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Home , Desoxymethyltestosterone, Drostanolone propionate, Ethylestrenol, Formestane, Metenolone enanthate, Prostanozol

Testing Androgenic Anabolic in Oral Fluid using Turbulent Flow Orbitrap 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 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 Developments with Western Slope Laboratory and beyond…. BACKGROUND IN ANABOLIC ANDROGENIC TESTING IN ORAL FLUID 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 – Same catalogue as 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 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 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 that mimic – They are lipids, but much different structure than the classical lipid – they are non-polar ∴ Need solvent based sample preparation probably SPE and high organic in mobile phases CH3 O

CH3 HC OH H3C CH CH CH Testosterone Derivatives 3 3 CH3 OH 3 3 OH CH CH3 OH H3C OH 3 HC CH 3 CH3 H3C 3 OH CH CH O CH 3 OH 3 3 H3C H3C

O O O O O Cl OH Cl O CH3 H3C O Dehydrochlormethyltestosterone 6b Hydroxyboldenone CH3 Common Sports Doping Steroids O

Metabolic Pathway CH3 O CH CH CH3 O CH3 OH CH CH3 CH O 3 3 CH OH 3 3 OH 3 CH OH CH 3 CH HC CH3 5 3 3 CH3 H3C 3 O OH CH CH HN HO 3 3 CH3 OH N HO O CH3 CH H3C O O O 3 HO CH3 DHEA CH 3 6 Methylandrostendione F 3 O O DHT Derivatives O CH 3 OH CH CH3 OH CH H3C 3 3 CH3 OH CH3 OH O OH HC CH3 3 CH H3C HC CH 3 O 3 HO 3 H3C H3C 1 CH3 HN HO N O O Receptor O O 4 5 Enanthate Testosterone CH O 3' Hydroxystanozolol HC CH OH 3 3 HO 3 OH CH H3C O 3 CH HO CH CH CH3 OH CH3 CH CH 3 3 3 CH 3 OH 3 CH3 CH H3C HC OH 3 CH3 CH 3 CH OH HO CH 3 3 H3C OH 3 OH CH3 H3C O F HO CH3 O Propionate 4 Androstenediol O O O O O HN OH 1 Testosterone O O Methyl 1 Testosterone Methandieone N 6b Hydroxyfluoxymesterone CH HC HO 3 O 3 OH CH3 OH CH3 O HC CH 3 CH CH 3 HC 3 CH3 3 OH CH3 HC 3 CH3 3 OH OH HC 3 O CH3 H3C CH HC 3 3 O O HO HO H3C CH3 OH 11b Hydroxyandrosterone CH3 CH3 19 Norandrostenediol 19 Norandrostenedione OH HC HO O O 7 2 3 CH CH 1 O 3 3 OH HC HO CH 3 OH CH CH OH 3 OH 3 3 Derivatives CH O HC CH3 O CH H3C 3 3 OH 4 Hydroxytestosterone OH CH3 CH CH3 3 O O HC OH HO 3 H3C OH H3C 16a Hydroetiocholanolone O O O HO 1 5a Androstanediol CH3 Nandrolone O O O OH 1= 3 Hydroxysteroid Dehydrogenase HO 2= 17b Hydroxysteroid Dehydrogenase CH O H3C OH O CH3 O 3 3= CH3 O CH CH3 CH 4= 5a Reductase 3 3 OH 5= 17, 20 Lyase OH H3C Norbolethone 6= 3b Hydroxysteroid Dehydrogenase CH CH3 CH CH 3 CH 7= 3 3 3 OH OH Cl CH CH3 3 O O CH3 OH NH N HO O O CH3 O 5a HO CH3 CH3 Methandrostenolone CH 19 Norandrosterone HN 3 Information Compiled forE. WSLA. Guice, by M.S. HO 2 H3C N Cl O Clenbuterol CH3 O

CH3 HC OH H3C CH CH CH Testosterone Derivatives 3 3 CH3 OH 3 3 OH CH CH3 OH H3C OH 3 HC CH 3 CH3 H3C 3 OH CH CH O CH 3 OH 3 3 H3C H3C Boldione

O O O O O Cl Desoxymethyltestosterone OH Cl O Methyltestosterone Boldenone CH3 Clostebol H3C O Dehydrochlormethyltestosterone Calusterone 6b Hydroxyboldenone CH3 Common Sports Doping Steroids Progesterones O Quinbolone

Metabolic Pathway CH3 O CH CH CH3 O CH3 OH CH CH3 CH O 3 3 CH OH 3 3 OH 3 CH OH CH 3 CH HC CH3 5 3 3 CH3 H3C 3 O OH CH CH HN HO 3 3 CH3 OH N HO O CH3 CH H3C O O O 3 HO CH3 DHEA Estradiol CH Stanozolol Ethisterone Norethandrolone 3 Androstenedione 6 Methylandrostendione F 3 O O DHT Derivatives O CH 3 OH CH CH3 OH CH H3C 3 Tetrahydrogestrinone 3 CH3 OH Progesterone Fluoxymesterone CH3 OH O OH HC CH3 3 CH H3C HC CH 3 O 3 HO 3 H3C H3C 1 CH3 HN HO N O O Agonists O O 4 5 Androstenediol Dihydrotestosterone Mesterolone Testosterone CH O 3' Hydroxystanozolol HC CH OH 3 3 HO 3 OH CH H3C O 3 CH HO CH CH CH3 OH CH3 CH CH 3 3 3 CH 3 OH 3 CH3 CH H3C HC OH 3 CH3 CH 3 CH OH HO CH 3 3 H3C OH 3 OH CH3 H3C O F HO CH3 O 4 Androstenediol O O O O O HN OH 1 Testosterone O O Methyl 1 Testosterone Gestrinone Methandieone N Epitestosterone Formebolone 6b Hydroxyfluoxymesterone CH HC HO 3 O 3 OH CH3 OH CH3 O HC CH 3 CH CH 3 HC 3 CH3 3 OH CH3 HC 3 CH3 3 OH OH Prostanozol HC 3 O CH3 H3C CH HC 3 3 O O HO HO H3C CH3 OH 11b Hydroxyandrosterone CH3 CH3 Stenbolone 19 Norandrostenediol 19 Norandrostenedione OH HC HO O O 7 2 3 CH CH 1 O 3 Methandriol 3 OH Mestanolone HC HO CH 3 OH CH CH OH 3 OH Methasterone 3 3 Nandrolone Derivatives CH O Androsterone HC CH3 O CH H3C 3 3 OH 4 Hydroxytestosterone OH CH3 CH CH3 3 O O HC OH HO 3 H3C OH H3C 16a Hydroetiocholanolone O Mibolerone O O HO 1 Danazol 5a Androstanediol CH3 Nandrolone O O O OH 1= 3 Hydroxysteroid Dehydrogenase Oxandrolone Oxymesterone HO Oxabolone 2= 17b Hydroxysteroid Dehydrogenase CH O H3C OH O CH3 O 3 3= Aromatase CH3 O CH CH3 CH 4= 5a Reductase 3 3 OH 5= 17, 20 Lyase OH H3C Norbolethone 6= 3b Hydroxysteroid Dehydrogenase CH CH3 CH CH 3 CH 7= Liver Metabolism 3 3 3 OH OH Cl CH CH3 3 O O CH3 OH NH N HO O O CH3 Trenbolone O 5a Androstanedione HO CH3 CH3 Methandrostenolone CH 19 Norandrosterone HN 3 Ethylestrenol Information Compiled forE. WSLA. Guice, by M.S. Formestane HO 2 H3C N Cl Furazabol O Clenbuterol Oxymetholone CH3 O

CH3 HC OH H3C CH CH CH Testosterone Derivatives 3 3 CH3 OH 3 3 OH CH CH3 OH H3C OH 3 HC CH 3 CH3 H3C 3 OH CH CH O CH 3 OH 3 3 H3C H3C Boldione

O O O O O Cl Desoxymethyltestosterone OH Cl O Methyltestosterone Boldenone CH3 Clostebol H3C O Dehydrochlormethyltestosterone Calusterone 6b Hydroxyboldenone CH3 Common Sports Doping Steroids Progesterones O Quinbolone

Metabolic Pathway CH3 O CH CH CH3 O CH3 OH CH CH3 CH O 3 3 CH OH 3 3 OH 3 CH OH CH 3 CH HC CH3 5 3 3 CH3 H3C 3 O OH CH CH HN HO 3 3 CH3 OH N HO O CH3 CH H3C O O O 3 HO CH3 DHEA Estradiol CH Stanozolol Ethisterone Norethandrolone 3 Androstenedione 6 Methylandrostendione F 3 O O DHT Derivatives O CH 3 OH CH CH3 OH CH H3C 3 Tetrahydrogestrinone 3 CH3 OH Progesterone Fluoxymesterone CH3 OH O OH HC CH3 3 CH H3C HC CH 3 O 3 HO 3 H3C H3C 1 CH3 HN HO N O O Androgen Receptor Agonists O O 4 5 Androstenediol Dihydrotestosterone Metenolone Enanthate Mesterolone Testosterone CH O 3' Hydroxystanozolol HC CH OH 3 3 HO 3 OH CH H3C O 3 CH HO CH CH CH3 OH CH3 CH CH 3 3 3 CH 3 OH 3 CH3 CH H3C HC OH 3 CH3 CH 3 CH OH HO CH 3 3 H3C OH 3 OH CH3 H3C O F HO CH3 O Drostanolone Propionate 4 Androstenediol O O O O O HN OH 1 Testosterone O O Methyl 1 Testosterone Gestrinone Methandieone N Epitestosterone Formebolone 6b Hydroxyfluoxymesterone CH HC HO 3 O 3 OH CH3 OH CH3 O HC CH 3 CH CH 3 HC 3 CH3 3 OH CH3 HC 3 CH3 3 OH OH Prostanozol HC 3 O CH3 H3C CH HC 3 3 O O HO HO H3C CH3 OH 11b Hydroxyandrosterone CH3 CH3 Stenbolone 19 Norandrostenediol 19 Norandrostenedione OH HC HO O O 7 2 3 CH CH 1 O 3 Methandriol 3 OH Mestanolone HC HO CH 3 OH CH CH OH 3 OH Methasterone 3 3 Nandrolone Derivatives CH O Androsterone HC CH3 O CH H3C 3 3 OH 4 Hydroxytestosterone OH CH3 CH CH3 3 O O HC OH HO 3 H3C OH H3C 16a Hydroetiocholanolone O Mibolerone O O HO 1 Danazol 5a Androstanediol CH3 Nandrolone O O O OH 1= 3 Hydroxysteroid Dehydrogenase Oxandrolone Oxymesterone HO Oxabolone 2= 17b Hydroxysteroid Dehydrogenase CH O H3C OH O CH3 O 3 3= Aromatase CH3 O CH CH3 CH 4= 5a Reductase 3 3 OH 5= 17, 20 Lyase OH H3C Norbolethone 6= 3b Hydroxysteroid Dehydrogenase CH CH3 CH CH 3 CH 7= Liver Metabolism 3 3 3 OH OH Cl CH CH3 3 O O CH3 OH NH N HO O O CH3 Trenbolone O 5a Androstanedione HO CH3 CH3 Methandrostenolone CH 19 Norandrosterone HN 3 Ethylestrenol Information Compiled forE. WSLA. Guice, by M.S. Formestane HO 2 H3C N Cl Furazabol O Clenbuterol Oxymetholone 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 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 suppression • Eliminate the need for extensive sample clean- up: – 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 amounts 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 TOF vs Orbitrap • Single Ion Monitoring would • Both can use CID to provide require the separation of fragment information isobars through • Also use the full scan chromatography spectrum for retention time • QQQ would require the use verification of Ion Ratios to determine • Biggest difference here is of specific compounds dynamic range and (isobars) spatial/temporal association of the chromatographic data Which instrument meets the needs?

SIM (LC/MS) QQQ Q-TOF Orbitrap (LC/MS/MS) (LC/HR-TOF) (LC/HR-MS)

Resolution of structurally similar compounds Sensitivity for low limits of detection Dynamic range requirements met Able to do online Maybe extraction 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: – 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 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 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. • If qual-quan peaks are used, it is encouraged that both have the appropriate amount of scans Why human growth DETAILS OF CURRENT STUDY 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 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

• C20 H26 O2 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 : – Goal is to include a number of children/adolescents – Also want to recruit persons currently taking hGH 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 – 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 MS Jaslow (2013) CBS News What is deer antler spray:IGF-1? www..com