Biomedical Engineering Reference
In-Depth Information
does not. Atropinesterase catalyses the stereoselective hydrolysis of
S
-hyoscyamine,
whereas
R
-hyoscyamine remains unaffected. Following procedure A the concentra-
tion of total hyoscyamine was determined and following procedure B only the
remaining
R
-hyoscyamine portion was measured.
S
-hyoscyamine was calculated by
the difference of both concentrations. Prepared plasma samples were separated on
an Atlantis T3 C18 column (150 mm × 4.6 mm I.D., 5 mm) applying an ACN gradi-
ent [
49
]. This application was based on a LC-ESI MS/MS method previously
described by John et al. for the simultaneous measurement of seven TA [
50
] . So far,
the enantioselective procedure was successfully applied to analyse hyoscyamine
concentrations in plasma of (a) a pesticide-poisoned patient under atropine therapy
[
49
] and (b) swine treated i.v. with atropine for a PK study [
47
] .
3.3
Mass Spectrometric Detection
Sections above discuss typical chromatographic methods applied to TA samples.
Beside sufficient separation, sensitive and selective detection is also required for
robust and reliable analysis. The following section is addressed to different ionization
interfaces, mass analysers and scan modes allowing valuable LC-MS method design.
3.3.1
Ionization
Most often positive ESI and only to a small extent positive atmospheric pressure
chemical ionization (APCI) were used as ion source (interface) to generate desol-
vated free ions of TTA or QTA suitable for MS or MS/MS detection. TTA were
detected as their proton adducts [M+H]
+
, whereas QTA were simply monitored as
the original cations [M]
+
thus not requiring adduct formation (Table
9
).
Statistical evaluation of the literature referred to in this chapter (Table
5
-
8
)
revealed a frequency of 2 % for thermospray (TSP), 4 % for offline FAB, 14 % for
APCI and 80 % for ESI (Fig.
5a
).
Beyer et al. compared the method characteristics of LC-APCI MS with LC-ESI
MS/MS for analysis of toxic alkaloids including atropine, scopolamine and deuter-
ated benzoylecgonine as IS in human plasma [
11
]. Following the same sample prep-
aration (SPE on mixed-mode C8) and gradient chromatographic separation
(acetonitrile/ammonium formate, pH 3.5) on a C8 base select column (Superspher
60 RP select B, Merck) eluates were subjected either to APCI or ESI prior to MS
detection. ESI MS/MS operating in the multiple reaction monitoring mode (MRM,
three transitions) was found to be more sensitive than APCI MS operated in the
selected ion monitoring (SIM) mode. This was most presumably due to the better
signal-to-noise ratio obtained in the MRM mode. Nevertheless, slightly higher
matrix effects causing suppression of atropine and scopolamine ionization were
obvious for ESI. Selectivity, accuracy and precision were comparable and satisfying
for both methods.
