Biomedical Engineering Reference
In-Depth Information
range should consist of at least six different concentration levels
[
70
,
71
]. During validation, the suitability of the curve range is
tested by preparing calibration curves for each analytical run in the
study. The suitability of the calibration curve is determined by
consistent and acceptable accuracy and precision of the calibration
samples, as well as acceptable accuracy of quality control (QC)
samples prepared at concentrations within the range of the assay.
QC samples are prepared independently of calibration samples at
low, mid, and high concentrations within the curve range, and are
used to assess both accuracy and precision of the method as well as
stability of the drug in matrix and in the prepared extracts.
It is advisable to prepare calibration standards in the same matrix as
the study samples to be analyzed. However, this will not always be
feasible due to the limited availability of control matrix on ethical
grounds for animal welfare. When this occurs, the choice of a
surrogate matrix of high similarity to the ocular study sample matrix
is recommended to allow for appropriate assay performance. If a
closely related surrogate matrix is not available and a less closely
related one is utilized (e.g., plasma as a surrogate matrix for ocular
tissues), use of a stable labeled internal standard becomes even
more important (when using LC/MS/MS). Also critical is a thor-
ough sample cleanup procedure, to ensure comparability of the
final extracted samples derived from the surrogate and study sample
matrices. This is important because of potential differences in the
recovery of drug and/or due to variable amounts of endogenous
compounds present in the surrogate and study sample matrices that
can affect quantification. A stable labeled internal standard is likely
to compensate for the differences. As discussed previously for
melanin-containing tissues, surrogate matrix may not yield compa-
rable drug peak response to that of the study sample matrix, but
with adequate internal standard correction, an assay can often be
developed to appropriately quantify drug in the tissues of interest
under these circumstances.
3.4 Use of a
Surrogate Matrix
After a method validation or qualification study has been completed
and the bioanalytical method has been shown to be acceptable for its
purpose, the sample analysis phase can be initiated. Acceptance cri-
teria are set for the calibration curve and for the QC samples used
during sample analysis based on the type of study, the performance of
the method during validation or qualification, and regulatory
requirements. Criteria are often set as per the regulatory guidance
documents [
70
,
71
] with calibration curves having at least 75 % of the
calibration standards within 15 % of nominal concentration (20 % at
the LLOQ) and two-thirds of QC samples within 15 % of nominal
concentration. It may be acceptable to widen the acceptance criteria,
especially during the early phases of drug development. Although
meeting the acceptance criteria is critical in the decision to report
3.5
Sample Analysis
