Biomedical Engineering Reference
In-Depth Information
broader peak shape. Quantitation is based on the responses (e.g., peak areas) of the
two peaks without the need for an internal standard. This technique has been used
mainly in the analysis of pesticide residuals in agricultural produces (e.g., fruits and
vegetables) by LC-MS, especially as the first screening measurement to identify
samples with residues above the maximum residue limits (MRLs).
Since it is always necessary to maintain a gap between the two peaks, the ability
of this technique to correct matrix effect will be impacted when there is a change in
matrix effect during the gap. Moreover, like postcolumn introduction of an internal
standard, any variations in sample preparation as well as in injection volume cannot
be corrected.
3
Performance of Internal Standards in LC-MS Bioanalysis
3.1
Stable Isotope Labeled Internal Standards vs. Structural
Analogue Internal Standards
As demonstrated by many studies, SIL internal standards outperform structural ana-
logue internal standards in terms of precision and accuracy provided the extraction
and LC separation are comparable [
8
,
25
-
30
]. In addition, SIL internal standards
can also extend linearity range, enable the usage of less reliable transitions (e.g.,
loss of water), and prolong in-processing and postpreparation stabilities [
8,
28
] .
3.2
Unexpected Results Using Deuterated Internal Standards
Despite the overall good performance of SIL internal standards, one must not take it
for granted due to the complexity of biological samples, particularly when deuterated
internal standards are used. Deuteration could cause differences in hydrophobicity,
reaction rates, and noncovalent interactions [
31,
32
]. It is usually observed that a deu-
terated internal standard elutes slightly earlier than the analyte does in reversed-phase
LC. This is even more pronounced with extensive deuteration and long retention time.
Sometimes, base-line resolution between an analyte and its deuterated internal
standard could be achieved. For example, when D
10
internal standards were used and
the retention time was longer than 15 min, pibutidine metabolites were completed
separated from their deuterated internal standards (Fig.
5
; [
33
] ).
Sometimes, even the slight separation between an analyte and its deuterated
internal standard could cause significant quantitation errors due to differential ion
suppression towards the analyte and its deuterated internal standard [
34,
35
] . As
shown in Fig.
6
, the elution of carvedilol and its internal standard (D
5
-carvedilol)
overlaps with the declining edge of a matrix suppression region in a matrix lot.
This resulted in more pronounced ion suppression for the slightly later eluted
