Biomedical Engineering Reference
In-Depth Information
solvent. After evaporating the methylene chloride fraction to dryness, extracts were
dissolved in MeOH for final LC-MS/MS analysis.
According to the limited tendency of charged molecules to be transferred into an
organic phase, only few examples can be found extracting QTA from plasma by
simple LLE. Dichloromethane was used for cimetropium [
61
] and
N
-butyl-
scopolamine [
79
] requiring a large relative excess of organic solvent (1:11.8 for
cimetropium; 1:8.6 for
N
-butyl-scopolamine) but yielding only in moderate recov-
eries of less than 70 % (Table
3
).
To improve extraction ef fi ciency, a modi fi cation of LLE—named ion pair
LLE—was established and applied to QTA extraction [
23,
24
] . Incubation mix-
tures containing liver microsomes and cimetropium were mixed with NaCl and
heptane sulfonic acid, which is a typical ion pairing reagent also used as HPLC
solvent additive in ion pair chromatography [
92,
93
] . Extraction was performed
twice by adding the 1.4-fold volume of an organic solvent. Diverse solvents were
tested yielding recoveries of 75 % for chloroform, 62 % for dichloromethane, 54 %
for Et
2
O and 32 % for EE [
23
] (Table
3
). It is noteworthy that chromatographic
separation was also carried out as ion pair chromatography and mass spectrometric
detection was done offline by fast atom bombardment (FAB).
An additional example for ion pair extraction of QTA (ipratropium) was
described by Tang et al. mixing equine urine with alkaline saturated borax buffer
prior to extraction with EE allowing to remove more lipophilic compounds.
Subsequently, the EE layer was discarded and an alkaline potassium iodide-glycine
solution was added to the aqueous phase. Afterwards, the ipratropium-ion pair
complex was extracted twice with dichloromethane yielding in a recovery of 82 %
[
24
] (Table
3
).
However, ion pair extraction for QTA did not achieve broad acceptance.
The addition of organic solvents to biological samples was also carried out to
extract TA from human hair. For forensic analysis hair represents a versatile speci-
men allowing to analyse incorporation of a compound [
94,
95
] . Segment-wise anal-
ysis of hairs further allows to estimate the duration and time point of exposure.
Kintz et al. successfully analysed hair by LC-MS/MS to prove the abuse of scopol-
amine and atropine [
56,
57
]. For sample preparation hair was initially washed
(decontaminated) twice with dichlormethane for 2 min. Subsequently, hair was seg-
mented and each segment was cut into small pieces (<1 mm). The chopped hair was
incubated for several hours in phosphate buffer (pH 8.4) containing deuterated atro-
pine as an internal standard (IS) prior to TTA extraction with organic solvent
(dichloromethane/iso-propanol/
n
-heptane 50:17:33). Extraction recovery was found
to be 67 % for scopolamine and 82 % for atropine [
57
] . Limits of quanti fi cation
were satisfying at 5 pg/mg hair.
In the following section SPE will be discussed as a third wide-spread method for
sample extraction.
