Biomedical Engineering Reference
In-Depth Information
1
Introduction
In many occasions of clinical, pharmaceutical, and biological analysis, a high
analytical sensitivity is desirable. For example, certain low-dose regimens, and
inhalation or intraocular injection (e.g., corticosteroids) may result in ultralow
drug concentration in body fluids (e.g., low-pg/mL range in plasma). Furthermore,
some highly active endogenous markers may present at very low levels in biologi-
cal matrices. For instance, the circulating levels of an important vitamin D (VitD)
metabolite, 1R, 25-dihydroxy-VitD
3
, are extremely low, which constitute a daunt-
ing challenge for quantification. Given these importance of analysis of such com-
pounds in clinical and pharmaceutical settings, a highly sensitive analytical method
is necessary. LC-MS/MS is considered one of the most powerful methods for the
clinical and pharmaceutical analysis. However, ultrasensitive quantification of
drug/markers presenting at low-pg/mL levels in plasma remains challenging even
for LC-MS-based methods, which usually carry typical sensitivities at high-pg/mL
to low-ng/mL levels.
As electro-spray ionization (ESI) MS is a concentration-dependent detector, it
seems to be plausible to employ low-flow-LC-MS for highly sensitive analysis. The
following rationale suggests that the high sensitivity of analysis can be achieved by
applying low-flow-LC-MS. During chromatographic separation, the dilution (
D
) of
an injected sample (
D = C
end
/C
inj
, where
C
end
is the concentration after chromatogra-
phy and
C
inj
is the concentration injected) is given by:
2
1/ 2
∈π
r
(1
+
k H
) ( 2
π
)
D
=
V
inj
where
∈
is the column porosity,
r
is the column radius,
k
is the retention factor,
L
is
the column length,
H
is the plate height, and
V
inj
is the injection volume [
1
] . If condi-
tions are otherwise equal,
D
is in direct proportion to the square of column radius.
Thus, compared to conventional HPLC, micro
-
LC increases the signal-to-noise
ratio (S/N) drastically when ESI-MS/MS is employed as the detector, because
micro
-
LC results in a much smaller dilution of peak concentration, and ESI-MS/MS
is a concentration-sensitive detector [
2,
3
]. Nevertheless, the micro-LC-MS falls
short in that the column loading capacity is proportionally small, which tends to
counteract the gain in sensitivity achieved through using low flow rate. When ana-
lyzing highly complex biological samples, as in clinical and pharmaceutical analy-
sis, micro-LC-MS analysis may encounter two primary problems when a relatively
large amount of samples are loaded onto the column: (1) mass overloading, where
chromatographic separation is severely compromised because a large amount of
compounds are loaded onto a small-diameter column and result in nonlinear adsorp-
tion, and (2) volume overloading, where the volume of the sample injected is so
large that the eluted peaks are markedly broadened. In order to circumvent these
problems, users of micro-LC-MS often employ very small injection volumes (
V
inj
).
As a result, while micro
-
LC-MS/MS greatly enhances the
absolute
sensitivity
