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Advantages of Ultra-High-Resolution Q Exactive Mass Spectrometer in Analysis of Unlimited Number of Compounds in Urine Quantitative Screening
Application for Forensics
Introduction
Implementation of ultra-high-resolution mass spectrometers for quantitative
forensic toxicology allows for unlimited number of analytes, short acquisition
times and simple sample preparation. At the same time, ultra-high-resolution
mass spectrometry provides high confidence in reported hits. Quantitative
screening in forensic toxicology applications is important because it allows
reporting of only those compounds with concentrations above specified threshold,
reinjection of samples following those with concentration above carry-over limit,
and appropriate sample dilution, if required, for confirmatory quantitative analysis.
Instrumentation
Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC system
Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap MS
Methods
Sample Preparation
Enzymatic hydrolysis followed by liquid-liquid extraction.
A 1 mL aliquot of urine (spiked calibrator, QC or donor sample) was spiked with
internal standard (Tolbutamide), and incubated with 10,000 U/mL beta-
glucuronidase enzyme in pH 5.5 buffer for 60 minutes at 60 °C. The resulting
mixture was basified with sodium carbonate and extracted with
et
hylacetate:hexane(1:1). The organic supernatant was evaporated to dryness
under nitrogen at 37 °C. The residue was reconstituted in 100 µL of 20%
methanol and 10 µL of the sample was analyzed by LC-MS.
LC Method
The column used was a Thermo Scientific™Hypersil™ GOLD PFP 100 x 2.1 mm,
5 µm. Mobile phase was 10 mM ammonium acetate in water (A) and methanol
(B). Both solvents were Fisher Scientific™ Optima™ grade. The LC gradient
was as follows:
Mass Spectrometry Method
The Q Exactive benchtop orbitrap mass spectrometer was equipped with a HESI
source and operated in positive ionization mode. The MS method consisted of 2
scan events: Full scan from 130–472
m/z
+ (R = 70K) and all ion fragmentation
(AIF) scan from 50–472
m/z
+ (R = 70K). The AIF spectra were collected with
stepped collision energy of 70 ± 50%
Method Validation
The method was validated for 37 representative compounds from different drug
classes (Table 1). Calibration standards (0.05–1000 ng/mL) and QC samples
(2, 10, 50 ng/mL) were prepared in pooled negative urine.
Matrix effects were evaluated by spiking urine from 15 different donors at
concentrations of 10 ng/mL (opioids) , 20 ng/mL (benzodiazepines) or 100 ng/mL
(amphetamines) and then processing the samples as described in sample
preparation above. Percent recovery was calculated against samples at the
same concentrations prepared in water instead of urine.
Data Analysis
Data was acquired and processed with Thermo Scientific™ TraceFinder™
s ftware version 3.1. Full-scan d ta (chromatogra s reconstr cted with
m/z
accuracy of 5 ppm) were used for analyte detection and quantification. AIF
FIGURE 1. Repre
diazepam in pool
chromatogram of
fragments from
AIF spectra for th
are reconstructed
across the peak.
Time (min)
%A
%B
Flow rate (mL/min)
0
95
5
0.75
0.5
95
5
0.75
3.6
60
40
0.75
6.1
5
95
0.75
6.15
0
100
0.85
7.1
0
100
0.85
7.15
95
5
0.85
9.0
95
5
0.85
RT:
2.62 - 3.56
SM:
5B
2.7 2.8 2.9 3.0 3.1
Time (min
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
3.11
3.10
3.
3.09
3.07
3.06
3.05
3.03
3.02
2.99
2.97
2.90
2.78
2.64
3.11
3.
3.09
3
3.08
3.07
3.05
3.04
3.03
3.01
2.99
2.96
2.62 2.70 2.84
Fragment
m/z
= 58.0653
Tramadol
m/z
= 222.1852
Confirmation
Identification,
quantitation
RT:
3.86 - 5.16
SM:
5B
4.0
4.2
4.4
Time (min
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
4.41
4.40
4.42
4.38
4.43
4.37
4.44
4.36
4.45
4.35
4.34
4.47
4.32
4.49
4.28
4.40 4.41
4.39
4.43
4.38
4.37 4.44
4.36
4.45
4.34
4.46
4.01 4.02
3.99
4.32
4.48
4.10
4.40 4.41
4.39
4.43
4.38
4.44
4.37
4.45
4.36
4.46
4.34
4.5
4.32
4.29
4.19
4.01
4.40 4.41
4.39
4.43
4.38
4.44
4.37
4.45
4.36
4.46
4.34
4.47
4.33
4.30
Identification, quantitation
Diazepam
m/z
= 285.0789
Confirmation
Fragment
m/z
= 193.0888
Fragment
m/z
= 154.0420
Fragment
m/z
= 222.1154
192.0 192.5 193.0 193.5 194.0 1
m/z
193.9983
193.0884
194.091
192.0803
192.9106
2.0491
194.11
FIGURE 2
.
Chrom
analytes as displ
Morph
Codei