Biomedical Engineering Reference
In-Depth Information
C
=
m
ULOQ / 5.
(2)
IS - min
Apparently, the
C
IS-max
must be equal to or greater than the
C
IS-min
, i.e.,
C
IS-max
³
C
IS-min
to simultaneously satisfy the criteria of purity for both the analyte and its internal
standard. Accordingly, the following formula is obtained.
ULOQ / LLOQ
<
100 / (
mn
).
(3)
In other words, the purity of analyte and IS reference standards determines the
maximum possible concentration span. Sometimes, it is impossible to simultane-
ously satisfy both (
1
) and (
2
), i.e.,
C
IS-min
>
C
IS-max.
In this case, either calibration
range needs to be adjusted (narrowed) or analyte or internal standard reference stan-
dards of higher purity must be used.
2.4.2
MS Sensitivity Towards an Analyte and Its Internal Standard
When the MS sensitivity towards an internal standard is higher than the analyte,
relatively lower IS concentration can be used. Otherwise, higher IS concentration is
necessary. The aim is to achieve S/N high enough for the internal standard so that
noises in IS signal is negligible, i.e., no inclusion of any variations that have not
been experienced by the analyte. For this reason, the IS concentration should be as
high as possible. On the other hand, the IS signal should not be so high that the
ratios for lower concentration standards are too low. When inappropriate regression
algorithm or calculation precision is used, unreliable or even incorrect regression
results could be obtained.
2.4.3
Ion Suppression or Enhancement
When matrix effect exists, it is usually preferable to coeluate the analyte and its
internal standard to better reduce the impact of matrix effect on quantitation. The
more their chromatographic peaks overlap, the better the correction is. Since the
concentration of the analyte varies while the amount of IS added is constant, a
choice must be made as to match which part of a calibration curve. Usually, the seg-
ment between 1/3 and 1/2 of the ULOQ is most important because this segment is
expected to cover the average
C
max
for most drugs and metabolites. This is probably
why other researchers have proposed to use IS concentrations around 1/3 or 1/2 of
the ULOQ of an analyte.
In addition, mutual ion suppression or enhancement may exist when an analyte
is coeluted with its internal standard. To maintain low detection limit, low IS con-
centration should be used. On the other hand, high IS concentration is necessary to
obtain good reproducibility when an internal standard is suppressed by the analyte
[
14,
18
]. Therefore, a balance needs to be made.