Biomedical Engineering Reference
In-Depth Information
10
Sample Preparation and Applications
Sample preparation prior to LC-MS analysis aims to reduce matrix effects by
removing potential interferences and to get the analyte into an amenable form for
the analysis. However, for drugs and low molecular mass compounds, coeluting
components such as proteins, lipids, and salts may cause variability in the efficiency
of analyte ionization [
65
]. It is common practice in the laboratory using appropriate
internal standards, better stable isotope-labeled internal standards (ISTDs), which
normalize signal excluding interferences. However, cases are reported in the litera-
ture in which a deuterated ISTD has different chromatographic properties [
90,
91
]
or interacted in different matrices differently to the unlabeled analyte [
92
] . In their
study Wang et al. [
92
] also suggested that ISTDs labeled with
13
C,
15
N, or
17
O may
be better at compensating for matrix effects than deuterium labeled ISTDs (although
they are often more expensive). When the cost of these labeled compounds is pro-
hibitive, for example in multianalyte procedures, it was demonstrated a possibility
to suitably select only some deuterium labeled ISTD, one by class of substance,
without compromising data quality [
66,
93
] .
10.1
Plasma, Serum, or Whole Blood
Serum, plasma, and even more whole blood are “dirty” matrices which need an
effective pretreatment. Different strategies have been applied for this purpose.
10.1.1
Protein Precipitation
The simplest approach is to promote protein precipitation (PPT). Blanchard et al. in
1981 [
94
] evaluated different precipitating solvents. However, after a simple treat-
ment such as PPT matrix effects may persist because of the endogenous compounds
(mainly phospholipids [
95
]): in some cases the supernatant was diluted, but sensi-
tivity got worse [
96
], or evaporated and reconstituted in mobile phase, with repro-
ducibility getting worse in this case [
55
]. However, the use of suitable internal
standards may be very helpful or even necessary, as demonstrated in a paper in 2009
by Sergi et al. [
93
]: the authors proposed an analytical procedure for the simultane-
ous determination in plasma and in OFs of 13 analytes belonging to different chemi-
cal and toxicological classes (amphetamine, methamphetamine, morphine, 6-MAM,
MDA, MDE, MDMA, cocaine, BEG, THC, THC-COOH, ketamine, and PCP). The
chromatographic run was also used as an online cleanup, allowing the selective elu-
tion of the analytes from the column and separating the whole injected solution into
three major fractions, of which only the second was analyzed by LC-MS/MS. The
use of a diverter valve enabled the maintenance of the source cleanliness for a long
period of time and prevented sudden reductions of sensitivity during the analysis of
