Biomedical Engineering Reference
In-Depth Information
observation is that K
+
and Na
+
salts do not co-elute with the target compounds when
sufficient chromatographic separation is employed.
The selective SPE procedure reduced matrix components in the cell lysate
samples and therefore permitted considerable concentration of the SPE eluate.
Nonetheless, the matrix components that co-elute with the analyte during SPE limit
the degree to which the eluate can be concentrated for mLC analysis. Large injection
volumes can contribute significantly to the sensitivity of quantification, but may
cause peak broadening as a result of both long sample injection times and the large
volume of sample solvent. Furthermore, the smaller column volume selected here
requires that
V
inj
be held to considerably smaller volumes in order to avoid column
overcapacity and fouling [
1
]. To address these issues, it is necessary to develop an
approach that would enable a high
V
inj
of the selectively extracted samples without
causing peak broadening and column fouling. A two-segment gradient was designed,
consisting of a focusing segment and a separation segment. The following theoreti-
cal rationale suggests the use of a low-organic mobile phase to load and focus a
large
V
inj
on the column. The hypothesis was that the complexity of cell lysates
would be reduced considerably by the selective SPE approach, and therefore a con-
siderably higher
V
inj
could be tolerated without compromising chromatographic
separation, provided the sample focusing strategy was also employed. To test this
hypothesis, the mLC performance was evaluated as a function of
V
inj
using cell
lysates spiked with the analyte. Typical chromatograms are shown in Fig.
9
[
5
] . The
retention times of the target compounds increased only slightly as
V
inj
was increased,
and peak broadening or peak shape deterioration was not observed over the range of
V
inj
from 0.2 to 8 mL. Furthermore, both the intensity and S/N for paclitaxel increased
roughly in proportion with
V
inj
. This confirmed that the selective SPE procedure, in
conjunction with the sample focusing strategy, permits a high
V
inj
on the m LC col-
umn without causing column overcapacity. A
V
inj
of 8 mL was selected for
quantification of paclitaxel in cell lysates, which is 20- to 40-fold higher than the
manufacturer's recommendation. Column durability was not compromised; after
approximately 300 injections of 8 mL of cell lysate samples on the mLC column, the
separation of the target analytes remained highly reproducible, with neither loss of
resolution nor increase in backpressure. The subsequent gradient steps constitute
the “separation” segment of the chromatographic strategy, in which a gradually
increasing percentage of mobile phases B elutes the compounds that were focused
on the column during the initial gradient step. To determine the optimal separation
conditions, we investigated chromatographic retention vs. S/N for cell lysate sam-
ples spiked with 250 pg/mL of paclitaxel and I.S. The S/N of the target analyte
improved considerably when their chromatographic retention increased, but beyond
a certain point, the S/N approached a maximum. This behavior has been observed
in the first example too, when a similar strategy was used for quantification of
corticosteroids in plasma.
The validated quantification range is between 5 and 6,250 pg/mL with good lin-
earity (
r
2
= 0.992). An ultralow detection limit of 0.5 pg/mL (defined as S/N = 3) was
achieved for paclitaxel in cell lysates. This detection limit is 200-500 folds lower
than conventional LC-MS/MS methods published previously. The LOQ was
