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The ToxSpec Analyzer includes a diverse and easily-
expandable MS/MS library of 300 compounds that it
screens using a single pre-configured method. In our
laboratory, we have expanded the library by more than 50
entries to date.
Sample preparation
The extraction procedure was performed by using
liquid/liquid extraction (LLE) with Toxi-Tube A
®
(Varian,
les Ulis, France). Details of the procedure are described
below.
• Vortex the Toxi-Tube A for 10 seconds.
• Add 1 mL of serum or urine into the Toxi-Tube A.
• Add 200 µL of a solution of internal standard
[haloperidol-d4, chlorpromazine-d3, and prazepam-d5
at the following concentrations: 100 ng/mL, 1 µg/mL
and 100 ng/mL, respectively, in 70/30 of A/B (A: water
containing 10 mM ammonium acetate and 0.1% formic
acid; B: acetonitrile containing 0.1% formic acid)].
• Add 5 mL of water.
• Vortex for 10 seconds.
• Mix for 5 minutes.
• Centrifuge for 5 minutes at 2700 rpm.
• Transfer the upper layer to a tube and evaporate to
dryness at 40 °C.
• Reconstitute the sample in 200 µL of 70/30 of A/B.
HPLC Conditions
Chromatographic analyses were performed using the
Thermo Scientific Accela UHPLC system. The
chromatographic conditions were as follows:
Column:
Thermo Scientific Hypersil GOLD PFP 5 µm,
150 x 2.1 mm
Flow rate:
0.2 mL/min
Mobile phase:
A: water containing 10 mM ammonium acetate and
0.1% formic acid;
B: acetonitrile containing 0.1% formic acid
Injection volume: 10 µL
Gradient:
T (min)
A (%)
B (%)
0.0
95
5
5.0
55
45
18.0
30
70
20.0
5
95
27.0
5
95
27.1
95
5
32.0
95
5
Mass Spectrometry Conditions
MS analysis was carried out on a our LXQ linear ion trap
mass spectrometer with an electrospray ionization (ESI)
source. The MS conditions were as follows:
Ion polarity:
Polarity-switching scan-dependent
experiment
Spray voltage:
5000 V
Sheath gas (N
2
) pressure:
30 (arbitrary units)
Auxiliary gas (N
2
) pressure:
8 (arbitrary units)
Capillary temperature:
275 °C
Microscan:
1
Wideband Activation
TM
:
Activated
Stepped normalized collision energy: 35% ± 10%
Results and Discussion
More than 150 real laboratory samples (serum and urine)
have been analyzed. Table 1 reports some of the data
obtained from both the REMEDi HS LC/UV system and
the ToxSpec Analyzer UHPLC/MS system. Among the 12
samples reported here, 22 compounds have been identified
using both the REMEDi HS and the ToxSpec Analyzer.
Notably however, the ToxSpec Analyzer system identified
24 additional compounds that were not detected with the
REMEDi HS due in most cases to a lack of sensitivity,
specificity, and coelution capability.
The ToxSpec Analyzer also provided a better response
for some classes of compounds, like benzodiazepines.
With the REMEDi HS system, the retention time for this
class of compounds was close to the dead volume of the
column. For that reason, the signals that interfered with
matrix components were rather difficult to identify. It was
also observed that haloperidol (sample #5) and paroxetine
(sample #10) gave a much better signal with the ToxSpec
Analyzer.