Biomedical Engineering Reference
In-Depth Information
Second, an internal standard must have adequate purity. Preferably, the
contribution of an internal standard to any analyte should be less than 20 % of
the corresponding lower limit of quantitation (LLOQ) of the analyte. Otherwise, the
significant amount of analyte from the added internal standard can bias the reported
signal to noise (S/N) ratio at the LLOQ and cause larger variability at low concen-
trations. The interference of an internal standard to other cointernal standards in a
multianalyte method is rare, but it should be also evaluated. Though there are no
reported criteria for this, it should be at least less than 15 % of the concentration of
a cointernal standard in a multianalyte method. In addition, an internal standard
should not correspond to any in vivo metabolic products of the analyte (e.g., hydrox-
ylated metabolite, N-dealkylation metabolite).
Lastly, an internal standard should be stable during sample processing and LC
separation. It should not significantly degrade, in particular not decompose to
components that can interfere with the determination of the analyte.
2.2
Types of Internal Standards
There are two main types of internal standards. The first ones are stable isotope
labeled (SIL) internal standards. They are compounds in which several atoms in the
analytes are replaced by their respective stable isotopes, such as deuterium (
2
H, D or d),
13
C,
15
N, or
17
O. Labeling with the first three isotopes are most common, particularly
labeling with deuterium (due to less difficulty in synthesis and therefore less expen-
sive). For examples, raloxifene-d
4
-6-glucuronide was used as the internal standard
for the determination of raloxifene-6-glucuronide [
5
] and 1, 2, 3, 4-
13
C
4
estrone
([
13
C
4
]E1) was used as the internal standard for estrone (E1) [
6
]. The usage of stable
isotope labeled internal standards in quantitative LC-MS or GC-MS analysis is
often termed as isotope dilution mass spectrometry (IDMS) [
7
] .
The second ones are structural analogues with different masses or even the same
mass. In the latter case, chromatographic separation between the analyte and its
internal standard must be achieved when distinctive MRM (multiple reaction moni-
toring) transitions could not be found. It is preferable that the key structure and
functionalities (e.g., -COOH, -SO
2
, -NH
2
, halogens, and heteroatoms) of an inter-
nal standard are the same as those of the analyte and differ only by C-H moieties
(length and/or position). Modifications in functionalities would result in significant
differences in ionization efficiency and extraction recovery [
8
] .
2.3
Selection of Internal Standards
An internal standard is expected to track the analyte in all the three distinctive stages
of LC-MS bioanalysis, i.e., sample preparation (extraction), chromatographic sepa-
ration, and mass spectrometric detection. Though the emphasis should usually be
