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Testing Androgenic Anabolic Steroids
in Oral Fluid using Turbulent Flow
Orbitrap Mass SpectrometryOrbitrap Mass Spectrometry
Erica A. Guice, Matt Pentis, and Brad Holden
Western Slope Laboratory, LLC
Erica A. Guice is the Director of Research and
Development at Western Slope Laboratory. Brad
Holden and Matt Pentis were employed as
Junior Analytical Chemists at Western Slope
Laboratory for the course of this research.
Funding for this research was provided by Funding for this research was provided by
Western Slope Laboratory.
Thermo Fisher Scientific supported this research
by providing instrumentation.
Outline
• Background in AAS testing OF
• Details of Method
• Details of Current Study
• Future Plans• Future Plans
BACKGROUND IN ANABOLIC
ANDROGENIC STEROID TESTING IN
ORAL FLUID
Developments with Western Slope Laboratory and beyond….
Foundation in Oral Fluid
• Western Slope Laboratory was founded on
oral fluid technologies and have continued
down that path
– Test for hundreds of analytes in oral fluid– Test for hundreds of analytes in oral fluid
– Same catalogue as urine analytes
– Low limits of detection and quantification
Oral Fluid-Matrix of the Future?
• Most toxicology laboratories now offer some
testing with the oral fluid matrix
• BUT, there are not any commercial
laboratories that offer oral fluid steroid laboratories that offer oral fluid steroid
testing….Western Slope Laboratory sought to
develop the method
DETAILS OF METHODOLOGY
Goal of testing for steroids:
o For forensics: low limits of detection for as
many compounds and their analogues
o For post mortem: large dynamic range for as
many compounds and their analoguesmany compounds and their analogues
o For clinical: low limits of detection for a
specific group of compounds
o For doping: large dynamic range for a specific
group of compounds
The Analytes
• Androgenic anabolic steroids are hormones
that mimic testosterone
– They are lipids, but much different structure than
the classical lipid the classical lipid
– they are non-polar
∴ Need solvent based sample preparation probably
SPE and high organic in mobile phases
Common Sports Doping SteroidsMetabolic Pathway
TetrahydrogestrinoneFluoxymesterone
OH
CH
CH3OH
OH CH3
CH3
CH3
CH3
OH
O
F
OHOH
O
CH3CH3
CH3
O
OH
CH3
CH3
DHEA
5
O
OCH3
CH3
Androstenedione
3
OH
OH
CH3
CH3
5 Androstenediol
1
OHCH3
CH3
O
OHCH3
CH3
Testosterone
4
3
OH
OHCH3
Estradiol
O
OHCH3
CH3
Dihydrotestosterone
O
OH
CH3
CH3
CH3
Mesterolone
OCH3
O
O
CH3
CH3
CH3
Metenolone Enanthate
O
OH
CH
CH3
CH3
Ethisterone
O
OH
CH3CH3
Norethandrolone6 Methylandrostendione
O
O
CH3
CH3
CH3
Progesterone
O
O
CH3
CH3
CH3
Progesterones
DHT Derivatives
Cl
OH
O
CH3CH3
CH3
Dehydrochlormethyltestosterone
OH
O
CH3
CH3
CH3
Methyltestosterone
OH
O
CH3CH3
CH3
CH3
Calusterone
Cl
OH
O
CH3
CH3
Clostebol
OH
CH3CH3
CH3
Desoxymethyltestosterone
Testosterone Derivatives
O
O
CH3
CH3
O
O
CH3
CH3
Boldione
Quinbolone
O
OHCH3
CH3
BoldenoneOH
O
OHCH3
CH3
6b Hydroxyboldenone
OH
CH3CH3
CH3
NH
N
Stanozolol
OH
OH
CH3CH3
CH3
NH
N
3' Hydroxystanozolol
OHCH3 CH3CH3
Androgen Receptor Agonists
CHCH3 CH
OCH3
O
CHCH3
CH3
Gestrinone
6b Hydroxyfluoxymesterone
Nandrolone
19 Norandrosterone
19 Norandrostenediol 19 Norandrostenedione
Nandrolone Derivatives
Oxabolone
Ethylestrenol
CH3 O
OH
CH3
CH3
OH
CH3OH
O O
CH3O
OH
O
O
OCH3
OH
OHCH3
17
O
CH
OH
O
F
3
CH3
OH
CH3
4 Androstenediol
OH
OCH3
CH3
Androsterone
O
O
CH3
CH3
5a Androstanedione
OH
OH
CH3
CH3
5a Androstanediol
2
1
OOH
OH
CH3
CH3
11b Hydroxyandrosterone
Epitestosterone
OH
O
CH3
CH3 CH3
CH3 OH
NH
N
Prostanozol
OH
CH
CH3
CH3
O
Danazol
OH
CH3CH3
CH3
N
O
N
Furazabol
O
OH
OH
CH3
CH3
16a Hydroetiocholanolone
OH
OH
CH3CH3
CH3
Methandriol
CH3
CH3
OH
OH
O
4 Hydroxytestosterone
O
O
OH
CH3
CH3
Formestane
O
CH3
1 TestosteroneO
OH3
CH3
Methyl 1 TestosteroneO
OH
CH3CH3
Methandieone
OH
O
O
OH
CH3CH3
CH3
Formebolone
CH3CH3
CH3
OH
O
Mestanolone
OH
O
CH3CH3
CH3CH3
Methasterone
Mibolerone
CH3OH
O
CH3
CH3
OH
O
CH3
CH3CH3
Stenbolone
O
CH3
CH3
Drostanolone Propionate
Oxandrolone
O
OH
CH3CH3
CH3
O
Norbolethone
CH3
CH3
OH
O
OH
O
OH
CH3CH3
CH3
OxymesteroneOH
O
CH3
Trenbolone
Oxymetholone
OH
CH3CH3
CH3CH3 OH
O
O
OHCH3CH3
CH3
Methandrostenolone CH3CH3
CH3
NH2
Cl
ClOH
NH
Clenbuterol
1= 3 Hydroxysteroid Dehydrogenase2= 17b Hydroxysteroid Dehydrogenase3= Aromatase4= 5a Reductase5= 17, 20 Lyase6= 3b Hydroxysteroid Dehydrogenase7= Liver Metabolism
Information Compiled for WSL by E. A. Guice, M.S.
Common Sports Doping SteroidsMetabolic Pathway
TetrahydrogestrinoneFluoxymesterone
OH
CH
CH3OH
OH CH3
CH3
CH3
CH3
OH
O
F
OHOH
O
CH3CH3
CH3
O
OH
CH3
CH3
DHEA
5
O
OCH3
CH3
Androstenedione
3
OH
OH
CH3
CH3
5 Androstenediol
1
OHCH3
CH3
O
OHCH3
CH3
Testosterone
4
3
OH
OHCH3
Estradiol
O
OHCH3
CH3
Dihydrotestosterone
O
OH
CH3
CH3
CH3
Mesterolone
OCH3
O
O
CH3
CH3
CH3
Metenolone Enanthate
O
OH
CH
CH3
CH3
Ethisterone
O
OH
CH3CH3
Norethandrolone6 Methylandrostendione
O
O
CH3
CH3
CH3
Progesterone
O
O
CH3
CH3
CH3
Progesterones
DHT Derivatives
Cl
OH
O
CH3CH3
CH3
Dehydrochlormethyltestosterone
OH
O
CH3
CH3
CH3
Methyltestosterone
OH
O
CH3CH3
CH3
CH3
Calusterone
Cl
OH
O
CH3
CH3
Clostebol
OH
CH3CH3
CH3
Desoxymethyltestosterone
Testosterone Derivatives
O
O
CH3
CH3
O
O
CH3
CH3
Boldione
Quinbolone
O
OHCH3
CH3
BoldenoneOH
O
OHCH3
CH3
6b Hydroxyboldenone
OH
CH3CH3
CH3
NH
N
Stanozolol
OH
OH
CH3CH3
CH3
NH
N
3' Hydroxystanozolol
OHCH3 CH3CH3
Androgen Receptor Agonists
CHCH3 CH
OCH3
O
CHCH3
CH3
Gestrinone
6b Hydroxyfluoxymesterone
Nandrolone
19 Norandrosterone
19 Norandrostenediol 19 Norandrostenedione
Nandrolone Derivatives
Oxabolone
Ethylestrenol
CH3 O
OH
CH3
CH3
OH
CH3OH
O O
CH3O
OH
O
O
OCH3
OH
OHCH3
17
O
CH
OH
O
F
3
CH3
OH
CH3
4 Androstenediol
OH
OCH3
CH3
Androsterone
O
O
CH3
CH3
5a Androstanedione
OH
OH
CH3
CH3
5a Androstanediol
2
1
OOH
OH
CH3
CH3
11b Hydroxyandrosterone
Epitestosterone
OH
O
CH3
CH3 CH3
CH3 OH
NH
N
Prostanozol
OH
CH
CH3
CH3
O
Danazol
OH
CH3CH3
CH3
N
O
N
Furazabol
O
OH
OH
CH3
CH3
16a Hydroetiocholanolone
OH
OH
CH3CH3
CH3
Methandriol
CH3
CH3
OH
OH
O
4 Hydroxytestosterone
O
O
OH
CH3
CH3
Formestane
O
CH3
1 TestosteroneO
OH3
CH3
Methyl 1 TestosteroneO
OH
CH3CH3
Methandieone
OH
O
O
OH
CH3CH3
CH3
Formebolone
CH3CH3
CH3
OH
O
Mestanolone
OH
O
CH3CH3
CH3CH3
Methasterone
Mibolerone
CH3OH
O
CH3
CH3
OH
O
CH3
CH3CH3
Stenbolone
O
CH3
CH3
Drostanolone Propionate
Oxandrolone
O
OH
CH3CH3
CH3
O
Norbolethone
CH3
CH3
OH
O
OH
O
OH
CH3CH3
CH3
OxymesteroneOH
O
CH3
Trenbolone
Oxymetholone
OH
CH3CH3
CH3CH3 OH
O
O
OHCH3CH3
CH3
Methandrostenolone CH3CH3
CH3
NH2
Cl
ClOH
NH
Clenbuterol
1= 3 Hydroxysteroid Dehydrogenase2= 17b Hydroxysteroid Dehydrogenase3= Aromatase4= 5a Reductase5= 17, 20 Lyase6= 3b Hydroxysteroid Dehydrogenase7= Liver Metabolism
Information Compiled for WSL by E. A. Guice, M.S.
Common Sports Doping SteroidsMetabolic Pathway
TetrahydrogestrinoneFluoxymesterone
OH
CH
CH3OH
OH CH3
CH3
CH3
CH3
OH
O
F
OHOH
O
CH3CH3
CH3
O
OH
CH3
CH3
DHEA
5
O
OCH3
CH3
Androstenedione
3
OH
OH
CH3
CH3
5 Androstenediol
1
OHCH3
CH3
O
OHCH3
CH3
Testosterone
4
3
OH
OHCH3
Estradiol
O
OHCH3
CH3
Dihydrotestosterone
O
OH
CH3
CH3
CH3
Mesterolone
OCH3
O
O
CH3
CH3
CH3
Metenolone Enanthate
O
OH
CH
CH3
CH3
Ethisterone
O
OH
CH3CH3
Norethandrolone6 Methylandrostendione
O
O
CH3
CH3
CH3
Progesterone
O
O
CH3
CH3
CH3
Progesterones
DHT Derivatives
Cl
OH
O
CH3CH3
CH3
Dehydrochlormethyltestosterone
OH
O
CH3
CH3
CH3
Methyltestosterone
OH
O
CH3CH3
CH3
CH3
Calusterone
Cl
OH
O
CH3
CH3
Clostebol
OH
CH3CH3
CH3
Desoxymethyltestosterone
Testosterone Derivatives
O
O
CH3
CH3
O
O
CH3
CH3
Boldione
Quinbolone
O
OHCH3
CH3
BoldenoneOH
O
OHCH3
CH3
6b Hydroxyboldenone
OH
CH3CH3
CH3
NH
N
Stanozolol
OH
OH
CH3CH3
CH3
NH
N
3' Hydroxystanozolol
OHCH3 CH3CH3
Androgen Receptor Agonists
CHCH3 CH
OCH3
O
CHCH3
CH3
Gestrinone
6b Hydroxyfluoxymesterone
Nandrolone
19 Norandrosterone
19 Norandrostenediol 19 Norandrostenedione
Nandrolone Derivatives
Oxabolone
Ethylestrenol
CH3 O
OH
CH3
CH3
OH
CH3OH
O O
CH3O
OH
O
O
OCH3
OH
OHCH3
17
O
CH
OH
O
F
3
CH3
OH
CH3
4 Androstenediol
OH
OCH3
CH3
Androsterone
O
O
CH3
CH3
5a Androstanedione
OH
OH
CH3
CH3
5a Androstanediol
2
1
OOH
OH
CH3
CH3
11b Hydroxyandrosterone
Epitestosterone
OH
O
CH3
CH3 CH3
CH3 OH
NH
N
Prostanozol
OH
CH
CH3
CH3
O
Danazol
OH
CH3CH3
CH3
N
O
N
Furazabol
O
OH
OH
CH3
CH3
16a Hydroetiocholanolone
OH
OH
CH3CH3
CH3
Methandriol
CH3
CH3
OH
OH
O
4 Hydroxytestosterone
O
O
OH
CH3
CH3
Formestane
O
CH3
1 TestosteroneO
OH3
CH3
Methyl 1 TestosteroneO
OH
CH3CH3
Methandieone
OH
O
O
OH
CH3CH3
CH3
Formebolone
CH3CH3
CH3
OH
O
Mestanolone
OH
O
CH3CH3
CH3CH3
Methasterone
Mibolerone
CH3OH
O
CH3
CH3
OH
O
CH3
CH3CH3
Stenbolone
O
CH3
CH3
Drostanolone Propionate
Oxandrolone
O
OH
CH3CH3
CH3
O
Norbolethone
CH3
CH3
OH
O
OH
O
OH
CH3CH3
CH3
OxymesteroneOH
O
CH3
Trenbolone
Oxymetholone
OH
CH3CH3
CH3CH3 OH
O
O
OHCH3CH3
CH3
Methandrostenolone CH3CH3
CH3
NH2
Cl
ClOH
NH
Clenbuterol
1= 3 Hydroxysteroid Dehydrogenase2= 17b Hydroxysteroid Dehydrogenase3= Aromatase4= 5a Reductase5= 17, 20 Lyase6= 3b Hydroxysteroid Dehydrogenase7= Liver Metabolism
Information Compiled for WSL by E. A. Guice, M.S.
Why Liquid Chromatography…
• Derivatization process can be quite lengthy and
often times irreproducible. In addition, this
process does not generally yield high conversion
of the steroid to the derivatized productof the steroid to the derivatized product
∴Liquid can reduce time and sample manipulation,
while still allowing for accurate analysis
∴We can take advantage of online sample
extraction systems i.e. turbulent flow
Why Turbulent Flow Online Extraction?
• Reduced ion suppression
• Eliminate the need for extensive sample clean-
up:
– Saving time, money, and people time– Saving time, money, and people time
– Very versatile
• Urine
• Saliva
• Plasma
Instrument requirements with steroids
• Resolution of structurally similar compounds, either by chromatography, mass, fragmentation, or combination
• Sensitivity to detect as low as femtogram• Sensitivity to detect as low as femtogramamounts on column; quantify picogram per milliliter solutions
• Dynamic range required for industry
• Ability to interface with online sample extraction
LC/MS vs LC/MS/MS vs LC/HR-TOF or MS
SIM vs QQQ
• Single Ion Monitoring would
require the separation of
isobars through
chromatography
TOF vs Orbitrap
• Both can use CID to provide
fragment information
• Also use the full scan
spectrum for retention time chromatography
• QQQ would require the use
of Ion Ratios to determine
of specific compounds
(isobars)
spectrum for retention time
verification
• Biggest difference here is
dynamic range and
spatial/temporal association
of the chromatographic
data
Which instrument meets the needs?
SIM (LC/MS) QQQ
(LC/MS/MS)
Q-TOF
(LC/HR-TOF)
Orbitrap
(LC/HR-MS)
Resolution of
structurally
similar
compoundscompounds
Sensitivity for
low limits of
detection
Dynamic range
requirements
met
Able to do
online
extractionMaybe
The Column: The Other “Instrument”
• With a turbulent flow-liquid chromatography
system set-up, there are two columns
selections that have a bearing on the results
of the method:of the method:
– The turbulent flow column
– The analytical column
• Column selection is affected by the pH and the
additives used.
The Turbulent Flow Column
• There are several chemistries that can be
employed including:
– C8, C18, Fluoro, Phenyl, and C2
• There are 0.5 and 1.0 mm diameter columns • There are 0.5 and 1.0 mm diameter columns
available as well; the lengths are all standard
The Analytical Column
• As usual with any chromatographic method,
you have to select the appropriate analytical
column chemistry, length, diameter, bead size,
and porousityand porousity
• Cation/Anion exchange can also be utilized
depending on the chemistry selected
Scans across the curve
• In order to quantify the amount of the
steroid(s) present in any given sample, an
appropriate number of scans under the peak
is required.is required.
• If qual-quan peaks are used, it is encouraged
that both have the appropriate amount of
scans
DETAILS OF CURRENT STUDY
Why human growth hormone
Where you are vs. Where you want to go
• We are able to detect endogenous and
exogenous compounds in urine and oral fluid
using the aforementioned method
• But one major group of compounds needed to • But one major group of compounds needed to
be considered: Hormones and Growth Factors
– Insulin-like Growth Factor-1 (Deer Antler Spray)
– Somatotrophin (GH Male/Vermont Maxi)
Targeted OF Sample
Untargeted OF Sample• C20H26O2 formula was given was a calculated mass of
298.193268 m/z of 299.25757
•Match to Norethindrone was given
•Donor questioned and admitted taking compound as birth
control
The compound and its major metabolites
were found in the sample.
Standard
1 yr Female Donor
66 yr Female Donor
FUTURE PLANS
Future Plans
• Conduct a clinical trial:
– Goal is to include a number of children/adolescents
– Also want to recruit persons currently taking hGH
recreationally (and other AASs)recreationally (and other AASs)
• Try to detect the parent (22kDa) hGH compound
• Look to detect the difference between
endogenous and exogenous hGH (preferably by
size)
Acknowledgements
• Western Slope Laboratory, LLC
– Matt Pentis
– Brad Holden
• Thermo Fisher Scientific• Thermo Fisher Scientific
– Jeff Zonderman
– Susan DiPietro
– Subodh Nimkar
Thank you for your attention
References
� Guice (2012) SOFT Workshop #4 Method
Development for Testing Androgenic Anabolic
Steroids in Oral Fluid and Urine using Turbulent
Flow Orbitrap MSFlow Orbitrap MS
�Jaslow (2013) CBS News What is deer antler
spray:IGF-1?
�www.bodybuilding.com