26
Forensic Toxicology use Only Analysis of ETG, ETS using the Thermo Scientific Exactive Mass Spectrometer Kent Johnson Fortes lab, Portland Oregon
Forensic Toxicology use Only
Analysis of ETG, ETS using theThermo Scientific Exactive Mass Spectrometer
Kent JohnsonFortes lab, Portland Oregon
2Forensic Toxicology use Only
For Forensic Toxicology Use Only
Goal
• Development of an easy to use, interference free, less than 4 min
analysis method for ETG and ETS in urine
• LOQ, 100ng/mL or better
• Method validation using QC’s and real samples
3Forensic Toxicology use Only
For Forensic Toxicology Use Only
Exactive Benchtop LC-MS
• Resolution100,000 at 1 scan per second 10,000 at 10 scans per second
• Mass accuracySub ppm
• Sensitivity500 fg Buspirone with S/N >10:1
• Dynamic range>10,000 within a spectrum
• Scan speedUp to 10 scans per second
• Mass rangem/z 50 - 4000
• Polarity switchingOne positive and one negative scan < 1 second (25K Resolution)
4Forensic Toxicology use Only
Ethinyl-Estradiol at Different Mass Resolutions
Resolution: 10k, 30k, 50k, 100k
279.12 279.14 279.16 279.18 279.20
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bundance
Ethinyl-Estradiol, 279.17434
Butyl-Phthalate, 279.15909
(ubiquitous background ion)
5Forensic Toxicology use Only
For Forensic Toxicology Use Only
ETG and ETS in Urine
6Forensic Toxicology use Only
For Forensic Toxicology Use Only
Standards, QC and sample preparation
+ 25uL int. std (IS) mix@2000ng/mL
100 uL urine
+ 900uL of mobile phase A
Sample preparation method
Inject 20uL
Vortex
Note: IS are ETG-D5 and ETS-D550 -100 x dilution with mobile phase recommended
ETG and ETS
7Forensic Toxicology use Only
LC/MS Method
• LC method
• Thermo Hypersil Gold 50 x 2.5 mm, 5u
• Mobile Phase A: 5mM Dihexylammonium acetate in LC DI H2O
• Mobile Phase B: 5mM Dihexylammonium acetate in CAN
• Mobile Phase C: ACN/IPA/Acetone = 45/45/10 v/v/v
• Column Temp: 50 Deg C
• Exactive MS method
• HESI source
• Full scan MS acquisition @ 100,000 Resolution
• External mass calibration
• Extract chromatogram for drugs of interest (m/z)@ better than 5ppm mass accuracy
• Confirm the identity using mass accuracy and RT
• Quantitate using peak area in the chromatogram
8Forensic Toxicology use Only
ETG-ETS LC gradient
Time (min) %A %B %C Flow
(mL/min)
0 95 5 0 0.8
0.1 95 5 0 0.8
3.0 64 36 0 0.8
3.01 0 100 0 0.8
3.5 0 100 0 0.8
3.51 0 0 100 0.8
4.0 0 0 100 0.8
4.01 95 5 0 0.8
5.0 95 5 0 0.8
9Forensic Toxicology use Only
For Forensic Toxicology Use Only
100 ng/mL EtG standard in urine
\\ussjo-sanserv1\Marketing\...\100ng_std 10/9/2009 11:26:01 AM
RT: 1.56 - 2.77 SM: 5G
2.0 2.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
1.89
1.67
2.722.02
2.532.38
2.16
1.89
2.691.682.60
2.13
2.29 2.441.61
NL: 3.29E7
TIC F: FTMS {0,0} -
p ESI Full ms
[123.00-230.00] MS
100ng_std
NL: 2.62E4
m/z=
221.0638-221.0682
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
100ng_std
NL: 1.91E5
m/z=
226.0957-226.1003
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
100ng_std
100ng_std #140 RT: 2.16 AV: 1 NL: 3.34E5
F: FTMS {0,0} - p ESI Full ms [123.00-230.00]
140 160 180 200 220
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
133.0502
226.0981
141.0165
212.9791
168.9890
202.9873
144.0662
159.0295194.9885
173.0564
100ng_std #140 RT: 2.16 AV: 1 NL: 1.53E5
F: FTMS {0,0} - p ESI Full ms [123.00-230.00]
220 222 224 226 228
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
226.0981
228.1606221.0667
223.0200
225.1609
227.1015
224.8035 229.1639
TIC
Spectrum 100- 250 amu
Peak width@ ½ ht0.0029 amu
XIC: ETG
XIC: ETG-D5
ETG IS
ETG
Very High Specificity
10Forensic Toxicology use Only
100 ng/mL EtS standard in urine
\\ussjo-sanserv1\Marketing\...\100ng_std 10/9/2009 11:26:01 AM
RT: 2.15 - 3.64 SM: 7G
2.5 3.0 3.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
3.25
2.72 2.902.532.38
3.60
2.71
2.24 3.21 3.323.12
2.69
2.16 3.12
NL: 3.09E7
TIC F: FTMS {0,0} -
p ESI Full ms
[123.00-230.00] MS
100ng_std
NL: 2.38E4
m/z=
124.9891-124.9915
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
100ng_std
NL: 2.15E5
m/z=
130.0204-130.0230
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
100ng_std
100ng_std #176 RT: 2.71 AV: 1 NL: 1.18E6
F: FTMS {0,0} - p ESI Full ms [123.00-230.00]
140 160 180 200 220
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
195.0521
222.9917
218.1033
127.0008
141.0165
209.0042
168.9890
155.0013 183.0296
100ng_std #176 RT: 2.71 AV: 1 NL: 1.72E5
F: FTMS {0,0} - p ESI Ful l ms [123.00-230.00]
124 126 128 130 132
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
127.0008 130.0223
131.0708
124.9909
129.0552
132.0180
TIC
Spectrum 100- 250 m/z
Peak width@ ½ ht0.0027amu
XIC: ETS
XIC: ETS-D5
ETS IS
ETS
ETS
Very High Specificity
11Forensic Toxicology use Only
Ion Suppression Test - Water vs Urine 250 ng/mL std
UrineWater
No Ion SuppressionPeak Areas SimilarAnalyte/IS ratio similar
c:\documents and settings\...\250ng_std 10/9/2009 11:32:26 AM
RT: 1.79 - 3.03 SM: 5G
1.8 2.0 2.2 2.4 2.6 2.8 3.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 1.98
MA: 219114
1.882.14 2.20 2.27 2.39 2.56 2.822.66 2.88
RT: 2.62
MA: 230996
2.32 2.751.82 2.442.01 2.06 3.022.931.94 2.17
NL: 5.75E4
m/z=
221.0638-221.0682
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
250ng_diw
NL: 6.25E4
m/z=
124.9891-124.9915
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
250ng_diw
RT: 1.82 - 3.09 SM: 7G
2.0 2.2 2.4 2.6 2.8 3.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 2.08
MA: 229884
2.23 2.751.96 2.542.48 2.83 3.012.902.64
RT: 2.70
MA: 240639
1.85 2.02 2.08 2.23 2.482.34 2.87 2.99
NL: 5.29E4
m/z=
221.0638-221.0682
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
250ng_std
NL: 5.54E4
m/z=
124.9891-124.9915
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
250ng_std
c:\documents and settings\...\250ng_std 10/9/2009 11:32:26 AM
RT: 1.79 - 3.03 SM: 5G
1.8 2.0 2.2 2.4 2.6 2.8 3.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100R
ela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 1.93
MA: 721112
2.11 2.322.20
RT: 2.59
MA: 744129
2.78 2.962.911.95 2.382.252.001.91
NL: 1.70E5
m/z=
226.0957-226.1003
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
500ng_diw
NL: 1.97E5
m/z=
130.0204-130.0230
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
250ng_diw
RT: 1.79 - 3.04 SM: 7G
1.8 2.0 2.2 2.4 2.6 2.8 3.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 1.93
MA: 723875
2.11 2.322.27
RT: 2.68
MA: 807336
1.95 2.812.07 2.992.171.79 2.34
NL: 1.55E5
m/z=
226.0957-226.1003
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
500ng_diw
NL: 1.96E5
m/z=
130.0204-130.0230
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
250ng_std
UrineWater
EtG-D5
EtS-D5
EtG-D5
EtS-D5
EtG
EtS
EtG
EtS
Analyte IS
Compound peak area water peak area urine
ETG 5.75 5.29
ETG D5 17 15.5
ETS 6.25 5.5
ETS D5 19.7 19.6
Water Urine
AnalyteWater
area
Urine
areaRatio Ratio
ETG 5.75 5.29 0.34 0.34
ETG-IS 17 15.5
ETS 6.25 5.5 0.32 0.28
ETS-IS 19.7 19.6
12Forensic Toxicology use Only
Calibration curves and LOQ
Ethyl_SulphateY = -0.0105889+0.0014412*X R^2 = 0.9837 W: 1/X^2
0 5000 10000 15000 20000
ng/mL
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
Are
a R
atio
ETS 25- 20,000 ng/mL
Ethyl_GlucuronideY = -0.0175482+0.00127676*X R^2 = 0.9907 W: 1/X^2
0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000
ng/mL
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
Are
a R
atio
ETG 100- 20,000 ng/mL C:\Xcalibur\...\Rawfiles\25ng_std 10/9/2009 1:01:38 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bun
dan
ce
RT: 1.94
MA: 106381.74
1.80
2.13
2.482.35 2.50
2.56 2.882.61
RT: 1.94
MA: 611241
2.29 2.35 2.632.23 2.81
NL: 7.35E3
m/z=
221.0638-221.0682
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
25ng_std
NL: 1.24E5
m/z=
226.0957-226.1003
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
25ng_std
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bun
dan
ce
RT: 2.59
MA: 19113
2.45 3.332.33 3.392.74 3.21
2.91 3.013.50 3.53 3.86
RT: 2.56
MA: 756109
2.962.772.332.21 3.44
NL: 4.42E3
m/z=
124.9891-124.9915
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
25ng_std
NL: 1.50E5
m/z=
130.0204-130.0230
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
25ng_std
25 ng/mL standards
ETG ETS
ETG-D5 ETS-D5
C:\Xcalibur\...\Rawfiles\50ng_std 10/9/2009 12:55:16 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
RT: 1.96
MA: 28930
1.77
1.75
2.45
2.392.272.13
2.49
2.552.58
2.722.90
RT: 1.93
MA: 590237
2.462.26 2.40 2.66
NL: 9.31E3
m/z=
221.0638-221.0682
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
50ng_std
NL: 1.15E5
m/z=
226.0957-226.1003
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
50ng_std
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
RT: 2.59
MA: 50721
3.352.352.13 3.252.99 3.462.90 3.49 3.84
RT: 2.56
MA: 768484
2.74 3.272.18 3.062.90
NL: 1.08E4
m/z=
124.9891-124.9915
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
50ng_std
NL: 1.52E5
m/z=
130.0204-130.0230
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
50ng_std
50 ng/mL standards
ETG ETS
ETG-D5 ETS-D5
13Forensic Toxicology use Only
100 ng/mL, 20 uL standards in urine
C:\Xcalibur\...\100ng_urine_dil_01 10/8/2009 4:32:39 PMjust 10 time urine dilution in mob phase A
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 2.11
MA: 62355
1.941.91
1.871.81 2.26 2.30 2.47 2.56 2.60 2.78 2.83
RT: 2.08
AA: 473813
2.22 2.44 2.57 2.93
NL: 1.41E4
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
100ng_urine_dil_01
NL: 8.66E4
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
ICIS
100ng_urine_dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 2.77
AA: 48429
2.112.26 2.41 2.93 3.04 3.28
RT: 2.74
MA: 414631
2.89 3.07 3.14 3.41 3.79
NL: 9.62E3
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
ICIS
100ng_urine_dil_01
NL: 7.73E4
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
100ng_urine_dil_01
ETGETS
ETG-D5 ETS-D5
14Forensic Toxicology use Only
C:\Xcalibur\...\Neg_control_urine_dil_01 10/8/2009 4:58:14 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
1.84 1.89
1.81
1.95 2.39
2.102.511.70
2.30
2.672.91
2.88
RT: 2.10
MA: 402077
1.75 2.802.722.52
NL: 3.34E3
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
Neg_control_urine_dil_0
1
NL: 7.40E4
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
Neg_control_urine_dil_0
1
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 2.77
MA: 13464
2.682.432.30
2.993.443.14 3.41
RT: 2.77
MA: 459122
2.93 3.11 3.252.32 2.61
NL: 3.11E3
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
Neg_control_urine_dil_0
1
NL: 8.82E4
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00] MS
Neg_control_urine_dil_0
1
Urine – Negative control
ETG ETS
ETG < LOD ETS present
ETG-D5 ETS-D5
15Forensic Toxicology use Only
C:\Xcalibur\...\QC375_urine_dil_01 10/9/2009 12:16:59 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 1.97
MA: 291523
1.86 2.12 2.29 2.38 2.50 2.64 2.79 2.881.78
RT: 1.95
MA: 656865
NL: 6.12E4
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC375_urine_dil_01
NL: 1.31E5
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC375_urine_dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.61
MA: 2052
3.36
2.382.35 2.972.77 3.02
3.563.80
RT: 2.59
MA: 747959
2.992.77 3.13 3.27
NL: 1.80E3
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC375_urine_dil_01
NL: 1.44E5
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC375_urine_dil_01
QC 375 ng/mL - ETG
ETGETS present
ETG-D5 ETS-D5
ETG=317 ng/mL, ETS present
16Forensic Toxicology use Only
QC 1250 ng/mL - ETG
C:\Xcalibur\...\QC1250_urine_dil_01 10/9/2009 12:23:22 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 1.99
MA: 921933
1.85 2.17 2.33 2.942.542.47 2.64 2.851.76
RT: 1.96
MA: 608934
2.11 2.39
NL: 1.77E5
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC1250_urine_dil_01
NL: 1.13E5
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC1250_urine_dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.63
MA: 204594
3.372.30 3.282.78 2.91 3.56 3.983.80
RT: 2.61
MA: 858670
2.91
NL: 4.15E4
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC1250_urine_dil_01
NL: 1.68E5
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
QC1250_urine_dil_01
ETGETS not expected found 190 ng/mL
ETG-D5 ETS-D5
ETG=1164 ng/mL, ETS=190 ng/mL
17Forensic Toxicology use Only
Urine sample
C:\Xcalibur\...\49443_urine_dil_01 10/8/2009 5:17:22 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
2.14
1.74 1.86
2.332.44 2.48
2.812.78 2.99
RT: 2.01
MA: 313437
2.46
NL: 5.30E4
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49443_urine_dil_01
NL: 6.41E4
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49443_urine_dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
2.19
2.302.33
2.43 2.683.27
2.51 3.24 3.30
3.19
2.82
3.152.88
2.96
RT: 2.67
MA: 336555
2.20 2.29 2.93 3.482.48 3.78
NL: 2.26E3
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49443_urine_dil_01
NL: 6.65E4
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49443_urine_dil_01
ETG < LOD ETS< LOD
ETG-D5 ETS-D5
ETG and ETS < LOD
18Forensic Toxicology use Only
Urine sample
C:\Xcalibur\...\49644_urine_dil_01 10/8/2009 5:23:45 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.02
MA: 115295053
2.752.65 2.931.76
RT: 2.01
MA: 275913
2.14 2.23 2.36
NL: 2.09E7
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49644_urine_dil_01
NL: 5.29E4
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49644_urine_dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.68
MA: 42796061
3.04 3.21 3.302.13 2.36 3.59 3.67 3.82
RT: 2.65
MA: 123292
2.26 2.313.303.07 3.38 3.62 3.79
NL: 7.24E6
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49644_urine_dil_01
NL: 2.31E4
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49644_urine_dil_01
ETGETS
ETG-D5
ETS-D5
ETG=312247, ETS=262149 ng/mL
19Forensic Toxicology use Only
C:\Xcalibur\...\49735_urine_dil_01 10/8/2009 5:36:32 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.06
MA: 36889901
2.37 2.42 2.541.75 1.85 2.782.70 2.94
RT: 2.04
MA: 449423
2.372.25
NL: 7.16E6
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49735_urine_dil_01
NL: 9.05E4
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49735_urine_dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.71
MA: 7904921
3.01 3.312.11 3.232.37 2.48 3.43 3.98
RT: 2.68
MA: 334297
2.84 2.962.39 3.202.16
NL: 1.62E6
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49735_urine_dil_01
NL: 7.08E4
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49735_urine_dil_01
Urine sample
ETG=68116, ETS=17789 ng/mL
ETG
ETS
ETG-D5
ETS-D5
20Forensic Toxicology use Only
C:\Xcalibur\...\49737_urine-dil_01 10/8/2009 5:42:54 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.03
MA: 6094109
1.67 1.79 2.30 2.52 2.772.69 2.97
RT: 2.00
MA: 359882
NL: 1.21E6
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS 49737_urine-dil_01
NL: 7.25E4
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS 49737_urine-dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.67
MA: 591111
2.34 2.383.03
3.18 3.33 3.51 3.74 3.94
RT: 2.66
MA: 403234
2.812.24 2.35 2.94 3.43 3.893.56
NL: 1.25E5
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS 49737_urine-dil_01
NL: 8.17E4
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS 49737_urine-dil_01
ETG ETS
Urine sample
ETG=14397, ETS=1176 ng/mL
ETG-D5 ETS-D5
21Forensic Toxicology use Only
C:\Xcalibur\...\49774_urine_dil_01 10/8/2009 5:49:17 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 2.02
MA: 223231
2.39
1.811.77
2.22
2.53 2.59 2.84 2.86
RT: 2.02
MA: 395246
2.25 2.34 2.46 2.59 2.79
NL: 5.81E4
m/z=
221.0638-221.0682 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49774_urine_dil_01
NL: 7.78E4
m/z=
226.0957-226.1003 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49774_urine_dil_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
RT: 2.70
MA: 85401
2.23
2.302.34
2.46
2.86 2.93
3.21 3.27
3.41 3.54 3.68 3.98
RT: 2.67
MA: 417998
2.82 2.992.11 2.26 2.44 3.89
NL: 2.42E4
m/z=
124.9891-124.9915 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49774_urine_dil_01
NL: 8.77E4
m/z=
130.0204-130.0230 F:
FTMS {0,0} - p ESI Full
ms [123.00-230.00]
MS
49774_urine_dil_01
ETG ETS
Urine sample
ETG=459.9 ETS=160.8 ng/mL
ETG-D5 ETS-D5
Passes lie detector test for alcohol but drinks 1 bottle of Nyquil every night
22Forensic Toxicology use Only
C:\Xcalibur\...\Rawfiles\50568 10/9/2009 12:36:07 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 1.97
MA: 553557
2.53
2.10
1.69 1.76 2.23 2.31 2.63 2.78
RT: 1.95
MA: 527518
2.57
NL: 1.24E5
m/z=
221.0638-
221.0682 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50568
NL: 1.08E5
m/z=
226.0957-
226.1003 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50568
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.62
MA: 666237
2.47 3.172.90 3.473.393.012.27 3.56
RT: 2.59
MA: 666379
2.23 2.912.37 3.09
NL: 1.35E5
m/z=
124.9891-
124.9915 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50568
NL: 1.35E5
m/z=
130.0204-
130.0230 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50568
ETG ETS
Urine sample
ETG=864.9 ETS=688.3 ng/mL
ETG-D5 ETS-D5
23Forensic Toxicology use Only
C:\Xcalibur\...\Rawfiles\50736 10/9/2009 12:42:30 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 1.96
MA: 344283
2.13
1.66 1.752.28 2.48
2.752.57 2.81
RT: 1.94
MA: 501545
NL: 8.03E4
m/z=
221.0638-
221.0682 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50736
NL: 1.06E5
m/z=
226.0957-
226.1003 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50736
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.60
MA: 576805
2.24 2.32 2.90 3.303.11 3.43 3.67
RT: 2.57
MA: 654381
2.21 2.792.36
NL: 1.17E5
m/z=
124.9891-
124.9915 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50736
NL: 1.35E5
m/z=
130.0204-
130.0230 F: FTMS
{0,0} - p ESI Full
ms [123.00-230.00]
MS 50736
ETG ETS
Urine sample
ETG=559.2 ETS=616.4 ng/mL
ETG-D5 ETS-D5
24Forensic Toxicology use Only
C:\Xcalibur\...\Rawfiles\Marta_01 10/9/2009 12:29:43 PM
RT: 1.63 - 2.99 SM: 5B
1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
2.12
1.841.72
2.382.44 2.49 2.81
2.77 2.91
RT: 1.97
MA: 608101
NL: 2.15E4
m/z=
221.0638-221.0682
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
Marta_01
NL: 1.22E5
m/z=
226.0957-226.1003
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
Marta_01
RT: 2.10 - 3.99 SM: 5B
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
RT: 2.63
MA: 18658
2.17 2.27 2.37
3.343.222.74
3.092.97 3.703.55 3.79
RT: 2.62
MA: 725622
2.12 2.41
NL: 4.34E3
m/z=
124.9891-124.9915
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
Marta_01
NL: 1.47E5
m/z=
130.0204-130.0230
F: FTMS {0,0} - p
ESI Full ms
[123.00-230.00] MS
Marta_01
ETGETS
Urine sample
ETG<LOQ, ETS< LOQ (~51.7 ng/mL)
ETG-D5 ETS-D5
25Forensic Toxicology use Only
Conclusions
• Assay based on Exactive ultra high resolution MS coupled to LC system is
• Well suited for accurate analysis of ETG and ETS
• Analysis time of 4 min for ETG and ETS
• ETG LOQ = 100ng/mL, LOD = 25ng/mL
• ETS LOQ = 25ng/mL, LOD = 10ng/mL
• Exactive with a resolution of 100,000 offers >/= SRM selectivity (SRM data comparison
not shown)
• Very easy for method development and routine operation
• Can be multiplexed for even higher throughput
26Forensic Toxicology use Only
Acknowledgements
• Kent Johnson, Fortes Lab Portland, Oregon
• Marta Kozak, Thermo Fisher Scientific
• Perkin Elmer (Steroid kit)
• LipoMed (EtS/EtS D5)
• Cerilliant (EtG/EtG D5)
• Thermo Scientific; Instrument, Technical, and application support
• TCI - Dihexylammonium acetate
