The Application of High Resolution Accurate

Mass LCMS to Sport Drug Screening (and

beyond)

Simon Hudson

7th June 2011

HFL Sport Science

• Long heritage in high integrity analysis (est. 1963)

• Only multisite sports testing lab in the world

• 60 people on our Fordham site 10 in Lexington, Kentucky

• State-of-the-art facilities (1997 Fordham, 2010 Lexington)

• Business areas

– Regulatory testing (sports authorities)

– Veterinary support (sales testing etc.)

– Sport supplement residue testing

– Nutritional, health and fitness biomarkers

– Forensic and environmental analysis

• Strong research focus

HFL – a brief history

HFL Sport Science, UK

HFL Sport Science Inc., USA

• Why we test? What we test for

• Challenges of meeting evolving customer

requirements

• High resolution accurate mass LCMS and how this

has helped

• Other applications of the technology

Overview

• Compliance with the Rules/Code of sport

• Prevent unfair advantage

• Maintain integrity of racing

• Animal welfare / safety of rider

• Protect breeding lines

Why is medication control

required?

Prohibited Substances

“ „Prohibited Substance‟ means a substance originating externally whether or not it

is endogenous to the horse which falls into any of the categories contained in the

List of Prohibited Substances published from time to time in the Racing Calendar.

„Substance‟ includes the metabolites of the substance and the isomers of the

substance and metabolites ”

Any substance capable at any time of acting on one or more of

the following mammalian body systems

the nervous system the cardiovascular system

the respiratory system the digestive system

the urinary system the reproductive system

the musculoskeletal system the blood system

the immune system except for licensed vaccines against infectious

agents

the endocrine system

In other words………potentially everything!

What is banned in racing?

The Problem - 2005Where we were Where we were heading

New drugs,

Changes in

sampling / testing

strategy

Assay sensitivity /

reproducibility

Screen 2005

SPE

Urine

Aliquot 1 Aliquot 2 Aliquot 3 Aliquot 4

SPESPE LLE

GCMSBasic drugs(full scan)

LCMSBasic drugs

(MRM )

GCMSAcid/Neuts(Full Scan)

GCMSAnabolic Steroids

(SIM)

LCMSMisc

compounds(trap dds)

LCMSCorticos(MRM)

GCMSVolatiles(full scan)

Accurate Mass LCMSWhere we were Where we are now

Excluding anabolic steroids /

large molecules

Selectivity of nominal massCompounds – all different elemental

compositions

Nominal Mass

Aminacrine (C13H10N2) 194

Aminohippuric acid (C9H10N2O3) 194

Anthranol (C14H10O) 194

Butylparaben (C11H14O) 194

Caffeine (C8H10N4O2) 194

Dimethyl phthalate (C10H10O4) 194

Galacturonic acid (C6H10O7) 194

Hexylresorcinol (C12H18O2) 194

Methylglucoside (C7H4O6) 194

Depreton (C8H10N4S) 194

Phenocoll (C10H14N2O2) 194

Solanone (C13H22O) 194

Temozolamide (C6H6N6O2) 194

Selectivity of accurate mass

Compound Accurate Mass

Aminacrine (C13H10N2) 194.08385

Aminohippuric acid (C9H10N2O3) 194.06859

Anthranol (C14H10O) 194.07262

Butylparaben (C11H14O) 194.09375

Caffeine (C8H10N4O2) 194.07983

Dimethyl phthalate (C10H10O4) 194.05736

Galacturonic acid (C6H10O7) 194.04210

Hexylresorcinol (C12H18O2) 194.13103

Methylglucoside (C7H4O6) 194.07849

Depreton (C8H10N4S) 194.06207

Phenocoll (C10H14N2O2) 194.10498

Solanone (C13H22O) 194.16652

Temozolamide (C6H6N6O2) 194.05467

Selectivity through resolution and

accurate mass measurement

C6H6N6O2*1.00 + C13H10N2*1.00 + C14H10O*1.00 + C11H14O3*...

193.6 193.7 193.8 193.9 194.0 194.1 194.2 194.3 194.4 194.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

194.0839C6H6N6O2*1.00 + C13H10N2*1.00 + C14H10O*1.00 + C11H14O3*...

193.6 193.7 193.8 193.9 194.0 194.1 194.2 194.3 194.4 194.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

194.0792

194.0547194.1050

C6H6N6O2*1.00 + C13H10N2*1.00 + C14H10O*1.00 + C11H14O3*...

194.00 194.05 194.10 194.15 194.20 194.25 194.30

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

194.0792

194.0547194.0421194.1050 194.1301 194.1665

194.0838

Selectivity through resolution and

accurate mass measurement

How we use accurate mass for

screening

• Combination of ability to separate ions and then measure them

accurately gives a high degree of selectivity.

• Use high selectivity in full scan to give very broad drug coverage.

MRM, even when scheduled, can maybe give coverage for 100-

200 analytes.

• Full scan accurate mass can realistically cover thousands of

compounds in a run if we combine mass with retention time.

• Do not need reference materials to set up screen.

Example – selectivity of full scan

accurate mass

328.2271 0.5amu

(327.7271 to 328.7271)

328.2271 0.001amu

(328.2261 to 328.2281)

Butorphanol 1ng/ml in

equine urine

Accurate Mass Drug Screens

• 2 x extraction methods (basic drugs and general screen) using

polymeric SPE cartridges – wide range of analytes extracted.

• 2 x Analysis on LTQ orbitrap (different columns / mobile phases)

full scan @ 30000 resolution. 7 minute cycle time.

• Data processing using ToxID.

• Follow up using same sample but with MSn on suspect analytes.

• MSn database in continuous development.

• Replaces 5 instrumental analyses.

Screen Before

SPE

Urine

Aliquot 1 Aliquot 2 Aliquot 3 Aliquot 4

SPESPE LLE

GCMSBasic drugs(full scan)

LCMSBasic drugs

(MRM )

GCMSAcid/Neuts(Full Scan)

GCMSAnabolic Steroids

(SIM)

LCMSMisc

compounds(trap dds)

LCMSCorticos(MRM)

GCMSVolatiles(full scan)

Screen After

SPE

Urine

Aliquot 1 Aliquot 2 Aliquot 3

SPESPE

LCMSBasic drugs

Full Scan (HRAM)

LCMSAcid/Neuts

Full Scan(HRAM)

GCMSAnabolic Steroids(SRM)

LCMSCorticos(MRM)

Coverage

• Basic Screen > 1100 analytes

• General Screen > 400 analytes

• MS2/MS3 libraries > 700 entries

• All databases constantly expanding – no impact upon instrumental method

Positive Control with 0.001 amu

mass windowT:\data\B_LCMS1250\499236-1BU-eqes 21/03/2011 18:47:56 499236-1BU

HFL

RT: 0.00 - 5.20

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time (min)

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

1800000000

Inte

nsity

3.63

3.18

3.283.10 3.770.89 4.23

2.982.282.920.71 2.26

2.601.27 1.472.101.74

3.94

4.30 4.40

4.64 4.93

NL:

1.91E9

TIC F: FTMS +

c ESI Full ms

[90.00-650.00]

MS

499236-1BU-

eqes

499236-1BU-eqes #380 RT: 3.72 AV: 1 NL: 5.52E6

F: FTMS + c ESI Full ms2 345.25@cid35.00 [50.00-360.00]

50 100 150 200 250 300 350

m/z

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

Inte

nsity

317.0897

327.2063274.1202 313.1295191.116558.5973 153.6920 258.8456134.028689.8527 223.7108

RT: 0.00 - 5.19

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

Re

lativ

e A

bu

nd

an

ce 0

50

100

0

50

100

0

50

100RT: 2.27

RT: 3.32

RT: 2.76

RT: 3.10 RT: 3.37RT: 2.66

RT: 3.31

RT: 2.25

RT: 3.23

RT: 4.43

RT: 3.34

RT: 3.24

NL: 2.75E7

m/z= 286.1433-286.1443 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

NL: 1.96E8

m/z= 267.1698-267.1708 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

NL: 1.58E8

m/z= 302.1745-302.1755 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

NL: 2.44E7

m/z= 289.1621-289.1631 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

NL: 5.88E6

m/z= 277.0864-277.0874 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

NL: 3.73E7

m/z= 271.0628-271.0638 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

NL: 2.53E6

m/z= 328.2266-328.2276 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

NL: 1.66E7

m/z= 377.9694-377.9704 F: FTMS + c ESI Full ms [90.00-650.00] MS ICIS jun30eq2

Atenolol 200ng/ml

Morphine 50ng/ml

Isoxsuprine 100ng/ml

D3 Morphine IM

Clenbuterol 4ng/ml

Nordazepam 50ng/ml

Butorphanol 1ng/ml

Dembrexine 50ng/ml

Mass stability / Mass accuracy

267.1698

267.1699

267.1700

267.1701

267.1702

267.1703

267.1704

267.1705

267.1706

267.1707

267.1708

267.1709

267.1710

Apr-09 May-09 Jun-09 Jul-09 Aug-09 Sep-09 Oct-09 Nov-09 Dec-09 Jan-10 Feb-10

Atenolol [M+H]+ = 267.1703 – 200ng/ml

1ppm

Data Processing

• Simple accurate mass

/ Rt database (csv file)

• Only reports what it’s

found (not what it

hasn’t found)

• Massive improvement

in efficiency compared

to previous multiple

GC and LC-MS

methods

• Use typically +/- 2mDa

mass window

• Room for further

improvement

Analytical Benefits• Broad full scan screen advantages of GCMS realised in the

more universal technique of LCMS. No derivatisation

• Fewer instruments/less labour to deliver improved service

• Easier to add drugs to screen – no mass spectral library

• Search against databases > 1000 compounds and only reports what it’s found (not what it hasn’t found)

• Retrospective data processing for new drugs

• Metabolomic type data review to search for designer compounds

• Turnaround times improved

Example data - Stanozolol

Example - formoterol

Metabolism Studies• Urine still the main matrix – normally detect metabolites

• Determine metabolites of new drugs

• Metabolism is largely predictable, appropriate mass can be determined theoretically

• Accurate mass alone sufficient to give some coverage

• But – potential for false hits

• Analysis of metabolites to determine retention time, is much preferred – very limited availability.

• Combination of in vivo/in vitro systems

• Orbitrap gives a rapid means of locating new metabolites

Chlorpromazine metabolism

Analyte Accurate Mass

Chlorpromazine

Desmethyl chlorpromazine

Desmethyl hydroxy chlorpromazine

Hydroxy chlorpromazine

Dihydroxy chlorpromazine

Desmethyl dihydroxy chlorpromazine

319.0130

305.0874

321.0823

335.0979

351.0929

337.0772

MW 318

Chlorpromazine metabolism

Desmethyl chlorpromazine

Desmethyl hydroxy

chlorpromazine

Hydroxy chlorpromazine

Dihydroxy chlorpromazine

F:\Basic_LCMS_IITP_06 25/04/2008 18:24:56 IITP

RT: 2.64 - 4.56

2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

1003.63

3.92

3.44

3.28

3.38

3.61 3.67 3.76 3.90 3.96 4.27 4.39

3.46

3.403.29 3.68 3.963.91 4.03 4.153.20 4.24 4.33 4.40

3.21

3.163.34 3.49 3.673.62 3.73 3.81 3.91 4.03 4.11 4.25 4.34 4.41

3.20

3.15 3.373.44 3.56 3.70 3.78

NL: 1.44E6

m/z= 305.08537-305.08937

F: FTMS + c ESI Full ms

[100.00-650.00] MS

Basic_LCMS_IITP_06

NL: 5.15E7

m/z= 321.08029-321.08429

F: FTMS + c ESI Full ms

[100.00-650.00] MS

Basic_LCMS_IITP_06

NL: 3.59E8

m/z= 335.09590-335.09990

F: FTMS + c ESI Full ms

[100.00-650.00] MS

Basic_LCMS_IITP_06

NL: 5.14E8

m/z= 351.09085-351.09485

F: FTMS + c ESI Full ms

[100.00-650.00] MS

Basic_LCMS_IITP_06

NL: 7.24E7

m/z= 337.07520-337.07920

F: FTMS + c ESI Full ms

[100.00-650.00] MS

Basic_LCMS_IITP_06

Desmethyl dihydroxy

chlorpromazine

Quantification - Salbutamol in

human urine

salbutamolY = -0.000415324+3.25746*X R^2 = 0.9997 W: 1/X

0.0 0.5 1.0 1.5 2.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Are

a R

atio

salbutamolY = -0.000415324+3.25746*X R^2 = 0.9997 W: 1/X

0.00 0.05 0.10 0.15 0.20 0.25 0.30

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Are

a R

atio

Hydrolysed human urine extracted in duplicate

D3-salbutamol IS. Full scan MS – 15,000 res

ug/ml

Concurrent full scan

confirmation RT: 0.00 - 5.20

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time (min)

0

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0

10

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50

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70

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Re

lative

Ab

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da

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2.55

2.79 3.47 3.61 4.121.491.32 4.612.55

2.83

2.071.16 3.462.95 3.640.70 1.30

NL: 6.99E7

m/z= 240.1554-240.1634 F: FTMS + c ESI Full ms [100.00-500.00] MS 106755-U-SPike-wb-2

NL: 1.55E6

TIC F: ITMS + c ESI Full ms2 240.00@cid35.00 [65.00-250.00] MS 106755-U-SPike-wb-2

106755-U-SPike-wb-7500 #238 RT: 2.45 AV: 1 NL: 8.07E6F: FTMS + c ESI w Full ms2 240.00@cid35.00 [65.00-250.00]

80 100 120 140 160 180 200 220 240

m/z

0

5

10

15

20

25

30

35

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45

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55

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70

75

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Re

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Ab

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148.0757C 9 H10 O N

166.0862C 9 H12 O2 N

222.1492C 13 H20 O2 N

204.1386C 13 H18 O N

133.0523C 8 H7 O N

120.0808C 8 H10 N

73.9593C O3 N 95.4740

180.0295C 8 H6 O4 N

Other applications

• Synthetic cannabinoids (spice)

• White powder analysis

• Niche environmental

• Quantitative analysis

• Metabolomics

Benefits of Orbitrap introductionPrevious Situation

• Service contract - £90K pa

• Capital to replace existing kit

£900K

• Space requirements high

• 5 complex data sets to

review

• Complex extraction process

Current Situation

• Service contract - £40K pa

• Cost of kit - £500K

• Space requirements low

• 3 simple data sets to review

• Fewer extraction streams

• More capacity with same

staff

Additional Benefits

• Easier to add drugs to screen

• Retrospective data processing for new drugs

• Rapid metabolic profiling

• New applications

Conclusion

• Investment in high resolution accurate

mass technology has had a major impact

on the delivery of a complex analytical

challenge

• Availability of technology has helped in

and opened up other business areas

Acknowledgents

• Steve Maynard

• Sarah Timbers