AI-64310-CRFT

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2 Two selected-reaction monitoring (SRM) transitions were
monitored for EtG, EtS and their deuterated internal
standards to provide ion ratio confirmations (IRC). The
scans were run in timed selected-reaction monitoring
(t-SRM) mode with a cycle time of 0.25 seconds. In this
mode, SRM transitions are given a retention time and
window in which the mass spectrometer acquires the
specified transitions only. This allows the instrument to
maximize the amount of time spent acquiring each
transition, while maintaining a consistent number of
data points across the chromatographic peak.
Data was acquired and processed with
Validation
Intra-assay precision and accuracy were determined by
analyzing a calibration curve along with six replicates of
quality control (QC) samples. Inter-assay precision and
accuracy were determined by analyzing a calibration
curve along with six replicates of QC samples on three
different days. Matrix effects were evaluated by observing
the internal standard signals in 23 different lots of human
urine.
Results
Both compounds were linear over a wide dynamic range.
EtG was linear from 50 to 50,000 ng/mL, while EtS had a
range of 25 to 50,000 ng/mL. Figure 2 shows representative
calibration curves for both compounds. Figure 3 shows
representative chromatograms for EtG and EtS at their
respective LOQs.
0
0.5
1
1.5
2
0 2000 4000 6000 8000 10000
Area Ratio
ng/mL
EtG
Y = 1.852e-4X + 1.709e-2; R^2: 0.9953; Origin: Ignore; W: 1/X; Area
0
0.05
0.1
0.15
0.2
0 200 400 600 800 1000
Area Ratio
Theoretical Calculated %Diff
50 54.962 9.92
100 97.747 -2.25
250 266.942 6.78
500 470.273 -5.95
1000 854.373 -14.6
5000 5450.4 9.01
10000 9705.303 -2.95
Figure 2. Representative calibration curves for EtG (top) and EtS (bottom) in urine
0
2
4
6
8
10
12
14
16
18
0 2000 4000 6000 8000 10000
Area Ratio
ng/mL
EtS
Y + 1.687e-3X + 2.021e-1; R^2: 0.9985; Origin: Ignore; W: 1/X; Area
Theoretical Calculated %Diff
25 22.269 -10.9
50 50.799 11.6
100 108.127 8.13
250 272.582 9.03
500 519.572 3.91
1000 890.546 -10.9
5000 4855.946 -2.88
10000 10204.859 2.05
0
0.5
1
1.5
2
0 2000 4000 6000 8000 10000
Area Ratio
ng/mL
EtG
Y = 1.852e-4X + 1.709e-2; R^2: 0.9953; Origin: Ignore; W: 1/X; Area
0
0.05
0.1
0.15
0.2
0 200 400 600 800 1000
Area Ratio
ng/mL
0
2
4
6
8
10
12
14
16
18
0 2000 4000 6000 8000 10000
Area Ratio
ng/mL
EtS
Y + 1.687e-3X + 2.021e-1; R^2: 0.9985; Origin: Ignore; W: 1/X; Area
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 200 400 600 800 1000
Area Ratio
ng/mL