testing androgenic anabolic steroids in oral fluid using...

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.

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

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