Biomedical Engineering Reference
In-Depth Information
corticosteroid-spiked plasma. The chromatographic performance on m LC became
unacceptable when more than 0.5 mL of an extracted plasma sample was injected on
the 300 mm I.D. column. This degradation of mLC performance probably resulted
from saturation of the capacity of the stationary phase of the column by the extracted
matrix materials, thus modifying the chemical characteristics of the stationary phase
and interfering with chromatographic separation.
To address this problem, it was necessary to develop a selective SPE procedure
to extract the target corticosteroids more selectively from plasma and simultane-
ously remove unwanted plasma components to the extent possible. Reversed-phase
SPE cartridges from several manufacturers were evaluated. The Oasis HLB car-
tridge appeared to exhibit the best performance for the extraction of the four corti-
costeroids. The plasma sample was mixed with 4 % phosphoric acid at a ratio of 1:1
to disrupt and/or inhibit drug-protein binding. Two wash steps were employed
before elution: Wash-1 used 2 % phosphoric acid to remove residual plasma pro-
teins, and Wash-2 used 0.1 % formic acid in a methanol-water mixture for the
removal of compounds that were more polar than the target compounds. Elution
employed a second, optimized methanol-water mixture (containing 0.1 % formic
acid) in order to elute the target compounds efficiently and leave compounds less
polar than the targets on the cartridges. Absolute analyte recoveries for the SPE
extraction step were investigated using Wash-2 steps of 20-65 % methanol and dilu-
tion steps of 60-100 % methanol, and results are shown in Fig.
2
[
4
] . The fi nal
solvent system selected for the wash cycle was 0.1 % formic acid in 50 % methanol,
and the elution solvent composition was 0.1 % formic acid in 85 % methanol in
order to complement with multiple corticosteroids.
Compared to conventional plasma sample preparation methods such as protein
precipitation and generic SPE, the selective SPE strategy described here has several
advantages. Protein precipitation using organic solvents dilutes the sample, and the
final solution contains a high percentage of organic solvent, which may cause peak
broadening problems during chromatography. Generic SPE, which is not optimized
for target analytes, nonspecifically extracts components from complex matrices and
results in a highly complex extract that may easily exceed mLC column capacity if
a relatively large injection volume was used. In contrast, the SPE strategy developed
here selectively concentrated target compound while simultaneously simplifying
the sample matrix greatly. As a result, this selective extraction approach enabled a
relatively high injection volume of plasma samples, yet avoided over capacity and
fouling of the mLC column. Representative chromatograms of corticosteroid
extracted from plasma using generic SPE vs. the optimized selective SPE condi-
tions are shown in Fig.
3
[
4
]. It was also observed that this approach improved the
S/N of the target compounds in plasma samples, probably by both lowering chemi-
cal noise and decreasing the ion suppression effect.
The MS and MS/MS behaviors of the four corticosteroids under positive ion ESI
was investigated using direct infusion of standard solutions into the mass spectrom-
eter. In preliminary experiments, it was observed that all four corticosteroids formed
strong [M + Na]
+
and [M + K]
+
ions along with relatively weaker [M+H]
+
ions in
methanol/acetonitrile/water. However, an acidic 2 mM ammonium acetate buffer
