Biomedical Engineering Reference
In-Depth Information
after three freeze-thaw cycles, and 3 % phosphoric acid was added in cell lysates in
order to further release protein bound paclitaxel into cell lysates. Optimized SPE
strategy was applied to cell lysates to clean up cell lysates matrices without losing
any target compound.
In order to extract paclitaxel and docetaxel (the I.S.) selectively from cell lysates,
while simultaneously reducing the quantity of matrix components that were also
derived from the samples, the SPE wash and elution conditions were optimized in a
manner similar as these described in the paradigm 1. Detailed experimental condi-
tions can be found in a previous publication [
5
] .
3.3
Results and Discussion
Optimization of the SPE procedure was carried out to meet three objectives. First,
the overall conditions selected would provide the highest and most consistent abso-
lute recovery of the target analyte. Second, the conditions for washing the SPE
cartridges containing bound analyte would remove unwanted matrix components
to the greatest extent possible without noticeably eluting the target analyte or I.S.
Third, elution conditions would subsequently recover the analyte efficiently while
minimizing the elution of less polar matrix components. To enable calculation of
the absolute recovery of paclitaxel through the SPE procedure, cell lysates were
spiked with known concentrations of paclitaxel, and the I.S. was added to the final
eluate. Because of the relatively high protein binding of paclitaxel and the structur-
ally similar I.S., phosphoric acid was added to the cell lysates to disrupt drug-
protein binding before loading onto SPE cartridges [
11,
12
] . After the SPE
cartridges were loaded with analyte-spiked cell lysates, they were washed in two
steps. First, 3 % phosphoric acid in 35 % methanol (“Weak-wash” step) was used
to remove residual cellular proteins and thus avoid precipitation and column block-
ing that could occur with subsequent washes that would employ higher-organic
solvent conditions. Second, a higher concentration of methanol, identified through
detailed optimization, was used to elute relatively polar matrix components with-
out loss of the analyte (“Strong-wash” step). Finally, the target analyte was eluted
quantitatively using a solvent composition optimized to recover it efficiently while
leaving more hydrophobic matrix components on the cartridge. The results of
absolute recovery of analyte through the SPE procedure are shown in Fig.
8
[
5
] . In
preliminary investigations [
13
], paclitaxel recovery from the SPE procedure was
very low even with 100% methanol. Here, after evaluating a number of mobile
phase modifiers in the present study, paclitaxel was eluted completely in 95 %
methanol with 0.1 % formic acid modifier (Fig.
8b
); the polarity of paclitaxel
increased by formic acid might be the reason. Based on the optimization results,
75 % methanol was chosen for Wash-2, and 0.1 % formic acid in 97.5% methanol
was chosen for the elution.
The development of the chromatographic separation conditions was approached
in terms of three interrelated variables: the column, the chromatographic separation
