Biomedical Engineering Reference
In-Depth Information
addition. Dried methanolic eluates were reconstituted in 5 mM ammonium acetate
buffer (0.1 % v/v formic acid) prior to mass spectrometric analysis (Table
6
). MRM
transitions used for trospium are given in Table
9
. In addition, Callegari et al. per-
formed in vitro binding studies to characterize the unbound (free) drug fraction in
brain homogenates using a 96-well equilibrium dialysis block. Analysis by LC-ESI
MS/MS was carried out by the same method. The authors found, that in contrast to
the tertiary TA scopolamine, the quarternary TA trospium and methyl scopolamine
did not show significant CNS penetration.
The three examples introduced above present applications of LC-MS-based meth-
ods to determine specific drug transfer into compartments relevant for systemic or
local activity and thus providing basic pharmacological or toxicological data.
Evaluation of biotransformation processes in vivo and in vitro is also an essential
issue for drug characterization that is addressed in the next section.
3.4.3
Biotransformation Studies
Biotransformation processes may partly convert a less polar parent drug into acti-
vated more polar forms (phase I) and subsequently into highly polar often charged
conjugates (phase II) that are excreted via liver and kidney into urine. Most TA
undergo such transformation even though not in quantitative yield. Evaluation of
drug metabolism in vivo is indispensable for understanding of pharmacokinetic and
pharmacological properties. Often laboratory animals were used as adequate mod-
els, e.g. rat [
5-
7,
37,
51,
59,
87
] , dog and monkey [
37
]. Despite its obvious limita-
tions several in vitro systems, e.g. organ homogenates (e.g. liver [
5,
6,
23,
37,
51,
82
] ), plasma [
49,
50
] , bacteria cultures [
87
] as well as pure isolated or engineered
enzymes [
5,
6,
23,
37,
51,
82
] were also used as valuable tools to clarify and describe
drug biotransformation.
Table
7
summarizes LC-MS-based methods that were applied to study metabo-
lism of TA and Fig.
7
depicts some selected metabolites identified by mass
spectrometry.
Chen et al. impressively presented the possibilities of modern LC-MS equipment
to elucidate metabolism of several TA [
5-
7,
51,
52,
87
]. The strategy was based on
the following considerations:
1. The general pathways of phase I and II metabolism are effective for TA.
2. Phase I and II metabolism add distinct and predictable chemical moieties to the
parent drug that cause defined changes in the molecular mass but keep the basic
structure (skeleton) unaltered (e.g. +16 Da for single oxidation, +32 Da for two-
fold oxidation, +30 Da for methoxylation, −18 Da for dehydration, −14 Da for
demethylation, +176 Da for glucuronidation, +80 Da for sulpho-conjugation).
3. The structural skeleton of the parent TA and its modified biotransformation prod-
ucts undergo similar mass spectrometric fragmentation by CID resulting in iden-
tical (diagnostic) or specifically shifted signals of product ions (e.g. cleavage of
the tropane moiety resulting in fragments at
m
/
z
124 and 93, Fig.
8
).
