Biomedical Engineering Reference
In-Depth Information
validated at 5 pg/mL; representative chromatograms of cell lysates spiked with this
concentration of drug are shown in Fig.
10c
and
d
.
In this study, a LOQ of 20 pg/mL in cell lysates would be needed in order to
quantify paclitaxel uptake by cells exposed to approx. 1 ng/mL drug, but the
detection limits of published LC-MS/MS based methods were in the range of
0.1-0.25 ng/mL [
10,
15-
21
]. Furthermore, concentrations of paclitaxel lower than
1 ng/mL are pharmacologically active, and therefore, a LOQ lower than 20 pg/mL
is desirable.
A121a cells were exposed to paclitaxel at concentrations of 0.2, 0.8, 2, and
5 ng/mL. Paclitaxel uptake by cells was able to be quantified at each time point
under four treatment conditions. The lowest measured intracellular accumulation
was 6.5 pg/106 cells, observed 10 min after the addition of 0.2 ng/mL paclitaxel to
cells. The detected concentration in this sample corresponded to approximately
9.6 pg/mL drug in the cell lysate, which is nearly twice the LOQ. Foremost con-
centrations of paclitaxel, it appeared that the intracellular drug concentrations
increased rapidly with exposure time and reached a plateau within 1-3 h. However,
for cells exposed to the lowest concentration (0.2 ng/mL), the maximum intracel-
lular drug concentration apparently was not achieved within 6 h, which was longest
time interval investigated. In future studies, these data will be expanded to include
additional concentrations and exposure times, and analyzed according to cellular
pharmacokinetic models such as those published previously [
12
] .
4
Paradigm 3: Ultrasensitive Quanti fi cation of Vitamin D
Metabolites in Human Plasma
4.1
Introduction
In this section, we show a paradigm for ultrasensitive quantification of endogenous
compounds in circulation system by the combination of selective SPE with m LC-MS.
VitD metabolites are clinically important and play a critical role in many impor-
tant biological processes, including maintenance of calcium homeostasis, immuno-
modulation, and cell differentiation [
22
]. VitD itself is not biologically active and
requires further metabolism that generates the active metabolites [
23
] . Studying
the physiological actions of VitD metabolites would contribute greatly to the mecha-
nism research, diagnosis, staging, and therapy of numerous diseases, such as multiple
sclerosis, diabetes, cancer, osteoporosis, microbial infections, and cardiovascular
diseases [
22,
24
]. For an example, the 25-hydroxyVitD (25(OH)VitD) metabolite is
clinically used as the marker for VitD deficiency. One of its metabolite, the 1R,
25-dihydroxy-VitD (1,25(OH)2VitD), is considered the primary biologically active
form of VitD and responsible for stimulating intestinal calcium absorption, mod-
ulating immune response, and maintaining calcium homeostasis [
25
] . Another
dihydroxyl metabolite, the 24(
R
), 25-dihydroxy-VitD (24,25(OH)2VitD), has been
