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target metabolite. A typical calibration curve is
shown in
Figure 1
.
Using this calibration curve, for example, the
actual concentration of the target metabolite in
a biological sample is determined by inverting
the equation for the
variation (CVs) of less than 15% for 80% or
more of the metabolites (
Figure 2
).
30,34
Such
approaches assume that the chosen SILIS
compounds have similar ionization behavior to
the metabolites under investigation. Another
approach uses structural analogues as internal
fitted calibration curve as:
¼
½
Park Area
ð
Metabolite
ÞO
Peak Area
ð
SILIS
Þ
0
:
0332
Metabolite Concentration
(1)
0
:
2474
where Peak Area (SILIS) is the peak area,
obtained from the multiple reaction mode detec-
tion, of the stable isotope-labeled internal stan-
dard spiked in the biological sample and 0.0332
and 0.2474 are the y-intercept and slope, respec-
tively, as shown in
Figure 1
.
A major drawback of this approach is the
often prohibitively high cost of stable SILIS
compounds and/or commercial unavailability
of isotope-labeled standards for many metabo-
lites. To circumvent this problem, many studies
have utilized a small set of representative SILIS
compounds and used them to analyze up to
200 or more metabolites with coef
standards that have similar structures and chem-
ico/physical properties as the targeted metabo-
lite class and are not a part of the endogenous
compounds. Tubercidin, for example, is thus
used as a structural analogue for the analysis of
nucleosides.
35,36
Quantitation using such struc-
tural analogues is performed by spiking biolog-
ical specimens with one or more structural
analogues as internal standards, and metabolite
concentrations are determined by comparison
of mass peak area with that of structural
analogue or by using a calibration curve similar
to that shown in
Figure 1
. CVs of 5% to 20% have
been obtained by this approach in the analysis of
cients of
4.0
3.5
y = 0.2474x + 0.0332
R² = 0.9994
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.0
5.0
10.0
15.0
Standard Metabolite Concentration (µM)
FIGURE 1
Typical calibration curve obtained using solutions with different standard metabolite concentration and fixed
internal standard (SILIS) concentration.
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