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

QQ

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