AI-64310-CRFT

427 / 658

3 Thermo Scientific Poster Note • PN64314-EN 1114S
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™
software version 3.1. Full-scan data (chromatograms reconstructed with m/z
accuracy of 5 ppm) were used for analyte detection and quantification. AIF
spectra were used for compound confirmation. Calibration ranges and LOQ’s
were evaluated based on concentration accuracy and data precision. Back-
calculated concentration had to be within 30% for the LOQ.
Results
 Examples of data collected with the method are presented in Figure 1.
 Representative chromatograms for the lowest calibration standards of selected
analytes are presented in Figure 2.
 Linearity ranges, method precision and % recovery in spiked urine samples
from 15 different donors are presented in Table 1.
 Examples of calibration curves are shown in Figure 3.
Codei
Prazepa
Ritalinic Ac
-Resolution Q Exactive Mass Spectrometer in Analysis of U
ntitative Screening Application for Forensics
, CA
ers for quantitative
tes, short acquisition
ultra-high-resolution
hits. Quantitative
because it allows
bove specified threshold,
above carry-over limit,
tory quantitative analysis.
stem
itrap MS
ample) was spiked with
00 U/mL beta-
60 °C. The resulting
d with
evaporated to dryness
in 100 µL of 20%
MS.
OLD PFP 100 x 2.1 mm,
ater (A) and methanol
de. The LC gradient
s equipped with a HESI
S method consisted of 2
FIGURE 1. Representative chromatograms and spectra of tramadol and
diazepam in pooled urine spiked at 100 ng/mL. Shown are the full-scan
chromatogram of parent compound for quantitation, chromatogram of
fragments from AIF spectra reconstructed with m/z accuracy of 5 ppm, and
AIF spectra for the scan collected at the apex of the peak. Chromatograms
are reconstructed in stick mode to demonstrate sufficient number of scans
across the peak.
Flow rate (mL/min)
0.75
0.75
0.75
0.75
0.85
0.85
0.85
0.85
RT: 2.62 - 3.56 SM: 5B
2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5
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
3.12
3.09
3.14
3.07
3.15
3.06
3.16 3.05
3.03 3.17
3.02
3.19
2.99
3.21 2.97 3.24 3.31 3.42 3.50 2.90 2.78 2.64
3.11
3.12
3.09
3.13
3.08
3.14
3.07
3.16
3.05
3.04 3.17
3.03 3.18
3.01
3.19
2.99 3.22 2.96 3.30 3.49 3.35 2.62 2.70 2.84
NL: 7.37E6
m/z=
264.1945-264.1971F:
FTMS + c ESIFull ms
[130.00-472.00] MS
100ng_01
NL: 3.45E6
m/z= 58.0650-58.0656
F: FTMS + c ESIFull
ms2
70.00
[50.00-472.00] MS
100ng_01
100ng_01# 414 RT: 3.11 AV: 1 NL: 4.20E6
F: FTMS + c ESIFull ms2 0.00 [50.00-472.00]
50 100 150 200 250 300 350 400 450
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
58.0653
149.0231
62.9291
116.9856
98.9839
230.0457 278.0593
333.1032
150.0264 240.2316 419.3143 307.0857
222.1847
334.1065 441.2962
AIF spectrum
Tramadol fragment
Fragment m/z = 58.0653
Tramadol
m/z = 222.1852
Confirmation
Identification,
quantitation
Tramadol confirmed with 1 fragment
Diazepam confirmed with 3 fragments
RT: 3.86 - 5.16 SM: 5B
4.0 4.2 4.4 4.6 4.8 5.0
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 4.52 4.76
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 4.79 4.81 4.76
4.48 4.97 4.10 4.99
4.40 4.41
4.39
4.43
4.38
4.44
4.37
4.45
4.36 4.46
4.34
4.55 4.58
4.32
4.64 4.29
4.19 4.01 4.68 5.03 4.99
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
NL: 2.75E6
m/z=
285.0775-285.0803F:
FTMS + c ESIFull ms
[130.00-472.00] MS
100ng_01
m/z=
193.0878-193.0898F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
m/z=
154.0412-154.0428F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
m/z=
222.1143-222.1165F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
100ng_01# 622 RT: 4.40 AV: 1 NL: 7.28E6
F: FTMS + c ESIFull ms2 0.00 [50.00-472.00]
50 100 150 200 250 300 350 400 450
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
230.0457
333.1033
62.9291 149.0231
176.0242
103.0752
64.9273
334.1062
184.0367
231.0489
335.0992 202.0272
285.0784 121.0282
240.2316
413.0356
287.0756 336.1031 436.0518
AIF spectrum
Identification, quantitation
Diazepam
m/z = 285.0789
Confirmation
Fragment m/z = 193.0888
Fragment m/z = 154.0420
Fragment m/z = 222.1154
192.0 192.5 193.0 193.5 194.0 194.5 195.0 195
m/z
193.9983
193.0884
194.0916 192.0803 195.0017
192.9106
2.0491 195.0871
194.1169 153.0 153.5 154.0 154.5 155.0 155.
m/z
153.0382
154.0415
52.9690
153.1270
154.9645
154.1224
154.9068 155.1423
220 221 222 223 224 225
m/z
222.1147
225.0629
221.1073
223.1181
222.1848
224.2001
1737
221.0031
225
FIGURE 3. Exa
for selected co
analytes.
Table1. Lineari
samples from 1
Compound Cali
r
(n
6-MAM 1–
7-aminoclonazepam 1–
Alprazolam 1–
Clonazepam 1–
Clorazepate 1–
Codeine 1–
Diazepam 1–
Dihydrocodeine 1–
Flurazepam 1–
Hydrocodone 1–
Hydromorphone 1–
Lorazepam 2–
MDA 50
MDEA 2–
MDMA 25
Meperidine 1–
Methadone 5–
Methamphetamine 5–
Methylphenidate 1–
* Precision obtained for
-Resolution Q Exactive Mass Spectrometer in Analysis of U
ntitative Screening Application for Forensics
, CA
ers for quantitative
t s, short cquisition
ultra- igh-resoluti
hits. Quantitative
because it allows
ove sp cified threshold,
above carry-over limit,
tory quantitative analysis.
stem
itrap MS
ample) was spiked with
00 U/mL beta-
6 °C. Th resulting
d with
evaporated to dryness
in 100 µL of 20%
MS.
OLD PFP 100 x 2.1 mm,
ater (A) and methanol
de. The LC gradient
s equipped with a HESI
S method consisted of 2
d all ion fragmentation
FIGURE 1. Representative chromatograms and spectra of tramadol and
diazepam in pooled urine spiked at 100 ng/mL. Shown re the full-sca
chrom togram f pare t compound for quantitation, chromatogram of
frag ents fro AIF spectra reco structed with m/z a curacy of 5 ppm, and
AIF spectra f r the scan collected a the apex of the peak. Chr matograms
are reconstructed in stick mode to demonstrate sufficient number f scans
cross the peak.
Flow rate (mL/min)
0.75
0.75
.
.
.8
.
0.85
.
RT: 2.62 - 3.56 SM: 5B
2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5
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
3.12
3.09
3.14
3.07
3.15
3.06
3.16 3.05
3.03 3.17
3.02
3.19
2.99
3.21 2.97 3.24 3.31 3.42 3.50
2.90 2.78 2.64
3.11
3.12
3.09
3.13
3.08
3.14
3.07
3.16
3.05
3.04 3.17
3.03 3.18
3.01
3.19
2.99 3.22
2.96 3.30 3.49 3.35 2.62 2.70 2.84
NL: 7.37E6
m/z=
264.1945-264.1971F:
FTMS + c ESIFull ms
[130.00-472.00] MS
100ng_01
m/z= 58.0650-58.0656
F: FTMS + c ESIFull
ms2
70.00
[50.00-472.00] MS
100ng_01
100ng_01# 414 RT: 3.11 AV: 1 NL: 4.20E6
F: FTMS + c ESIFull ms2 0.00 [50.00-472.00]
50 100 150 200 250 300 350 400 450
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
58.0653
149.0231
62.9291
116.9856
98.9839
230.0457 278.0593
333.1032
150.0264 240.2316 419.3143
307.0857
222.1847
334.1065 441.2962
AIF spectrum
Tramadol fragment
Fragment m/z = 58.0653
Tramadol
m/z = 222.1852
Confirmation
Identification,
quantitation
Tramadol confirmed with 1 fragment
Diazepam confirmed with 3 fragments
RT: 3.86 - 5.16 SM: 5B
4.0 4.2 4.4 4.6 4.8 5.0
Time (min)
2
4
6
80
100
2
4
6
80
100
2
4
6
80
100
Rel tiv Abundance
2
4
6
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 4.52 4.76
4.40 4.41
4. 9
4.43 4.38
4.37 4.4
4.36 4.45
4.34
4. 6
4.01 4.02
3.99 4.32 4.79 4.81 4.76
4.48 4.97 4.10 4.99
4.40 4.41
. 9
4. 3
4.38
4.44
4.37
.45
4.36 4.46
4.34
4.55 4.58
4.32
4.64 4.29
4.19 4.01 4.68 5.03 4.99
4.40 4.41
4.39
4.43
4.38
4.4
4.37
4.45
4.36
4. 6
4.34
4.47 4.33
4.30
NL: 2.75E6
m/z=
285.0775-285.0803F:
FTMS + c ESIFull ms
[130.00-472.00] MS
100ng_01
NL: 1.56E5
m/z=
193.0878-193.0898F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
NL: 1.20E5
m/z=
154.0412-154.0428F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
00ng_01
NL: 4.77E4
m/z=
222.1143-222.1165F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
100ng_01# 622 RT: 4.40 AV: 1 NL: 7.28E6
F: FTMS + c ESIFull ms2 0.00 [50.00-472.00]
50 100 150 200 250 300 350 400 450
m/z
1
1
20
2
30
3
40
45
5
5
60
65
70
7
80
8
90
95
100
Rel tiv Abundance
230.0457
333.1033
62.9291 149.0231
176.0242
103.0752
64.9273
334.1062
184.0367
231.0489
335.0992 202.0272
285.0784 121.0282
240.2316
413.0356
287.0756 336.1031 436.0518
AIF spectrum
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 94.5 195.0 195
m/z
193.9983
193.0884
194.0916 192.0803 195.0017
192.9106
2.0491 195.0871
194.1169 153.0 153.5 154.0 154.5 155.0 155.
m/z
153.0382
154.0415
52.9690
153.1270
154.9645
154.1224
154.9068 155.1423
220 221 222 223 24 225
m/z
222.1147
225.0629
221.1073
223.1181
222.1848
224.2001
1737
221.0031
225
FIGURE 3. Exa
for selected co
analytes.
Table1. Lineari
s mples from 1
Compound Cali
r
(n
6-MAM 1–
7-aminoclonazepam
Alprazolam 1–
Clonazepam –
Clorazepate 1–
odeine
Diaz pam –
ihydrocodeine
Flurazepam 1–
Hydrocodone
r morphone 1–
Lorazepam 2
MDA 50
EA 2–
MDMA 25
eperidine 1–
thado 5
t mphetamine
t ylphenidate 1
* Precision obtained for
-Resolution Q Ex ctive Mass Sp ctrometer in Analysis of U
ntitative Screening Application for Forensics
, CA
ers for quantitative
t s, short cquis tion
ultra- igh-resolution
hits. Quantitative
because it allows
bove sp cified threshold,
above carry-ove limit,
tory quantitative analysis.
stem
itrap MS
ample) was spiked with
00 U/mL beta-
6 °C. Th resulting
d with
evaporated to dryness
in 100 µL of 20%
MS.
OLD PFP 100 x 2.1 mm,
ater (A) and methanol
de. The LC gradie t
s equipped with a HESI
S method consisted of 2
d all ion fragment tion
a were collected with
FIGURE 1. Representative chromatograms and spectra of tramadol and
diazepam in pooled urine spiked t 100 ng/mL. Shown re he full-sc
chromatogram f pare t comp und fo quantitation, chr matogram of
fr gments fro AIF spectra reconstructed with m/z a curacy of 5 ppm, and
AIF sp ctra f r the sca ollected a the apex of the peak. Chr m tograms
are reconstructed in tick mod to demonstrate sufficient number f sc
cro s the peak.
Flow rate (mL/min)
0.75
8
.
RT: 2.62 - 3.56 SM: 5B
2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5
Time (min)
0
10
20
30
40
50
60
7
8
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel tiv Abundance
3.11
3.10
3.12
3.09
3.14
3.07
3.15
3.06
3.16 3.05
3.03 3.17
3.02
3.19
2.99
3.21 2.97 3.24 3.31 3.42 3.50
2.90 2.78 2.64
3.11
3.12
3.09
3.13
3.08
3.14
3.07
3.16
3.05
3.04 3.17
3.03 3.18
3.01 3.19
2.99 3.22 2.96 3.30 3.49 3.35 2.62 2.70 2.84
NL: 7.37E6
m/z=
264.1945-264.1971F:
FTMS + c ESIFull ms
[130.00-472.00] MS
100ng_01
NL: 3.45E6
m/z= 58.0650-58.0656
F: FTMS + c ESIFull
ms2
70.00
[50.00-472.0 ] MS
100ng_01
100ng_01# 414 RT: 3.11 AV: 1 NL: 4.20E6
F: FTMS + c ESIFull ms2 0.00 [50.00-472.00]
50 100 150 00 250 300 350 400 450
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel tiveAbundance
58.0653
149.0231
62.9291
116.9856
98.9839
230.0457 278.0593
333.1032
150.0264 240.2316 419.3143 307.0857
222.1847
334.1065 441.2962
AIF spectrum
Tramadol fragment
Fragment m/z = 58.0653
Tramadol
m/z = 222.1852
Confirmation
Identification,
quantitation
Tramadol confirmed with 1 fragment
Diazepam confirmed with 3 fragments
RT: 3.86 - 5.16 SM: 5B
4.0 4.2 4.4 4.6 4.8 5.0
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 .49 4.28 4.52 4.76
4.40 .41
4.39
4. 3 4.38
4.37 4.44
4.36 .45
4.34
4.46
4.01 4.02 3.99 4.32 4.79 4.81
4.76 4.48 4.97
4.10 4.99
4.40 4.41
4.39
4.43
4.38
4.44
4.37
4.45
4.36
4.46
4.34 4.55 4.58
4.32
4.64 4.29
4.19 4.01 4.68 5.03 4.99
4.40 4.41
4.39
4.43
4.38
4.4
4.37
4.45
4.36
4.46
4.34
4.47 4.33
4.30
NL: 2.75E6
m/z=
285.0775-285.0803F:
FTMS + c ESIFull ms
[130.00-472.00] MS
100ng_01
NL: 1.56E5
m/z=
93.0878-193.0898F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
NL: 1.20E5
154.0412-154.0428F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
NL: 4.77E4
222.11 3-222.1165F:
FTMS + c ESIFull ms2
70.00
[50.00-472.00] MS
100ng_01
100ng_01# 622 RT: 4.40 AV: 1 NL: 7.28E6
F: FTMS + c ESIFull ms2 0.00 [50.00-472.00]
50 100 150 200 250 300 0 400 450
m/z
0
5
1
15
2
25
3
35
40
45
50
55
6
65
7
7
80
85
9
95
100
RelativeAbundance
230.0457
333.1033
62.9291 149.0231
176.0242
10 .0752
64.9273
334.1062 184.0367
231.0489
335.0992
202.0272
285.0784 121.0282
240.2316
413.0356
287.0756 336.1031 436.0518
AIF spectrum
Identification, quantitation
Diazepam
m/z = 285.0789
Confirmation
Fragment m/z = 193.0888
Fragment m/z = 154.0420
Frag e t /z= 222.1154
192.0 192.5 193.0 193.5 194.0 194.5 195.0 195
m/z
193.9983
193.0884
194.0916
192.0803 195.0017
192.9106 2.0491 195.0871
194.1169 153.0 153.5 154.0 154.5 155.0 155.
m/z
153.0382
154.0415
52.9690
153.1270
154.9645
154.1224
154.9068 155.1423
220 221 222 23 224 225
m/z
222.1147
225.0629
221.1073
223.1181
222.1848
224.2001 1737
221.0031
225
FIGURE 3. Exa
for selected co
analyt s.
Table1. Lineari
s mples from 1
Compound Cali
r
(n
6-MAM 1–
7-aminoclonazepam 1–
Alprazolam 1–
Clonazepam 1–
Clorazepate 1–
Codeine 1–
Diazepam 1–
Dihydrocodeine 1–
Flurazepam 1–
Hydrocodone 1–
Hydromorphone 1–
Lorazepam 2–
MDA 50
MDEA 2–
MDMA 25
Meperidine 1–
Methadone 5–
Methamphetamine 5–
Methylphenidate 1–
* Precision obtained for