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(Clenbuterol) Contaminations

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Hair Testing …to provide additional evidence in doping cases of potential (clenbuterol) contaminations Detlef Thieme Institute of Doping Analysis, Dresden, Germany Beijing, Oct 2017 [M. Jahn (www.kreischa.net)] 0. Principles of Hair Testing 1. Cases: Long-Term Detection of Drugs 2. Clenbuterol: Field Study in Mexico 3. Clenbuterol WADA Project: Administration Study 4. Clenbuterol: Real Doping Cases 0. Principles of Hair Incorporation Decomposition External UV-light Wash Contamination (Steroids) Out (Cocaine) (Cortisol) pH~5 Incorporation Melanin Binding Incorporation from (Clenbuterol) from Bloodstream sweat and sebum pH=7.4 Neutral and acidic drugs Basic drugs (Steroids) (Amfetamines) 0. Segmental Analysis Positive Segment 6 cm Segment 2 3 cm Segment 1 (proximal) Time Post 1 Week 1 Months 6 Months Administration: 4 0. Clenbuterol-Application – 4 Horses Dose: 2x 0,8µg/kg KG/d, oral (10 Days) Hair collected 4 months post administration 25 BlackSchw arz1 1 BlackSchw arz2 2 20 BlackSchw arz3 3 BrownBraun 1 15 10 Konz. [pg/mg] 5 0 2 4 6 8 10 12 14 Length [cm]Haarlänge ~ Time [ months[cm] ] Individual Growth Rate Hair Color 1. Amitriptyline Case (~40 children) 120 100 80 N 60 40 hair conc. conc. hair [pg/cm] 20 0 0 5 10 15 20 Distance from root [cm] 1. Single administration of Mifegyne® • Progesterone receptor antagonist • Used for early terminating of pregnancy 1. Single administration of Mifegyne® http://www.womenonwaves.org/en/page/702/how-to-do-an- abortion-with-pills--misoprostol--cytotec HOW TO GET MISOPROSTOL In some countries women can get Misoprostol at their local pharmacies and use it alone. … Sometimes it is sold over the counter without a prescription, or sometimes a prescription is required … A woman can of course also make her own prescription (see example) 1. Single administration of Mifegyne® Single Administration of 200mg Mifepristone Could be Detected in Hairs of Two Victims XIC of +MRM (Mifepristone 10pg/mg) 5.73 1000 or LC-MS/HRMS 800 Intensity XIC of +TOF Product (430.2): 372.203 to 372.253 Da from Sample 1 (14h048/1) 5.67 600 , cps 100 400 80 200 0 60 5.0 5.5 6.0 6.5 7.0 Time, min by LC-MS/MS 40 20 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Time, min 96 Hair Tests in Bodybuilding Compound Hair Conc (pg/mg) Urine 29 y.o. Clenbuterol 2 n.d. Male Boldenone 3 n.d. bodybuilder Boldenone Undecylenate 400 bleached Drostanolone 4 Drostanolone hair 0-3 cm Drostanolone Enantate 30 Metandienone 220 Metandienone-Mb ~160 Nandrolone doping agents Phenylpropionate 145 Norandrosterone confiscated Stanozolol 43 OH-Stano Testosterone Enantate 500 Trenbolone 15 Epi-Trenbolone Trenbolone Acetate 10 Trenbolone Enantate 270 Tamoxifen 150 n.d. Anastrozol 85 Anastrozol Testosterone 500 T/E Fluoxymesterone n.d. Mb. Findings in Hairs of Presumptive Steroid Users No of cases Anabolic Steroid 2500 96 Hair Samples Confiscations Testosterone Enantate 29 561 Metandienone 24 206 Testosterone Propionate 24 558 Clenbuterol 8 59 Trenbolone Acetate 8 174 Stanozolol 7 116 Trenbolone Enantate 7 194 Drostanolone Propionate 4 35 Boldenone Undecylenate 3 125 Nandrolone Decanoate 3 134 Testosterone Decanoate 3 64 Testosterone Phenylpropionate 3 57 Drostanolone Enantate 2 45 Nandrolone Phenylpropionate 1 36 Testosterone Cypionate 1 81 Correlation between Urine and Hair (96 Cases) Hair (214 findings) pos neg Urine pos 56.5 % 27.1 % neg 16.4 % (9 cases) 2. Field Study Mexico (FIFA) Samples Team- positive findings Average conc. Max collected Classification (%) [pg/mg] [pg/mg] [n] U20 19 18 (94.7) 0.18 1.29 U17 24 20 (83.3) 0.37 1.79 local 20 18 (90.0) 0.49 1.90 2. Clenbuterol Hair Concentrations 26 25 20 16 15 10 10 number of findings 7 5 3 1 0 hair clenbuterol [pg/mg] 2. Field Study Mexico Clenbuterol Profiles long-term contamination! 3. Administration Study (WADA) ADI (JECFA, 1998) 0.004 µg/kg/d Admin Study (Average) 0.02 µg/kg/d Therapy 0.4 µg/kg/d Abuse (?) 1-2 µg/kg/d 3. Dosage vs. Serum Level Therapeutic~300-600pg/mL 100 90 80 70 60 50 serum Clenbuterol [pg/ml] 40 30 20 10 0 000 000 000 000 000 000 000 000 000 000 000 applied Clenbuterol µg/kg body weight 3. Clenbuterol Hair Concentrations 4.0 3.0 2.0 Hair Hair Clenbuterol [pg/mg] 1.0 .0 study person 4. Clenbuterol Cases (before 2012, LOD=0.9 pg/mg) Code Discipline Urine Hair Hair Concentration 12D002 Cycling 02.09.11 04.01.12 0-2,5cm: neg 50pg/ml Head Hair (dark brown); 2,5-5cm: neg 5-7,5cm: neg 10D171 Table Tennis ? 06.10.10 0-2cm: neg Head Hair (brown); 2-5cm: neg 10D151 Table Tennis ? 24.09.10 0-2cm: neg Head Hair (dark blond); 2-4cm: neg 4. Clenbuterol Cases (after 2012, LOD=0.02 pg/mg) Code Discipline Urine Hair Hair Concentration 14h082 Cycling ? 01.12.14 0-1,5cm: neg Head Hair (dark brown); 1,5-4cm: neg 14h026 (FIFA, ? 22.04.14 0-2cm: trace Guatemala) Head Hair (dark brown); 14h027 (FIFA, ? 22.04.14 0-3cm: 0,5pg/mg Guatemala) Head Hair (dark brown); 13h0039 (FIFA, ? 29.03.13 0-0,5cm: neg Guatemala) Head Hair (black); 13h0040 (FIFA, ? 29.03.13 0-3cm: neg Guatemala) Head Hair (dark brown); 13h0041 (FIFA, ? 29.03.13 0-5cm: neg Guatemala) Head Hair (dark brown); 13h0042 (FIFA, ? 29.03.13 0-3cm: 0,03pg/mg Guatemala) Head Hair (dark brown); 13h0043 (FIFA, ? 29.03.13 0-3cm: 0,06pg/mg; Guatemala) Head Hair (black); 3-6cm: 0,06pg/mg; 6-9cm: 0,08pg/mg 4 Cases Track Cycling 1. Female Urine (OOC, 11.12.13): Clenbuterol 0,02 ng/ml Hair (22.01.14): Head Hair, brown 0-2 cm / 2-4 cm / 4-6 cm all segments negative 2. Male Urine (OOC, 12.12.13): Clenbuterol 0,08 ng/ml Hair (22.01.14) Head-hair, dark brown 0-2 cm: 0,08 pg/mg Clenbuterol (November 2013 – January 2014) 2-4 cm: negative 3. Male Urine (23.01.14): Clenbuterol 0,13 ng/ml Hair (17.03.14): Pubic Hair, dark blond 0-2 cm: negative 4. Discrimination Power of Hair Testing for Clenbuterol 30 ADMIN~5*ADI 25 20 15 10 Abuse Cases: Lit: 15-122 pg/mg 5 IDAS: 10-90 pg/mg 0 0.09 0.5 1 2 5 10 20 50 100 4. Discrimination Power of Hair Testing for Clenbuterol 30 MEX_FIELD 25 ADMIN~5*ADI 20 15 10 Abuse Cases: Lit: 15-122 pg/mg 5 IDAS: 10-90 pg/mg 0 0.09 0.5 1 2 5 10 20 50 100 4. Discrimination Power of Hair Testing for Clenbuterol 30 MEX_FIELD ADMIN~5*ADI 25 CASES 20 15 10 Abuse Cases: Lit: 15-122 pg/mg 5 IDAS: 10-90 pg/mg 0 0.09 0.5 1 2 5 10 20 50 100 4. Clenbuterol Cases Code Discipline Urine Hair Hair Concentration 15h001 Weightlifting 13.11.14 10.01.15 0-2cm: 20pg/mg; (WM Women) 38 pg/ml Head Hair (brown, 2-4cm: 18pg/mg colorized); 15h002 Weightlifting 10.11.14 10.01.15 0-2cm: 23pg/mg; 50 pg/ml Head Hair (dark brown); 2-4cm: 18pg/mg 15h003 Weightlifting 09.11.14 10.01.15 0-2cm: 86pg/mg; 100 pg/ml Head Hair (black); 2-4cm: 81pg/mg Hair Testing potentially suitable to confirm Clenbuterol Doping at low urinary concentrations!!! Case report: Possession of… • Testosterone-enantate, Testosteronepropionate, Testosteronecypionate, Testosteroneisocaproate, Testosteronephenylpropionate, Testosteronedecanoate • Trenboloneacetate, Trenbolone-enantate, Trenbolonehexahydrobenzoate • Metenolone-enantate, • Nandrolonedecanoate, • Boldenoneundecylenate, • Oxandrolone, • Metandienone, • Oxymetholone, • Fluoxymesterone, • Methyltestosterone • Stanozolol • Tamoxifen • Clenbuterol • Somatropin, GH-Fragment 176-191, mod- GRF Urine Test • Elevated TE (microbial degradation) • LH < 0,1 mIU/ml; • Norandrosterone, Noretiocholanolone (high) • Drostanolone (metabolite,low) Hair Test • Boldenone (6 pg/mg), • Clenbuterol (6 pg/mg) • Metandienone (9 pg/mg), • Nandrolone (7 pg/mg), • Testosteron-Propionate (7 pg/mg) • Testosterone 6 pg/mg > normal Summary • Hair Testing lacks sensitivity but gains retrospection. Follow up possible weeks after urine test (if hair is available) • (Dark) hair concentration of clenbuterol are comparatively well dose related. • High Discrimination Power between Low (5*ADI) and Hi (Abuse) cases. • A proposed threshold of 1…5 pg/mg clenbuterol in hair could contribute to discriminate most of the pending clenbuterol cases. • Increasing reporting levels of clenbuterol would cause potential disregarding real (even excessive) cases. • Estimated Clenbuterol Contamination Dosages in high risk areas (Mexico) correspond to administrations in the order of magnitude of 2 * ADI. Acknowledgements • Administration Study was funded by WADA • Field Study funded by FIFA (logistically supported by Mexican Football Federation) • Administration study was accomplished by Lena Gfrerer at the Institute of Legal Medicine (Munich University) • Analytical work was carried out by Aniko Krumbholz and Patricia Anielski (IDAS Dresden). .
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  • Influence of the Anabolic-Androgenic Steroid Nandrolone on Cannabinoid

    Influence of the Anabolic-Androgenic Steroid Nandrolone on Cannabinoid

    Neuropharmacology 50 (2006) 788e806 www.elsevier.com/locate/neuropharm Influence of the anabolic-androgenic steroid nandrolone on cannabinoid dependence Evelyne Ce´le´rier a, Therese Ahdepil b, Helena Wikander b, Fernando Berrendero a, Fred Nyberg b, Rafael Maldonado a,* a Laboratori of Neurofarmacologia, Facultat de Cie´ncies de la Salut i de la Vida, Universitat Pompeu Fabra, C/Doctor Aiguader 80, 08003 Barcelona, Spain b Department of Pharmaceutical Biosciences, University of Uppsala, Box 591 Biomedicum, S751 24 Uppsala, Sweden Received 8 April 2005; received in revised form 29 November 2005; accepted 29 November 2005 Abstract The identification of the possible factors that might enhance the risk of developing drug addiction and related motivational disorders is crucial to reduce the prevalence of these problems. Here, we examined in mice whether the exposure to the anabolic-androgenic steroid nandrolone would affect the pharmacological and motivational effects induced by D9-tetrahydrocannabinol (THC), the principal psychoactive component of Cannabis sativa. Mice received nandrolone using pre-exposure (during 14 days before THC treatment) or co-administration (1 h before each THC injection) procedures. Both nandrolone treatments did not modify the acute antinociceptive, hypothermic and hypolocomotor effects of THC or the development of tolerance after chronic THC administration. Nandrolone pre-exposure blocked THC- and food-induced condi- tioned place preference and increased the somatic manifestations of THC withdrawal precipitated by the CB1 cannabinoid antagonist rimona- bant (SR141617A). The aversive effects of THC were not changed by nandrolone. Furthermore, nandrolone pre-exposure attenuated the anxiolytic-like effects of a low dose of THC without altering the anxiogenic-like effects of a high dose in the lit/dark box, open field and elevated plus-maze.
  • Ameliorative Effects of Lycopene Against Boldenone Undecylenate Toxicity in Albino Rats

    Ameliorative Effects of Lycopene Against Boldenone Undecylenate Toxicity in Albino Rats

    BENHA VETERINARY MEDICAL JOURNAL, VOL. 35, NO. 2: 237-249, DECEMBER , 2018 BENHA UNIVERSITY FACULTY OF VETERINARY MEDICINE Ameliorative Effects of Lycopene against Boldenone Undecylenate Toxicity in Albino Rats Sabra H.M., El-Shawarby R.M., Abosalem M., Nabila M. A., Ibrahim S.S. Department of Forensic Medicine & Toxicology. Faculty of Veterinary. Medicine. Benha University. A B S T R A C T Although anabolic androgenic steroids like boldenone (BOL) may increase lean muscle mass, strength, and the ability to train longer and harder, the serious side effects of steroid are many and may not be reversible. So, the present study was designed to investigate the role of lycopene in improving the oxidative stress of boldenone in male albino rats. Twenty eight male adult rats were equally divided into four groups.1st group (control) that received olive oil once a week (5 mg/kg intramuscularly). 2nd group includes rats received lycopene daily (orally, 60mg/kg body weight). 3rd group includes animals that received intramuscular injections of boldenone once a week (5 mg/Kg body weight). 4th group was co-treated with boldenone and lycopene with the same previous doses of both, the experiment extended for 5 weeks. Intramuscular injection of rats with caused significant increase in total weight gain (TWG), serum total protein and globulin, malandialdehyde (MDA), Lactate dehydrogenase (LDH), creatinine kinase (CK), total protein in skeletal muscle, when compared with the control group. On the other hand, a significant decrease in albumin, Albumin/Globulin (A\G) ratio and superoxide dismutase (SOD) in boldenone group when compared with the control group.