Biomedical Engineering Reference
In-Depth Information
3.1.3
Solid-Phase Extraction
SPE is a frequently used procedure for extraction and clean-up of analytes from
fluids by prepacked cartridges. Perculating the sample solution through the precon-
ditioned sorbent enables analyte adsorption to the solid phase and elution of unbound
matrix components. Subsequent washing steps may be performed by means of
reduced pressure to remove interfering compounds prior to analyte elution with an
appropriate mixture of organic solvents. The eluate requires evaporation to remove
the organic solvent that might cause fatal matrix effects in chromatography. At last,
the dried residue has to be re-dissolved for LC-MS analysis. Functionalized sor-
bents are commercially available in high quality providing excellent reproducibility.
Typical materials include diverse hydrophobic reversed-phase (RP) sorbents
(C
4
-C
18
) but also cation exchanger or mixed-mode solids that will be addressed
below. In comparison to LLE, SPE avoids disposal of larger volumes of organic
solvents and prevents from incomplete phase separation and thus optimizes recov-
ery. Therefore, SPE represents a modern, but multi-step sample preparation proce-
dure that is capable for automation to moderate costs.
For TTA and QTA extraction from urine, plasma, incubation mixtures and organ
homogenates, SPE has been successfully applied as summarized in Table
4
.
Extraction of anisodamine [
6
] , anisodine [
5
] , atropine [
51,
52
] and scopolamine
[
7,
87
] and their biotransformation products was done with AccuBOND
II
C18 car-
tridges (Agilent). This sorbent consists of irregular silica particles that were deriva-
tized with octadecyl chains to obtain a non-polar, non-end-capped, reversed-phase
material well suited for non-polar, rather weak hydrophobic analytes. Accordingly,
end-capped Sep-PAK C18 (Waters) was also used for atropine extraction [
96
] .
With respect to Ingelse et al. compounds with a log
P
> 0 are considered as more
or less polar and water soluble but can be extracted by RP-SPE in contrast to mol-
ecules with log
P
< 0 [
28
] . Log
P
values of TTA mentioned above (Table
1
) are
between 0.64 (anisodine) and 1.77 (atropine) and thus confirm the assumption of
extractability. Surprisingly, C
18
-SPE was also applied to extract the charged polar
QTA compounds cimetropium [
23,
62
] and tiotropium [
81
] from biological samples
with excellent recoveries of 100 and 92 %, although corresponding log
P
values
were calculated to be negative (−2.06 and −1.94, respectively, Table
1
). Kajbaf et al.
investigated optimum extraction procedures for cimetropium from liver microsome
incubation mixtures by comparing precipitation with ACN, LLE with diverse sol-
vents and RP-SPE [
23
]. By far the highest recovery was obtained after protein pre-
cipitation (100 %) and SPE (100 %), whereas LLE yielded in much smaller rates
(e.g. 32 % for EE to 75 % for chloroform).
Besides RP-sorbents, cation exchange material was also applied appropriate for
extraction of positively charged protonated TTA or QTA. Atropine and scopolamine
were extracted from human plasma at pH 3.0 using a C8-SCX (strong cation
exchange) mixed mode sorbent (Bond Elute certify, Agilent/Varian) [
11
] . This solid
phase is an irregular silica-based, non-end-capped sorbent combining reversed-
phase C8 and SCX (benzoylsulfonic acid) properties. In addition, the QTA Ipra was
separated from human whole blood [
54
] and equine urine [
78
] by the use of CBA
