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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 [email protected] [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
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
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 [email protected] [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 [email protected] [65.00-250.00]
80 100 120 140 160 180 200 220 240
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
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