Calibration curve issues [Bioanalytics]
Dear Sri!
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) AUCt ≥80% AUC∞.
The ULOQ should be chosen based on the expected Cmax 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
Example: LLOQ 10, ULOQ 500, n=6–8
Now you have to adjust these values for practicability (while keeping the LLOQ and ULOQ fixed).
❝ 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) AUCt ≥80% AUC∞.
The ULOQ should be chosen based on the expected Cmax 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.
Ci = Ci-1 × (Cn/C1)1/(n-1)
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).
—
Dif-tor heh smusma 🖖🏼 Довге життя Україна!
Helmut Schütz
The quality of responses received is directly proportional to the quality of the question asked. 🚮
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Dif-tor heh smusma 🖖🏼 Довге життя Україна!
Helmut Schütz
The quality of responses received is directly proportional to the quality of the question asked. 🚮
Science Quotes
Complete thread:
- Regarding US FDA guidelines sri 2009-07-09 13:01 [Bioanalytics]
- Calibration curve issuesHelmut 2009-07-09 13:48
- Calibration curve issues ElMaestro 2009-07-09 18:38
- Weighing Helmut 2009-07-09 19:24
- Weighing ElMaestro 2009-07-09 20:06
- Weighing Helmut 2009-07-09 21:00
- Weighing or weighting ? Ohlbe 2009-07-10 10:33
- Weighting! Helmut 2009-07-10 14:07
- Weighting! yjlee168 2009-07-16 12:05
- Weighting! Helmut 2009-07-10 14:07
- Weighing or weighting ? Ohlbe 2009-07-10 10:33
- Weighing Helmut 2009-07-09 21:00
- Weighing ElMaestro 2009-07-09 20:06
- Weighing Helmut 2009-07-09 19:24
- Calibration curve issues ElMaestro 2009-07-09 18:38
- Calibration curve issuesHelmut 2009-07-09 13:48