Biomedical Engineering Reference
In-Depth Information
preparation in a 96-well format [
45,
46
]. Sample preparation for LC analysis is
considered to be less time-consuming than for GC analysis as no derivatization of
the compounds is required.
After extraction of the drugs of interest from the sample matrix, the selection
of the most appropriate ionization conditions is required. Electrospray Ionization
(ESI) is used in the vast majority of published methods whilst Atmospheric-
Pressure Chemical Ionization (APCI) is less common [
40,
45,
47,
48
] . Since all
APs carry at least one nitrogen-group, ionization generally takes place in posi-
tive mode. In 2007 following a study using morphine, Dams et al. [
49
] concluded
that ESI is more prone to matrix effects than APCI, but both ionization tech-
niques are affected by this potentially significant analytical issue. Accordingly,
new techniques should not be accepted for use unless sufficient matrix effect
studies have been conducted as part of the method validation, using one of the
two internationally accepted approaches either by Bonfiglio et al. [
50
] or
Matuszewski et al. [
51
] .
The use of an appropriate IS is mandatory when developing new LC-MS(/MS)
detection methods. The IS must not be in therapeutic use, as the possibility of a
patient being comedicated with this drug can never be fully excluded. Ideally a
deuterated IS from a different drug class should be incorporated, rather than
taking the risk of overestimating the peak area of the IS by using a nondeuter-
ated compound from the same class [
52
]. Examples for recommended IS are
9OH-risperidone-d
4
[
53
] , haloperidol-d
4
[
41,
54
] , and olanzapine-d
3
[
32
] .
All published methods for the detection and quantification of APs in biological
matrices use reversed phase (RP) stationary phases with C
8
or C
18
chains. The pre-
dominantly hydrophobic APs are prone to bind to the hydrophobic column in a
polar mobile phase. A decrease in polarity of the mobile phase by introduction and
increase of a nonpolar solvent, results in desorption of the ionized compounds
from the stationary phase over time and elution of the APs of interest. Isocratic and
gradient elution are both common, using different combinations of an aqueous
buffer and an organic solvent. Run times vary from 2 to 20 min. Shorter run times
may seem beneficial in laboratories with a large sample throughput but can also
create two major problems. First, Saar et al. [
41
] highlight the importance of
sufficient run-times in order to distinguish compounds included in the method that
show identical Q1 masses and share the first two transitions despite having
different chemical structures such as the butyrophenone derivatives haloperidol
(Fig.
1
X) and pipamperone (Fig.
1
XIII). Making a clear distinction between these
two typical APs is only possible by having a sufficiently long run time or adding a
third transition. Second, unknown compounds or matrix components can co-elute
and cause ion-suppression or enhancement, resulting in an over- or underestima-
tion of a concentration [
55
] .
Multiple Reaction Monitoring (MRM) methods are most commonly used in
analytical methods as they provide the opportunity for fast and simple detection
and quantification. However, a large number of published methods do not fulfill
the international requirement of at least two MRM transitions for reliable
identification of an analyte [
56-
58
] which can cause problems; particularly
