Use of Equine Hair as a New Matrix for Anti-Doping Control

Transcription

1 Use of Equine air as a New Matrix for Anti-Doping Control Scott D. Stanley, Ph.D. Professor University of California Davis School of Veterinary Medicine

2 Contents The Maddy Lab AQA zero tolerance policy toward Clenbuterol Principles of air Analysis air Segment Analysis

3 Maddy Equine Analytical Chemistry Lab Drug Detection Determination, Identification, Confirmation Pharmacokinetics T 1/2, AUC, Cl Metabolite ID Cl C 3 N C C 3 2 N C C C 3 Cl Clenbuterol m/z 240 Proteomics Erythropoietin, Growth ormone, Cobratoxin

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5 Clenbuterol

6 v1 Gallon = 1043 doses Compounded Clenbuterol vsuspension stated concentration for all products was 100 µg/ml

7 Compounded Clenbuterol v Clear Regular Strength v Red Double Strength

8 Incorporation of Drugs into air Each hair follicle has its own blood supply After the drug enters the body, it is absorbed into the blood stream As hair grows drugs are incorporated into the hair Drugs will remain in the hair almost permanently

9 Incorporation of Drugs into air

10 Incorporation of Drugs into air

11 Incorporation of Drugs into air

12 Growth Rates & istory of Use orse hair grows at approx. 2 cm per month (12 cm section = 6 months) Consecutive sections provide a retrospective history of drug usage Drug use history can be attained as long as the hair allows (i.e., 24 cm of hair = 1 year)

13 Sample Collection & Preparation Collection Mane or tail hair, cut from the proximal end (closest to the horse) air is assigned a barcode #, bagged and sealed Washing Removes external contaminates Extractions Chemical procedures isolates drugs from hair

14 air Collection

15 Chain of Custody air sample collection materials are part of the chain of custody Important that all relevant fields are completed i.e., hair color, medications, collector info The sample is tracked at every stage from collection to the issue of results The robustness of the chain of custody procedure is legally defensible in court

16 air Samples

17 air Sample Preparation

18 air Analysis Unique long window of detection May differentiate between regular administration versus single exposure Drugs remain locked in hair Simple & quick sample collection Not appropriate for recent admin. detection, time line in months not days

19 Segmental Analysis

20 Drug Groups 1 of 4 Anabolic Steroids Endogenous Androstenedione, DEA, Dihydrotestosterone, Nandrolone, Testosterone Exogenous Bolasterone, Boldenone, Boldione, Danazol, Drostanolone, Ethylestrenol, Furazbol, Methyltestosterone, Methylandrosterenediol, Methandienone, Norethandrone, xandrolone, Stanozolol, Tetrahydrogestrinone (TG), Trenbolone

21 Drug Groups 2 of 4 Corticosteroids (steroid hormones) Exogenous Becomethasone, Betamethasone, Budesonide, Dexamethasone, Flumethasone, Fluticasone, Isofluperdone, Methylprednisolone, Prednisolone, Triamcinolone Endogenous - (ydrocortisone)

22 Drug Groups 3 of 4 β-2 Agonist - Bambuterol, Clenbuterol, Fenoterol, Ractopamine, Xamoterol, Zeranol, Zilpaterol Selective Androgen Receptor Modulators (SARMs) Andarine (S-4), starine (S-22)

23 200 mg of pulverized hair UPLC MS/MS LC-RMS GC-MS/MS Steroid Esters Basic Drugs Acid Drugs Anabolic Steroid Steroid

24 Acknowledgements orse Industry Support: CRB, Los Alamitos Race Track, NMRC, The Jockey Club, Breeder s Cup, Keeneland Association, AQA, TC, CTT, PCQA, RMTC, and ARCI University of California - Davis Dr. eather Knych

25 Questions! AAEP, San Antonio 2011

26 AAS Impact on Performance and ealth Efficacy: AAS administration in the horse for therapeutic treatment of conditions is limited. Performance: Used to promote muscle growth ealth effects: Effects on reproduction have been studied with a reversible suppression of reproductive function.

27 AAS Impact on Steroid 1. Steroidogenesis is tightly regulated 2. The impact of AAS usage on altering steroid profiles in the horse is incomplete and inconclusive: Roser, J (2008) a) Exogenous treatments of decreased testosterone

28 Typical Anabolic-Androgenic Steroids Derived from Testosterone 1,2-oxidation 2-methylation 1-methylation desmethylation methylation Testosterone 7-methylation 4,5-reduction 4-chlorination 27

29 Steroidogenesis S Bold P4 17P4 A4 P5 17P5 DEA Test E1 βe2 6αA4 E2S NS S S BS S TS NG TG S DEAS 19dione ADD Nan αe2 ENan 19EA 19A 5αDN ETest 5αE2 5αDT 5βDT Adiol Ediol 5βDP AP Pdiol

30 Prohibited Substances Anabolic steroids 16β-ydroxystanozolol 1-Androstenedione 4-Estrene-3,17-dione Altrenogest Androstadienone Bolasterone Boldenone Boldione Calusterone Clostebol acetate Danazol Deoxycortone Dimethisterone Ethyltestosterone Gestrinone ydroxytestosterone Medroxyprogesterone acetate Methyltestosterone Nandrolone Norbolethone Norethandrolone xyguno Propyltrenbolone Stanozolol Stenbolone Testosterone Testosterone propionate Trenbolone Turinabol α- & β-agonists Albuterol Bambuterol Brombuterol Bromchlorbuterol Cimaterol Cimbuterol Clenbuterol Clencyclohexerol Clenhexerol Clenisopenterol Clenpenterol Clenproperol Denopamine Dobutamine Etafedrine Etilefrine Fenoterol Formoterol Guanabenz Guanfacine ydroxydetomidine Mabuterol Mapenterol Methoxamine Methoxyphenamine Naphazoline Phenylpropanolamine Procaterol Pseudoephedrine Ractopamine Romifidine Salmeterol Terbutaline Tuaminoheptane Tulobuterol Xamoterol Xylazine Xylometazoline Analgesic/Anesthetic 4-Methylaminophenazone Anileridine Buprenorphine Butorphanol Cocaine Demorphin Dihydrocapsaicin Etorphine Ketamine Lidocaine Mepivacaine Methadone Midazolam Nalbuphine N-Norpropoxyphene Nonivamide Noroxymorphone -Desmethyltramadol xycodone xymorphone Paracetamol Procaine Thebaine Tramadol Zolazepam

31 Plasma Concentration Curve Plasma or tissue drug level Time for drug effect to begin Intensity of drug effect Minimum Therapeutic Drug Level Time (t)

32 Metabolism of AAS Phase I P450 mediated ydroxylation De-methylation 5α-Reducatase and 5β-Reductase mediated ydroxy Steroid Dehydrogenase mediated 3 and 17 hydroxy positions Phase II Glucuronidation Sulfation

33 Urine Analysis Good for detecting drugs over 1-45 days Difficult to collect, time consuming and requires multiple steps for sample preparation Requires refrigeration for transportation and storage Minimal withdrawal can be employed to avoid most detection

34 bjectives Explain the principles of equine hair testing Provide an overview of compounded clenbuterol ighlight the benefits of hair analysis over urine and plasma analysis Provide an overview of drug groups and their metabolites Describe hair testing limitations

35 Drug Groups 4 of 4 Peptide ormones and Growth Factors Growth ormone, Growth ormone Releasing Factor-2, GRF-6, Insulin-Like Growth Factor-1, Fibroblast Growth Factor, Machano Growth Factor, Vascular Endothelial Growth Factor

36 Q Exactive MS Benchtop rbitrap MS igh resolution (140,0000 at m/z 200) igh Mass Accuracy (<5 ppm, external) Full scan MS igh Sensitivity Advanced LC-MS Proteomics Metabolomics Small Molecule and Structure Elucidation Ultra-trace Level Analysis Sub-fmol Sensitivity

37 The rbitrap The axial oscillation frequency follows the formula Where w = oscillation frequency k = instrumental constant m/z = mass/charge ratio w = k m / z m/z

38 MS method: basic compounds Data acquisition mode: full scan in m/z range Resolution: 70 K

39 Autosampler method: Basic compounds Injection volume: 40 ul

40 Stanozolol: LQ =10 pg/ml

41 16--Stanozolol: LQ = 10 pg/ml

42 Theoretical Experimental Name Formula Mass (M+) Mass Mass Error (ppm) 19-norandrostendione C b-hydroxytestosterone C Bolasterone C Boldenone C Boldenone-16 C Clostebol C Cl Danazol C N Dianabol C Epi-testosterone C Fluoxymesterone C F Gestrinone C Methyltestosterone C Nandrolone C xandrolone C Stanozolol C N Stanozolol-16 C N Testosterone C Tetrahydrogestrinone (TG) C Trenbolone C Turinabol C Cl

43 Plasma sample ~ 18 pg/ml Blank Plasma sample Spiked sample 25 pg/ml Stanozolol Administration

44 Stanozolol air + 4 weeks

45 Stanozolol Urine + 4 weeks

46 Method Parameters MS: Thermo TSQ Vantage Triple Quadrupole Ionization: eated Electrospray Ionization (ESI) ESI Temp: 200 C Scan Mode: SRM Q1 0.1 m/z at FWM Q3 0.7 m/z at FWM Scan Width: 0.01 m/z Scan Time: 50 millisecond

47 Selected Reaction Monitoring Fixed m/z Pass All Fixed m/z l l Q1 Q2 Q3 0.7 m/z for SRM 0.1 m/z for SRM l l 0.7 m/z for SRM 0.7 m/z for SRM

48 Mass Spec Method Compound Segment / ESI Mode Formula T R S-Lens Precursor Ion Product Ions Collision Energy Relative Abundance 6aA4 1/+ C , 209.1, , 20, , 90, 75 E2S 1/- C S , 80.0, , 40, , 2, 5 NS 1/- C S , 80.2, , 72, , 18, 1 BS 1/- C S , 95.9, , 52, , 52, 47 NG 1,2/+ C , 109.0, , 33, , 70, 90 TS 1,2/- C S , 80.1, , 80, , 10, 1 TS-d3 1,2/- C D 3 5 S ADD 2/+ C , 77.0, , 49, , 25, 31 TG 2/+ C , 97.1, 109.0, , 34, 34, , 25, 25, 26 DEAS 2/- C S , 80.1, , 80, , 15, 1 19dione 2/+ C , 109.1, , 25, 16 25, 85, 100 Bold-d3 2,3/+ C 19 D Bold 2,3/+ C , 91.1, , 43, , 30, 38 A4-d7 2,3/+ C D A4 2,3/+ C , 109.1, , 21, , 70, 22 Nan 3/+ C , 145.1, , 21, , 50, 62 E1 3/+ C , 157.0, , 19, 21 82, 100, 87 βe2 3/+ C , 133.1, , 18, , 30, 17 αe2 3/+ C , 133.1, , 18, , 30, 17 Test-d3 3/+ C D Test 3/+ C , 109.0, 79.1, , 27, 43, , 90, 28, 16 ENan 3/+ C , 145.1, , 21, , 70, 60 17P4 3/+ C , , 28, , 85, 41 19EA 4/+ C , 145.1, , 18, , 38, 20 DEA 4/+ C , 197.1, , 19, 11 44, 26, P5 4/+ C αDN 4/+ C , 91.1, , 43, , 28, 8 ETest 4/+ C , 109.0, 79.1, , 27, 43, , 92, 34, 18 5αE2 4/+ C , 91.1, , 38, , 65, 38 5αDT 4/+ C , 273.2, , 7, , 18, 24 19A 4,5/+ C , 145.1, , 18, , 34, 22 5βDT 4,5/+ C , 273.2, , 7, , 54, 20 P4 5/+ C , 109.1, , 26, , 95, 28 Adiol 5/+ C , 91.1, , 40, , 78, 94 Ediol 5/+ C , 91.1, , 43, , 78, 94 P5 6/+ C , 159.1, , 23, , 28, 24 5αDP 6/+ C , 105.1, , 36, , 22, 24 AP 6/+ C , 91.1, , 43, , 92, 92 Pdiol 6/+ C , 203.1, , 15, , 90, 80

49 air Analysis Limitations Not currently possible to determine drug purity, dose used, or frequency of administration Unable to determine the route of administration Cannot pinpoint administration time such as to exactly what day

50 Conclusions BA has Zero Tolerance for anabolic steroids for North American horses imported into England Anabolic steroid abuse is still a significant concern Routine LC-MS/MS has significantly improved ease of detection Routine screening in the low part per trillion air analysis offers potential for long term detection of many historic drugs of abuse Segmental analysis may provide a time line for drug administration

51 Anabolic Androgenic Steroid: Stanozolol 3' N N C 3 Many modifications possible! Details on fragmentation of paramount interest

52 ESI-MS/MS Spectrum of 100 Stanozolol ' N N C 3 16 Relative Abundance m/z

53 ESI-MS/MS Spectrum of Stanozolol (LTQ-rbitrap) Relative Abundance C 5 7 N ppm C ppm m/z C N ppm

54 ESI-MS/MS Spectrum of 16-hydroxystanozolol C 3 Relative Abundance N N m/z

55 Relative Abundance ESI-MS/MS Spectrum of Stanozolol MS C 5 7 N ppm C ppm m/z C N ppm

56 ESI-MS 3 Data of Fragments derived from m/z 203 Fragment (m/z) Precursor m/z 329 Precursors m/z measured mass (u) elemental composition error (ppm) Fragment (m/z) measured mass (u) elemental composition error (ppm) C 4 5 N C 4 5 N C 5 7 N C 5 7 N C C 8 9 N C 8 9 N C C 8 11 N C 8 11 N C C 9 11 N C 9 11 N C C N C N 2-1.5

57 Dexamethasone: LQ = 5 ng/ml

58 Dexamethasone: LQ= 5 pg/ml Acid_2nd_C4 - m/z= SM: 5 RT: NL: 8.62E5 F: FTMS + p ESI Full ms [ ] 100 RT: Time (min)

59 Screening for Abuse Sample Load: undreds Time Frame: 1-2 days Requirements: Sensitive Quantitative Selective igh Throughput Minimal Sample Preparation

60 Androgen Production in the orse Primary circulating (serum/plasma) androgens are: 0-1 year old Colt: pg/ml a,e Filly: Testosterone >3 years old 0 40 pg/ml a References: a Lemazurier et al (2001) b Soma et al (2008) c Gastal et al (2007) d Silberzahn et al (1989) e Brown-Douglas et al (2004) Androstenedione 0-1 year old Colt: pg/ml a Filly: pg/ml a >3 years old Male: pg/ml d

61 Endogenous Production of AAS Boldenone Plasma/Serum Stallion: none reported a Mare: none reported a Soma et al (2008) b Sterk et al (1989) c Grace et al (2008) Nandrolone Plasma/Serum Stallion: pg/ml a Mare: 0 -?? pregnancy b

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63 Steroids Monitored: LD and LQ Analyte LD pg/ml LQ pg/ml Analyte LD pg/ml LQ pg/ml 6α-Androstenedione 500 2,500 Epi-Nandrolone Boldenone Sulfate Progesterone 500 1,250 Nandrolone Sulfate Norepiandrosterone 500 1,250 17β-Estradiol Sulfate Dehydroepiandrosterone 1,250 2,500 Nandrolone Glucuronide 750 2, Pregnenolone 2,500 5,000 Testosterone Sulfate α-Dihydronandrolone 750 1,250 1,4-Androstadien-3,17-one Epi-Testosterone Testosterone Glucuronide α-Estran-3b,17α-diol 750 1, Norandrostendione 250 1,250 5α-Dihydrotestosterone 500 1,250 Dehydroepiandrosterone Sulfate Norandrosterone 500 1,250 Boldenone β-Dihydrotestosterone 250 1,250 Androstenedione Progesterone Nandrolone α-Androstane-3α,17β-diol 2,500 10,000 Estrone Etiocholan-3α,17β-diol 10,000 25,000 17β-estradiol 2,500 5,000 Pregnenolone 1,000 2,500 17α-Estradiol 1,000 5,000 5α-Dihydroprogesterone 2,500 5,000 Testosterone Allopregnanolone 2,500 5,000 Pregnanediol 5,000 10,000

64 Background Performance horses have the longest established, most elaborate broad-based, and technically accurate systems for drug detection of any competitive sport.

65 Blood Flow

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67 Mechanism of Action SP SP A R A R GPCR A R A R Down Stream Signaling A R A R

68 Implications of Route of Administration Brain D Lungs * G Routes of Administration A C Arterial Flow eart Liver * Intestines * Gut lumen Kidneys B A. Intravenous B. ral C. Intraperitoneal D. Inhalation E. Intramuscular F. Subcutaneous G. Intraarterial Venous Flow * First- Pass rgans Site of Sampling Elimination (Metabolism Excretion) Muscle Fat Skin E F

69 Absorption, Distribution, Excretion of Drugs/Toxicants Ingestion Inhalation Intravenous Intraperitoneal Gastrointestinal Tract Liver Bile Lung Portal Blood Blood and Lymph Subcutanous Intramuscular Dermal Target Site Extracellular Fluid Fat Kidney Lung Secretory Structure rgans Bladder Alveoli Soft Tissue Bone Feces Urine Expired Air Secretions

70 Potential of Segmental Analysis Distal (oldest growth ) segment contained highest concentrations Proximal (newest growth) segment contained lowest concentrations

71 Program Elements Analytical Chemist and a Veterinary Pharmacologist Scientific and Industry Advisory Boards to assist in program development and direction Able to draw on expertise of faculty in numerous fields at UC-Davis

72 The Sungate Saga

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74 Frontpage News Testing In Training visit 45 plasma and hair samples collected 4 positive for Stanozolol Matched by positives in corresponding hair samples

75 Frontpage News Second inspection 3 weeks after initial visit 203 samples, only plasma collected 11 positive for Stanozolol 3 of 4 original horse still positive Concentrations dropped to ~10% of initial findings air samples would likely have given more information

76 BA announced a new zero tolerance policy regarding anabolic steroids starting in 2015 The headline elements of the policy: A horse must not be administered an anabolic steroid at any point in its life Any horse administered an anabolic steroid will face a mandatory stand down period from training for 12 months and ineligible to start in any race in Britain for 14 months. All horses must be available for testing at any time, regardless of physical location and whose care the horse is under, from the time it is first registered with Weatherbys

77 BA announced a new zero tolerance policy regarding anabolic steroids starting in 2015 All GB bred horses must be registered with Weatherbys within 12 months of birth, phased to six months in two years. Permanently imported horses must be registered with Weatherbys within three months of arrival in Britain accompanied by a sample that shows no evidence of anabolic steroid administration. Due to their mirror policies, horses imported from Ireland, France and Germany which have spent 12 months under their equivalent policies will be exempt from this requirement. Likewise, runners from Ireland, France and Germany will be treated as British runners and sampled as per the standard testing policy. All other foreign runners must be in Britain (and the BA notified of their whereabouts) a minimum of 14 days in advance of their intended race to facilitate post-arrival sampling and analysis, the results of which will be received prior to the horse running.

78 The Sungate Ò Saga Testing In Training visit 28 plasma samples collected 9 positive for stanozolol picograms per ml Trainer reported use of Sungate Ò (Stanozolol, 5 mg/ml) in medication records Recommended by Veterinarian

79 Factors Affecting Drug Incorporation into air Melanin content - The most common form of biological melanin in hair is eumelanin. Lipophilicity - Refers to the ability of a drug compound to dissolve in fats, oils, and lipids. Alkalinity - The quantitative capacity of an aqueous solution to neutralize an acid.

80 orse Racing Industry Integrity Industry Reputation $917.5 million in gross sales 7,161 yearlings ($430 million) igh Profile Events Challenge Championship Breeder s Cup Purses = $28 M orse s Reputation Sept $2.2 million Nov $3.9 million

81 Clenbuterol 10x

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