Biomedical Engineering Reference
In-Depth Information
and accuracy, especially when an endogenous counterpart of the drug is present. In
these cases, the PK assay could detect positivity in normal donor and baseline
patient's samples. In such cases, using normal or baseline samples for LOD assess-
ment would artificially increase LOD and make the PK assay appear to be of poor
sensitivity. An alternative approach would be to use samples depleted of endogenous
target analyte for assessment of LOD. Other potential interference factors could also
contribute to background positivity, which need to be examined. Defining minimal
sample dilution is necessary.
11.3.3 Lower Limit of Quantification
A separate set of samples at low concentrations is prepared and assessed in multiple
repeats for lower limit of quantification (LLOQ) assessment. The lowest interpolated
concentration that meets precision (CV
20%) and accuracy (recovery 80-120%)
criteria is considered assay LLOQ. If minimal sample dilution is needed, the dilution
factor needs to be accounted for in calculating assay LLOQ. Limit of quantification at
high concentrations also needs to be defined if a prozone effect (or hook effect) is seen.
In these cases, drug at high concentrations shows decreased signal and the sample
result would be misreported. For samples above the upper quantification range,
repeated testing using diluted samples is recommended.
11.3.4 Linearity of Sample Dilution
If high concentrations (above the upper quantification range) of the analyte are
expected, samples may need to be diluted prior to testing. Samples are prepared at
high concentrations (at least 10-fold higher than the upper limit of the standard curve
point) and tested at different dilutions. Recovery of samples tested at different
dilutions is expected to be within 80-120% from the theoretical value. The dilution
curves should be plotted with the standard curve to demonstrate continuity and
linearity. The slope from the linearity study curve should be within 10% of the slope
from standard curve. Lack of hook effect should be demonstrated. Serial and parallel
dilutions will be both examined to demonstrate sample dilution linearity.
11.3.5 Stability
Sample stability is examined for benchtop (ambient temperature), short-term 4
C and
long-term
80
C storage using samples at H, M, andL concentrations. Sample
recovery between 80-120% from theoretical value (or baseline value) is considered
acceptable. Cell-based assays generally are prone to inherent variability and this can
also occur with flow cytometric assays. Consequently, it is not appropriate to use a
single data value at time 0 as the reference value for evaluating sample stability. It is
advisable to establish a reference baseline value using multiple runs within a short
timeframe. Alternatively, the use of the “true” value (spiked concentrations at H, M,
and L) could also be considered as reference baseline value for stability studies.
20 to
Search WWH ::
Custom Search