Biomedical Engineering Reference
In-Depth Information
moieties. On the other hand, valacyclovir is not as good because of the introduction
of an extra amine moiety and the absence of the hydroxy moiety.
2.4
Concentration of Internal Standards
It is generally believed that as long as the same amount of an internal standard is
added to all the samples in a batch (run), i.e., calibration standards, quality controls,
and unknown samples, the concentration of an internal standard is not important.
This is probably why not much information exists as how to determine an appropri-
ate concentration for an internal standard. Some researchers proposed that the
concentration of an internal standard should be approximately half of the upper
limit of quantitation (ULOQ) of the analyte [
13,
14
] or even higher than the ULOQ
[
2
], while others suggested a relatively lower concentration corresponding to about
the first third of the calibration range, in order to minimize potential interferences
with the analyte due to potential impurities from SIL internal standards [
15
] .
Unfortunately, none of these were followed by more detailed theoretical consider-
ations or supporting experimental data.
Based on our experience, it is difficult to set a clear-cut guideline as what IS
concentration should be used. Instead, all of the following factors should be taken
into consideration while determining an appropriate concentration for an internal
standard with the emphasis on the first factor.
2.4.1
Purity of Reference Standards and the Targeted Calibration Range
An internal standard may contain trace amount of the analyte of interest, especially
a SIL internal standard due to similar synthesizing routes. In this case, a maximum
IS concentration can be obtained based on its impurity (e.g.,
n
% of the analyte in
the IS reference standard) and the ±20 % acceptance criterion for bias at the LLOQ
level [
13, 16-
17
]. In this regard, the concentration of an IS should be therefore kept
as low as possible to reduce its contribution to analyte concentration.
C
=
20 LLOQ /
n
.
(1)
IS - max
On the other hand, the analyte or analyte reference standard may contribute to
the response of an internal standard either due to impurity or because of natural
abundance of stable isotopes (say
m
%). Therefore, the concentration of the internal
standard must not be too low. Otherwise, the contribution from the analyte or its
reference standard would be significant, e.g., more than 5 % of IS concentration or
response [
16
], and linearity and accuracy could be greatly affected. Accordingly, a
minimum IS concentration could be obtained based on the ULOQ for the analyte.
For this reason, a high IS concentration is preferred.
