Biomedical Engineering Reference
In-Depth Information
7.2.7
Limits
Limit of detection (LOD) is the lowest concentration of an analyte that the
bioanalytical procedure can reliably differentiate from background noise [
58
] .
SOFT/AAFS guidelines establish that in chromatographic assays, the LOD might
be the smallest sample concentration of a drug needed to give a peak height three
times the noise level of the background signal from a blank sample. Limit of
quantification (LOQ) can be determined experimentally as the lowest concentration
for which an acceptable coefficient of variation can be routinely achieved. LOQs
may be administratively defined in terms of the concentration of the lowest calibra-
tor [
56
]. FDA indeed defines lower limit of quantification (LLOQ) as the lowest
amount of an analyte in a sample that can be quantitatively determined with suitable
precision and accuracy (not more than 20 % in terms of CV). The upper limit of
quantification (ULOQ) is the maximum analyte concentration of a sample that can
be quantified with acceptable precision and accuracy (bias). In general, the ULOQ
is identical with the concentration of the highest calibration standard.
7.2.8
Matrix Effects
A mandatory requirement when validating an LC-MS(-MS) based method accord-
ing to the FDA guidelines is to evaluate and attempt to minimize the incidence of
matrix effects [
58
]. Matrix effects refer to the direct or indirect alteration or interfer-
ence in response due to the presence of interfering substances in the sample [
52
] . It
can either reduce the analyte response (ion suppression) or enhance it (ion enhance-
ment). Both can considerably compromise the accuracy of quantification and ion
suppression may in the worst case even lead to decrease sensitivity and to false
negative results.
It is acknowledged that ESI is more susceptible to matrix effects compared with
APCI or APPI, and that ionization in the negative mode is more selective than the
positive mode [
55
] .
Two main methods exist for matrix effect evaluation and are widely cited. The
method proposed by Bonfiglio et al. uses postcolumn infusion of target analytes to
qualitate highlight regions of suppression/enhancement in a chromatogram [
65
] ,
whilst that proposed by Matuszewski et al. [
64
] suggests a method to quantify the
degree of matrix effects for particular analytes using peak area/height ratios from
analyses carried out with and without the presence of matrix components. Another
method to evaluate matrix effect has been used in literature: a comparison is per-
formed between two calibration curves: one created with blank samples, fortified
after the extraction process, and the other with standard solutions. The value of
matrix effects, intended as ion suppression, is calculated by comparing the slopes of
those curves, this method directly highlighted influence of matrix components on
the sensitivity of the method [
66,
67
] .
The use of adequate internal standard or better isotopic labeled internal standard
can minimize the matrix effect.
