Biomedical Engineering Reference
In-Depth Information
(i.e., sensitivity expressed in terms of the amount of the target on column) compared
to conventional LC-MS/MS, the
relative
sensitivity (i.e., sensitivity expressed in
terms of the concentration of the target in sample), which is more important for
pharmaceutical/biological analysis, typically does not improve drastically unless a
high
V
inj
is feasible. In order to achieve high concentration sensitivity for pharma-
ceutical and biomedical analysis, our lab has developed a suite of approaches using
a high
V
inj
while overcoming the problems of mass/volume overloading [
4-
6
] .
Selective sample preparation strategies, such as a selective SPE procedure, was
applied to simplify the complex biological matrices and to alleviate the mass over-
loading problem. SPE washing and elution conditions were optimized to reduce the
amount of matrix components, as well as to decrease matrix effects and endogenous
interferences from biological samples. Using such a SPE strategy, a high sample
loading volume on the mLC column is feasible without causing mass overloading
[
4-
6
]. In one preliminary experiment, we observed when plasma samples were
extracted using generic SPE methods, a relatively high
V
inj
of the extract easily
exceeded micro-LC column capacity and deteriorated chromatography perfor-
mance. By comparison, after a selective extraction, it was feasible to achieve high
concentration sensitivity by employing a high
V
inj
, without mass overloading [
4
] .
An on-column focusing strategy was employed to overcome volume overloading
[
4-
6
]. By using low-organic mobile phase for sample loading, the target compounds
are focused on the front end of column prior to separation. For a typical m LC system
that is running at low flow rates, a large
V
inj
leads to a long sample loading time and may
result in undesirable consequences such as severe band-broadening and lag of gradient
changes. Those problems can be avoided by applying on-column focusing strategy. In
the above works, the combination of selective SPE and on-column focusing strategy
enabled ultrasensitive quantification of clinically or pharmaceutically important com-
pounds such as corticosteroids, VitD metabolites, and anticancer agents.
The detailed procedure of this method is described in the following three
paradigms.
2
Paradigm 1: Ultrasensitive Quanti fi cation of Corticosteroids
in Plasma After Intravitreous Injection
Low-dose regimens frequently result in very low corticosteroid concentrations in
body fluids (e.g., low pg/mL range in plasma). PK analysis that is often necessary
to manage these therapies is hindered by the inadequate sensitivity by most estab-
lished analytical methods. In addition, certain drug delivery strategies, such as inha-
lation or intraocular injection, result in systemic levels too low to be detected by
current methods. Furthermore, administration of some corticosteroid prodrugs, such
as these in the forms of acetates/propionate, often results in sustained, low concen-
trations in plasma. Therefore, a highly sensitive and selective analytical approach is
necessary for cases in which sustained low concentration of corticosteroids may be
present systemically or in tissues.
