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High-Resolution, Accurate-Mass Forensic Toxicology Screening in Blood Samples Using a Q Exactive Mass Spectrometer
Overview
Purpose:
To evaluate the Thermo Scientific™ Q
Exactive
™ High
-Resolution Mass
Spectrometer in Forensic Toxicology Screening for whole blood analysis and make a
comparison with Targeted Screening on a Triple Quadrupole MS using the SRM
(Selected Reaction Monitoring) mode and also UPLC/Diode Array Detection (DAD).
Methods:
Blood samples were spiked with internal standards and extracted with
TOXI-
TUBES™ A (Agilent Technologies, Santa Clara, CA). LC separation was
performed with a 30 minute gradient. Mass spectrometry data were acquired in Full
Scan and MS
2
mode using the Q Exactive MS.
Results:
Data collected show benefits of high-resolution screening over both the triple
quadrupole approach and DAD detection.
Introduction
Forensic scientists and forensic toxicologists need to identify an unlimited number of
compounds in complex matrixes with the capability of retrospective data analysis for
quick and confident analysis. The major challenge is to separate the analytes of
interest from the matrix and accurately identify them. Here we evaluated the Q
Exactive MS, a bench-top quadrupole-Orbitrap
™ ultra
-high resolution mass
spectrometer routinely capable of better than 5 ppm mass accuracy and 140,000
FWHM resolution, with Thermo Scientific™
ExactFinder
™ data
processing software,
for forensic toxicology screening in blood samples. We will also compare the results
with those obtained by forensic targeted screening using an SRM approach and DAD
detection.
Methods
Sample Preparation
500 µl of each blood sample was spiked with 20 µl of an internal standard solution
(Flurazepam at1 mg/L) and extracted with TOXI-
TUBES A™ (Agilent Technologies).
The organic layers were transferred, evaporated to dryness, reconstituted in 2.5 ml of
a mixture containing 70% of mobile phase A and 30% of mobile phase B, and injected
onto the Q Exactive MS. For triple quadrupole analysis and DAD detection, the sample
was reconstituted in 500 µl and 100 µl, respectively, of the mixture described above.
Liquid Chromatography
The U-
HPLC comprises Thermo Scientific™
Accela
™ 1250 pumps with an
Accela
Autosampler. Mobile phases are 10 mM Ammonium formate and 0.1% Formic acid in
water (A) and 0.1% Formic acid in Acetonitrile (B). The LC separation was performed
on a Thermo Scientific™
Hypersil
™ GOLD PFP column 150 x 2.1 mm
3µm.
FIGURE 1. HPLC Gradient Method
Mass Spectrometry
Compounds are detected on a Q Exactive mass spectrometer equipped with an
Orbitrap mass analyzer. A schematic diagram of the Q Exactive MS is illustrated in
Figure 2. A Heated Electrospray Source Ionization (HESI) probe was used as an ion
source. The instrument was operating in alternating positive and negative full scan
mode. Each Full Scan was followed by 8 high-resolution MS
2
scans in positive mode
and 3 high-resolution MS
2
scans in negative mode. Precursor selection was done in
the data-dependent operation mode where the most intense ion of the previous scan
was selected for fragmentation. Resolution was set to 70,000 FWHM for each full scan
mode and 17,500 FWHM for MS
2
scan acquisition.
FIGURE 2. Schematic d
Accurate-Mass Instrum
Data Analysis
All MS data have been p
analytes is performed u
isotopic distribution and t
Results
Data Processing
Chromatograms were re
to identify compounds b
Confirmation was perfor
obtained from each prec
for processing. Figure
(extracted ion chromat
accuracy (a), isotopic pat
Start (min)
Flow (mL/min)
%A
%B
0.00
0.2
95
5
5
0.2
55
45
18
0.2
30
70
20
0.2
5
95
27
0.2
5
95
27.1
0.2
95
5
32
0.2
95
5
FIGURE 3. Scan Param
Mass Spectrometer
Parameter
Full MS
Microscans
Resolution (FWHM)
AGC Target
Maximum IT
Scan Range
MS
2
Experiments
Microscans
Resolution
AGC Target
Maximum IT
NCE
MS
2
spectra were acquir
scan and source parame
DAD Detection
Data have been acquire
equipped with a DAD det
performed using a 15 mi
Triple Quadrupole Dete
Six different targeted LC/
(Selected Reaction Moni