/(n-1)

where

i index of the respective calibrator (2, 3, …, n), n number of calibrators, Ci calculated calibrator at i, Ci-1 previous calibrator, C1 lowest calibrator (LLOQ), and Cn highest calibrator (ULOQ).

Example: LLOQ 10, ULOQ 500, n=6–8
 6    7    8
-------------
 10   10   10
 22   19   17
 48   37   31
105   71   53
229  136   94
500  261  164
     500  286
          500
Now you have to adjust these values for practicability (while keeping the LLOQ and ULOQ fixed).