Forensic Toxicology Use Only
Quantitative Screening of 46 Illicit Drugs in Urine using
Exactive Ultrahigh Resolution and Accurate Mass system
Kevin MchaleThermo Fisher Scientific, San Jose CA
2Forensic Toxicology Use Only
Presentation Overview
• Anabolic androgenic steroids landscape
• Triple quadrupole MS/MS characteristics and performance
• AAS behavior in ESI-MS
• Sample preparation methods
• Orbitrap high resolution mass spectrometry
• Quantitative analysis of AAS in OF using Q Exactive
• Testosterone in serum using TurboFlow and Q Exactive
• Summary
3Forensic Toxicology Use Only
Presentation overview
• Exactive system introduction
• Principle of operation
• Method overview and salient features
• Urine analysis results
• Conclusions
• Appendix
4Forensic 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)
5Forensic Toxicology Use Only
Orbitrap – Principle of Operation
{ })/ln(2/2
),(222
mm RrRrzk
zrU ⋅+−⋅=
z
φ
Hyper-logarithmic potential distribution: “ideal Kingdon trap”
r
12
2
−
=
R
Rmzωωϕ 2
2
−
=
R
Rmzr ωω
qm
kz
/=ω
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
� Characteristic frequencies:• Frequency of rotation ωφ• Frequency of radial oscillations ω
r
• Frequency of axial oscillations ω
z
6Forensic Toxicology Use Only
Method overview and salient features
• Negative standard urine diluted with 25% ACN spiked with 46 drugs at
different concentrations.
• Isotopically labeled internal standards at constant 50 ng/mL.
• No sample preparation. A 10 uL aliquot directly injected onto reverse phase column.
• Exactive operated at a resolution of 50,000 FWHH
• External calibration mass accuracy better than 5 ppm.
• Analysis time <10 minutes
Note: LC/MS method details in appendix
7Forensic Toxicology Use Only
Dilute with 25% ACN
Negative urine sample
Spike 46 drugs at different concentrations
Spike isotopically labeled int standards
at 50 ng/mL
Inject 10 uL
Hypersil GOLD™ PFP
2.1 x 100 mm, 3 µm
Operated at res. 50,000 w/ Ext. Calib.
Mass accuracy better than 5 ppm
Method overview and salient features
Note: LC/MS method details in appendix
8Forensic Toxicology Use Only
Results - XIC@ ± 5 ppm at LOQs in urine
R T : 0 .50 - 4 .50 S M : 7G
1 2 3 4
T im e (m in)
0
200 00
0
200 00
400 00
0
500 0
0
500 0
0
200 000
400 000
0
200 00
0
200 00
0
500 0
AA : 15 961 AA : 187 81
AA : 11 446 5
A A: 7 511 8
A A: 95 964
A A: 16 41 610
A A: 5 295 8
AA : 360 98
A A: 1 452 7
AA : 65 25AA : 37 32
AA: 231 75
AA : 15 406 1
A A: 1 107 77
AA : 636 32
A A: 2 498 0
R T : 4 .00 - 7 .00 S M : 7G
4 5 6 7
T im e (m in)
0
200 00
400 00
0
200 00
0
500 00
100 000
0
100 00
200 00
0
200 00
0
100 00
200 00
0
500 00
0
100 00
200 00
AA: 7 37 68
AA: 245 518
A A: 7 500 0
A A: 8 906 9
A A: 92 81 9
A A: 6 219 5
AA: 344 575
AA: 6 264 0
AA : 10 573 7
A A: 18 74 08
R T : 4 .00 - 5 .50 S M : 7G
4.0 4 .5 5.0 5.5
T im e (m in )
0
2 000 0
4 000 0
6 000 0
0
1 000 0
2 000 0
0
2 000 0
4 000 0
6 000 0
0
5 000 0
0
2 000 0
0
2 000
4 000
0
1 000 0
2 000 0
AA : 68 965
A A: 15 19 4
A A: 95 14 3
AA: 2 13 492
A A: 19 327 5
AA : 57 388
A A: 1 798 96
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
A
Note: Drug names and LOQs on slide 9 & 10
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Results - XIC @±±±± 5ppm at LOQs in urine.
RT: 7 .75 - 9 .00 S M: 7G
8 .0 8.5 9.0
Time (min)
0
500 00
100 000
0
500 00
100 000
0
200 00
0
500 00
100 000
0
200 00
400 00
0
200 00
0
500 00
0
200 000
400 000
AA : 17 777 78
A A: 1 726 25
A A: 79 129
AA: 135 478
AA: 4 064 01
AA : 80 280
AA : 37 469 8
AA: 3 588 98
RT: 7 .25 - 8 .25 S M: 7G
7 .4 7 .6 7 .8 8 .0 8 .2
Time (min )
0
2 000 0
4 000 0
6 000 0
0
1 000 0
2 000 0
0
5 000
1 000 0
0
2 000 0
4 000 0
0
2 000 0
0
1 000 0
2 000 0
0
5 000
1 000 0
A A: 4 384 4
AA : 46 502
AA : 764 58
A A: 1 644 73
A A: 3 491 7
A A: 6 548 2
AA: 158 753
AA: 9 034 9
RT: 6 .25 - 7 .75 S M: 7G
6.5 7.0 7.5
Time (min)
0
100 00
0
500 00
100 000
0
100 00
200 00
0
200 00
0
200 00
0
200 00
400 00
0
500 00
0
500 00
AA : 21 467 5
A A: 1 837 64
A A: 1 103 82
A A: 8 031 5
A A: 8 413 0
A A: 49 967
AA: 371 001
A A: 3 517 4
AA
BB
CC
DD
EE
FF
GG
HH
II
JJ
KK
LL
MM
NN
OO
PP
RR
SS
TT
UU
VV
WW
Note: Drug names and LOQs on slide 9 & 10
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List of drugs and mass errors @ LOQ
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List of drugs monitored and mass errors @ LOQ
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Need for 50,000 resolution
163.10 163.15
m/z
0
50
100
0
50
100
0
50
100
0
50
100
163.1326
C 8 H 19 O 3
163.1229
C 10 H 15 N 2
163.1329
C 8 H 19 O 3
163.1230
C 10 H 15 N 2
163.1328
C 8 H 19 O 3
163.1222
C 10 H 15 N 2
163.1296
C 4 H 15 O N 6
NL:
1.53E5
drugs_abuse_ur ine_50k_313#18
1 RT: 1.40 AV: 1 T: FTMS {0,0} + p ESI Full ms [110.00-800.00]
NL:5.67E4
C 8 H 18 O 3 *1.00 + C 10 H 14 N 2 *0.50 +H: p (gss,
s /p:15) Chrg 1
R: 50000 Res .Pwr . @FWHM
NL:5.74E4
C 8 H 18 O 3 *1.00 + C 10 H 14 N 2 *0.50 +H: p (gss,
s /p:15) Chrg 1
R: 20000 Res .Pwr . @FWHM
NL:6.91E4
C 8 H 18 O 3 *1.00 + C 10 H 14 N 2 *0.50 +H: p (gss,
s /p:15) Chrg 1
R: 10000 Res .Pwr . @FWHM
Nicotine mass spectra at experimental (Exp) and
different simulated (Sim) resolutions (FWHM)
Exp: 50,000
Sim: 50,000
Sim: 20,000
Sim: 10,000
Note: Experimental Nicotine at RT 1.40 min; exact mass = 163.1230
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Conclusions
• The method is capable of easy confirmatory and quantitative analysis of 46 illicit drugs at LOQs of 0.5 – 5 ng/mL in urine.
• Ultrahigh mass resolution of 50,000 FWHM provided the necessary selectivity to identify 46 drugs in urine in 9 minutes with minimal sample preparation
• Analyte confirmation is achieved by utilizing better than 5 ppm mass accuracy with external mass calibration.
• Good quantitative accuracy achieved by isotopically-labeled standard.
• Nicotine example demonstrates the need for ≥50,000 resolution in addition to exact masses.
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• Appendix
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LC/MS Method
HPLC system• Accela UHPLC™ Pump, PAL autosampler (CTC Analytics)
• LC Column• 2.1 x 100 mm, 3 µm Hypersil GOLD™ PFP
• Injection Volume = 10 µL
• Mobile Phase• (A) 5 mM NH4OOCH + 0.1% HCOOH, (B) ACN + 0.1% HCOOH
• Flow Rate• 0.35 mL/min
• MS system• Exactive™ (Thermo Scientific)
• Heated ESI; Polarity: Positive; Scan Range: m/z 110 – 800
• Resolution: 50,000 FWHM;
• External mass calibration
