Biomedical Engineering Reference
In-Depth Information
placed on tracking the analyte in mass spectrometric detection due to the possibility
of ion suppression or enhancement from coeluting component(s), all these three
stages should be considered as a whole while choosing an internal standard. In other
words, these three stages affect the selection of an internal standard interactively.
For example, when a sample preparation method produces extremely clean extracts,
then the emphasis should be shifted from tracking the analyte in MS detection to
tracking the analyte in extraction. On the other hand, if the extracts from the sample
preparation contain coeluting matrix components that cause ion suppression or
enhancement, then tracking the analyte during MS detection to correct matrix effects
becomes more important. Moreover, different extraction methods may have different
requirements on the internal standards. Apparently, the requirement on internal
standards for tracking an analyte during a simple “dilution-and-shoot” treatment
would be less stringent than that for liquid-liquid extraction (LLE) or solid-phase
extraction (SPE) methods.
Among the two types of internal standards, SIL internal standards, particularly
those labeled with
13
C and/or
15
N, are most effective and should therefore be used
whenever possible [
9
]. The molecular weight (MW) of an SIL internal standard
should be at least 3 Da (ideally 4 or 5 Da) higher than that of the analyte, though this
is not an absolute necessity. For example, norethindrone-
13
C
2
has been successfully
used for the determination of norethindrone over the concentration ranges of 2.5-
500 pg/mL and 0.05-10 ng/mL [
10
]. In addition, the location of stable isotope
atoms should be given consideration in synthesizing a deuterated internal standard,
so that deuterium exchange would not occur during sample preparation. It is also
preferable that the stable isotope atoms are included in the product ions as well to
avoid possible cross-contamination during mass spectrometric detection, though it
is rare in modern mass spectrometers.
Despite the very desirable performances of SIL internal standards, they are not
always available or are very expensive, especially when seeking exclusively non-
deuterium-labeled internal standards. Then, structural analogues can be used. In this
case, coelution of an analyte and its internal standard is preferred in order to reduce
matrix effect [
11
] or to expand linearity range [
12
] .
There are many different ways to find structural analogues, such as literature
search or key chemical structure search. Merck index is a valuable reference source.
Usually, internal standards can be found from the same therapeutic class as the ana-
lyte. For example, to find a potential internal standard for penciclovir, acyclovir,
ganciclovir, and valacyclovir could be considered because all of them belong to
guanine analogue antiviral drug, i.e., containing a key moiety of guanine (Fig.
1
).
Using skeleton structure search is another useful way to find an internal standard.
Alternatively, structural analogues can be synthesized by adding a nonessential
group, such as an extra methylene group (-CH
2
-).
Once potential structural analogues are found, their physical-chemical proper-
ties, such as log
D
(hydrophobicity) vs. pH, can be calculated and compared with
those of the analyte using software (e.g., Pallas) prior to being experimentally tested.
As shown in Fig.
2
, both acyclovir and ganciclovir could be used as the internal
standard for penciclovir, particularly the latter due to the same number of hydroxy
