Biomedical Engineering Reference
In-Depth Information
Leave the fractions, that are more
hydrophobic ( )than the target, on the
cartridge
Wash out the fractions that are
more hydrophilic
(
)than the
target
Hydrophobicity
Selectively elute the fraction containing
our target (
) for
m
LC/MS analysis
Fig. 1
Selective SPE rationale. The fraction that contains the target compound can be selectively
collected, while simultaneously reducing the quantity of matrix components from complex sam-
ple, using optimized wash/elution strategy
Traditional analytical approaches such as GC-MS, HPLC fluorescence, and
conventional LC-MS, are not sufficiently sensitive to quantify the low-pg/mL levels
of corticosteroids in bodily fluids [
4
]. The purpose of this work was to develop an
ultrasensitive and selective approach to quantify corticosteroids present at
extremely low, but biologically meaningful concentration in body fluids. A selec-
tive solid-phase extraction (SPE) and reversed-phase capillary LC coupled to
tandem mass spectrometry (mLC-MS/MS) strategy was employed to achieve a
high concentration sensitivity.
2.1
Experimental
To concentrate target corticosteroids and to reduce or eliminate matrix components
and interfering substances in plasma, a selective SPE procedure was developed.
Figure
1
shows the rationale for selective SPE strategy. Oasis HLB cartridge was
selected. Cartridges were conditioned with 1 mL of methanol followed by 1 mL of
water. One-mL portions of pooled porcine plasma containing 200 pg/mL each of
DEX, TACA, BUD, and DEX-AC were mixed 1:1 with 4 % phosphoric acid to
displace the corticosteroids from plasma proteins and then respectively loaded onto
18 cartridges at a rate of approximately 1 mL/min, followed by washing with 1 mL
2 % phosphoric acid. The cartridges were divided into two groups: for group 1,
individual cartridges were washed with 1 mL of 0.1 % formic acid in one of follow-
ing concentrations of methanol: 20, 30, 35, 40, 45, 50, 55, 60, or 65 %. The absorbed
corticosteroids were eluted from all nine cartridges with 1 mL methanol containing
0.1 % formic acid. For group 2, all nine cartridges were washed with 1 mL of meth-
anol with 0.1 % formic acid, and the absorbed analytes were then elute with 1 mL
of 0.1 % formic acid in one of the following concentrations of methanol: 60, 65, 70,
75, 80, 85, 90, 95, or 100 %. As an internal standard (I.S.), d7-TACA was then
added to each eluate at a concentration of 100 pg/mL. The eluates were evaporated
