Biomedical Engineering Reference
In-Depth Information
complex ocular tissue assays. There are two types of internal
standards, structural analogues and stable isotopically labeled mole-
cules. Typically, an analogue internal standard will differ only
slightly in chemical structure from the drug and will have similar
extractability and chromatographic behavior. Analogue internal
standards can be used with LC/MS/MS; however, stable labeled
internal standards are superior and are the internal standard of
choice. Besides almost identical extractability, a stable labeled inter-
nal standard will have nearly identical chromatographic character-
istics to the drug and will elute at approximately the same retention
time. This is important in controlling ionization effects in LC/MS/
MS methods caused by matrix components which can suppress or
enhance peak response. Under certain conditions, with highly effi-
cient LC columns, drug and stable labeled internal standard can
chromatographically separate, which may not be desirable.
The % recovery of a drug (and internal standard) is an impor-
tant component of bioanalytical method development. Recovery
can be estimated by spiking the drug and the internal standard into
control matrix (e.g., quality control samples) and comparing assay
response after sample processing to that of a reference sample at
theoretical concentrations representing 100 % recovery. Recovery
of drug across several concentrations as well as that of the internal
standard at the concentration used for extraction should be similar.
For LC/MS/MS, the reference sample should be prepared in an
extracted blank of the biological matrix of interest to account for
any ion suppression or enhancement of mass spectrometer response
(matrix effects) by endogenous components of the biological
matrix. With tissues, it is difficult to determine the true recovery
of a drug [
62
,
63
]. Homogenates can be prepared and spiked with
drug, but recovery estimation in homogenates only addresses the
extraction component of the assay. Therefore, it is important to
have recovery assessments from intact tissues, when feasible, to
allow for the measurement of drug loss through the homogeniza-
tion and extraction procedures. However, it must be recognized
that exogenous fortification of a control tissue sample does not
necessarily equate with biologically incurred drug present in tissue
samples following drug administration. Nonetheless, assessments
of recovery from both homogenates and intact tissues can better
lead to the development of an acceptable method which can enter
into assay qualification or validation studies.
Bioanalytical assays used in studies for submissions to regulatory
agencies must meet certain requirements to be acceptable and must
be assessed prior to use. Method assessments of assay performance
generally fall into two categories, the more rigorous method vali-
dation and the abbreviated method qualification. Method qualifi-
cations are validation-like studies that assess a subset of components
normally required for a validation. For bioanalytical assays
3.3 Method
Qualification/
Validation
