Biomedical Engineering Reference
In-Depth Information
to remove the organic layer that is inapplicable for direct injection to reversed-phase
chromatography. Therefore, LLE is more laborious and time consuming but often
enables efficient extraction, good separation from deteriorating matrix ingredients
and effective analyte concentration with satisfying recovery (Table
3
).
The partition equilibrium depends on the solubility of the analyte in the organic
solvent but more critical on the analyte's polarity that is determined by the func-
tional groups and charge. As discussed above (
Lipophilicity of TA
) the log
P
value
is a measure of extractability into an organic phase. The higher the log
P
value the
more lipophilic is the compound (Table
1
).
Besides substance lipophilicity, the polarity of the organic solvent also deter-
mines extraction efficiency premising sufficient solubility. Polarity of liquids can be
characterized by the Snyder polarity index (
P
¢) sorting solvents from smallest polar-
ity (pentane,
P
¢0), over small (methyl-
tert
-butyl ether, MTBE,
P
¢ 2.5; diethylether,
Et
2
O
P
¢2.8; dichloromethane
P
¢3.1), and medium (chloroform
P
¢ 4.1; ethylacetate,
EE
P
¢4.4) to high polarity (dimethylsulfoxide, DMSO
P
¢7.2; water
P
¢ 10.2) [
91
] .
Therefore, diverse organic solvents were used for LLE of TA including pentane,
chloroform, very often dichloromethane, most frequently EE, sometimes Et
2
O and
seldom MTBE as listed in Table
3
.
In case of TTA extraction efficiency requires to avoid acidic pH to minimize the
grade of protonation of the weak basic N-atom of the tropane moiety. Resulting
uncharged TTA are predominantly transferred into the organic layer. The charged
protonated form exhibits better solubility in aqueous media and often causes insol-
ubility in non-polar aprotic solvents, e.g. Et
2
O, chloroform. Therefore, a slight
basic 0.001 % Na
2
CO
3
solution was added to rat faeces prior to LLE of aniso-
damine [
6
] , anisodine [
5
] , atropine [
52
] and scopolamine [
87
] and their biotrans-
formation products using ethylacetate (Table
3
). Xu et al. added 1 M NH
4
OH to
human plasma prior to extraction of atropine with dichloromethane (recovery
110 %) [
90
] and Ahmed et al. also alkalized human plasma for LLE of scopol-
amine with MTBE (recovery not specified) [
88
] (Table
3
). A recovery of 98 % for
granisetron was obtained after addition of 0.1 M NaOH to plasma before LLE with
EE [
71
] .
Intending to improve extract purity of atropine and scopolamine from horse
urine, Gerber et al. performed an excessive sequence of consecutive LLE steps [
14
] .
Initially, urine was incubated overnight at pH 5.0 with b-glucuronidase from
E. coli
to hydrolyze glucuronide-conjugates of both TTA and thus enhance the concentra-
tion of structurally unaltered compounds (Table
3
). Afterwards, the incubation mix-
ture was alkalized and extracted for the first time with dichloromethane/iso-propanol
4:1 (v/v) for 15 min. The liquid organic layer containing the analytes was separated
from the frozen aqueous phase after freezing in an ice/alcohol bath. Subsequently,
the organic layer was acidified with HCl and extraction was performed a second
time for 15 min transferring protonated TTA back into the aqueous layer. After
separating both layers the aqueous solution was extracted with EE to remove any
kind of undesired non-polar matrix components. Subsequent to discarding the EE
phase, the aqueous layer was alkalized again (pH 9.0) by addition of NH
4
OH fol-
lowed by LLE with dichloromethane for 15 min thus transferring TA into the organic
