Il controllo antidoping da Atene 2004 a Torino 2006

Transcription

1 !" # $% Il controllo antidoping da Atene 2004 a Torino 2006 Francesco Botrè Laboratorio Antidoping, FMSI, Rome, Italy

2

3

4 LOCATION

5

6

7 Prohibited classes and methods (The last WADA list, effective Jan 1st 2005) Prohibited classes of substances Stimulants, narcotics, anabolic agents (anabolic androgenic steroids & beta-2-agonists), diuretics, peptide hormones, mimetics and analogues, agents with anti-oestrogenic activity, masking agents, glucocorticoids, cannabinoids Prohibited methods Enhancement of oxygen transfer, pharmacological, chemical and physical manipulation, gene doping Classes of prohibited substances in certain sports Alcohol, beta-blockers

8 Law n. 376/2000 ( Legge antidoping, ) I farmaci ( ) sono ripartiti in classi ( ) approvate con decreto del Ministero della Sanità, d intesa con il Ministero per i Beni e le Attività Culturali, su proposta della Commissione. ( )

9 Analytical laboratory Clinical chemistry: Matrices: any suitable biological material Aim: diagnostic tests test report based on: detection and quantitation of specific markers multiparametric profiles doubtful=positive (further investigation) Doping control analysis: Matrices: only urine for officially recognized tests Aim: to supply evidence test report based on: unambiguous identification of specific drugs/metabolites doubtful=negative (no further investigation)

10 The 33 ISO-IOC-WADA laboratories Africa: South Africa (Bloemfontein), Tunisia (Tunis) Americas: Brazil (Rio de Janeiro), Canada (Montreal), Colombia (Bogota), Cuba (La Habana), United States (Los Angeles) Asia: China (Beijing), S. Corea (Seoul), Japan (Tokyo), Malaysia (Penang), Thailand (Bangkok) Europa: Austria (Seibersdorf), Belgium (Ghent), Czech Republic (Prague), Finland (Helsinki), France (Paris), Germany (Cologne, Kreischa), Greece (Athens), Italy (Rome), Norway (Oslo), Poland (Sofia), Portugal (Lisbon), Russian Federation (Moscow), Spain (Barcelona, Madrid), Sweden (Stockholm), Switzerland (Lausanne), Turkey (Ankara), United Kingdom (Cambridge, London) Oceania: Australia (Sydney)\

11 Doping control laboratories must obtain and maintain accreditation ISO accreditation IOC-WADA accreditation ISO-IOC-WADA harmonization compliance with IOC-WADA ethical code

12 WAADS World Association of Anti-Doping Scientists Formed in 2001 All IOC accredited laboratories represented

13 SAMPLE RECEIPT - ALLOCATION REGISTRATION ( internal identification code) transport SAMPLE COLLECTION PROCEED ONLY IF CRITERIA FOR ACCEPTANCE ARE MATCHED, OTHERWISE INFORM THE RELEVANT AUTHORITIES (IOC-MC, WADA) { OPENING OF TRANSPORT BAGS AND OF EXTERNAL SEALS VISUAL INSPECTION (col, V, sediments ) / BOTTLE OPENING, ALIQUOTING, BATCHING NEW ALIQUOTING - SAMPLE PRETREATMENT SCREENING ANALYSIS SUSPECT SAMPLE SPECIFIC PRETREATMENT CONFIRMATION ANALYSIS NO CRITERIA FOR REVIEWING OF RESULTS CONFIRMATION MATCHED? YES NEGATIVE INTERNAL IDENTIFICATION OF SAMPLE (INTERNAL/EXTERNAL CODE) TEST REPORT

14 Updates/upgrades of the last four years (IOC-MC) (IOC-MC - WADA) (WADA) (WADA)

15 2002 (IOC MC) Anabolic Agents: Anabolic Androgenic Steroids (AAS): exogenous boldenone, clostebol, danazol, dehydrochlormethyltestosterone, drostanolone, fluoxymesterone, formebolone, mesterolone, metandienone, metenolone, methyltestosterone, nandrolone, norethandrolone, oxymesterone, oxandrolone, oxymetholone, stanazolol, trenbolone Anabolic Agents: Anabolic Androgenic Steroids (AAS): endogenous dihydrotestosterone, epitestosterone, testosterone Other Anabolic Agents clenbuterol Peptide hormones choriogonadotropin (hcg) Beta-2-agonists fenoterol, salbutamol, terbutaline Diuretics & Masking agents acetazolamide, bendroflumethiazide, bumetanide, chlortalidone, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, spironolactone, triamterene, canrenone Stimulants amfepramone, amphetamine, bromantan, caffeine, carphedon, cathine, cocaine, cropropamide, crotethamide, ephedrine, etamivan, etilefrine, fencamfamin, fenetylline, fenfluramine, heptaminol, mefenorex, mesocarb, methamphetamine, methoxyphenamine, MDA, methylephedrine, methylphenidate, nikethamide, pemoline, phendimetrazine, phentermine, pholedrine, phenylephrine, pipradol, prolintane, pseudoephedrine, selegiline, strychnine Narcotics + Cannabinoids buprenorphine, methadone, morphine, pentazocine, pethidine, THC Beta-blockers acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol

16 2003 (IOC - WADA) Anabolic Agents: Anabolic Androgenic Steroids (AAS): exogenous bolasterone, boldenone, clostebol, danazol, dehydrochlormethyltestosterone, drostanolone, fluoxymesterone, formebolone, mesterolone, metandienone, metenolone, methyltestosterone, mibolerone, nandrolone, norethandrolone, oxymesterone, oxandrolone, oxymetholone, stanazolol, trenbolone Anabolic Agents: Anabolic Androgenic Steroids (AAS): endogenous dihydrotestosterone, epitestosterone, testosterone Other Anabolic Agents clenbuterol Peptide hormones choriogonadotropin (hcg) Beta-2-agonists bambuterol, fenoterol, salbutamol, terbutaline Diuretics & Masking agents acetazolamide, amiloride, bendroflumethiazide, bumetanide, chlortalidone, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, spironolactone, triamterene, canrenone, HES Stimulants amfepramone, amphetamine, bromantan, caffeine, carphedon, cathine, clobenzorex, cocaine, cropropamide, crotethamide, ephedrine, etamivan, etilefrine, fencamfamin, fenetylline, fenfluramine, fenproporex, heptaminol, mefenorex, mesocarb, methamphetamine, methoxyphenamine, MDA, MDMA, methylephedrine, methylphenidate, nikethamide, pemoline, phendimetrazine, phenmetrazine, phentermine, pholedrine, phenylephrine, pipradol, prolintane, propylhexedrine, pseudoephedrine, selegiline, strychnine Narcotics + Cannabinoids buprenorphine, methadone, morphine, pentazocine, pethidine, THC Beta-blockers acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol Anti-oestrogens clomiphene, cyclofenyl, tamoxifen

17 2004 (WADA) Anabolic Agents: Anabolic Androgenic Steroids (AAS): exogenous bolasterone, boldenone, clostebol, danazol, dehydrochlormethyltestosterone, drostanolone, fluoxymesterone, formebolone, mestanolone, mesterolone, metandienone, metenolone, methyltestosterone, mibolerone, nandrolone, norbolethone, norethandrolone, oxymesterone, oxandrolone, oxymetholone, quinbolone*, stanazolol, stenbolone*, THG, trenbolone, 4- hydroxytestosterone*, Anabolic Agents: Anabolic Androgenic Steroids (AAS): endogenous androstenediol, androstenedione, DHEA, dihydrotestosterone, epitestosterone, testosterone Other Anabolic Agents clenbuterol, zeranol Peptide hormones choriogonadotropin (hcg), erythropoietin (EPO) + RSR13 Beta-2-agonists bambuterol, fenoterol, formoterol, salbutamol, salmeterol, terbutaline Diuretics & Masking agents acetazolamide, amiloride, bendroflumethiazide, bumetanide, chlorothiazide, chlortalidone, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, spironolactone, triamterene, canrenone, HES Stimulants amfepramone, amiphenazole, amphetamine, amphetaminil, benzphetamine, bromantan, caffeine, carphedon, cathine, clobenzorex, cocaine, cropropamide, crotethamide, DMA, ephedrine, etamivan, ethylamphetamine, etilefrine, fencamfamin, fenetylline, fenfluramine, fenproporex, furfenorex, heptaminol, mefenorex, mesocarb, methamphetamine, methoxyphenamine, MDA, MDMA, mephentermine, methylephedrine, methylphenidate, modafinil, nikethamide, pemoline, phendimetrazine, phenmetrazine, phentermine, pholedrine, phenylephrine, pipradol, prolintane, propylhexedrine, pseudoephedrine, selegiline, strychnine Narcotics + Cannabinoids buprenorphine, methadone, morphine, pentazocine, pethidine, THC Beta-blockers acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bunolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol Anti-oestrogens aminoglutethimide, anastrozole*, clomiphene, cyclofenyl, exemestane*, formestane*, letrozole*, tamoxifen Glucocorticoids* beclomethasone, betamethasone, budesonide, desonide, dexamethasone, fludrocortisone, flumethasone, flunisolide, fluocortolone, fluticasone, methylprednisolone, prednisolone, triamcinolone

18 2005 (WADA) Anabolic Agents: Anabolic Androgenic Steroids (AAS): exogenous bolasterone, boldenone, boldione, clostebol, danazol, delta1-androstene-3,17-dione* dehydrochlormethyltestosterone, drostanolone, ethylestrenol*, fluoxymesterone, formebolone, mestanolone, mesterolone, metandienone, metenolone, methyldienolone*, methyltrienolone*, methyltestosterone, mibolerone, nandrolone, norbolethone, norclostebol*, norethandrolone, oxymesterone, oxandrolone, oxymetholone, quinbolone*, stanazolol, stenbolone*, THG, trenbolone, 4-hydroxytestosterone*, 18a-homo-17b-hydroxyestr-4-en-3-one*, 4-hydroxy-19- nortestosterone Anabolic Agents: Anabolic Androgenic Steroids (AAS): endogenous androstenediol, androstenedione, DHEA, dihydrotestosterone, epitestosterone, testosterone Other Anabolic Agents clenbuterol, zeranol Peptide hormones choriogonadotropin (hcg), erythropoietin (EPO) + RSR13 Beta-2-agonists bambuterol, fenoterol, formoterol, salbutamol, salmeterol, terbutaline Diuretics & Masking agents acetazolamide, amiloride, bendroflumethiazide, bumetanide, chlorothiazide, chlortalidone, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, spironolactone, triamterene, canrenone, HES

19 2005 (WADA) Stimulants amfepramone, amiphenazole, amphetamine, amphetaminil, benzphetamine, bromantan, caffeine, carphedon, cathine, clobenzorex, cocaine, cropropamide, crotethamide, DMA, ephedrine, etamivan, ethylamphetamine, etilefrine, fampropazone, fencamfamin, fenetylline, fenfluramine, fenproporex, furfenorex, heptaminol, mefenorex, mesocarb, methamphetamine, methoxyphenamine, MDA, MDMA, mephentermine, methylephedrine, methylphenidate, modafinil, nikethamide, pemoline, phendimetrazine, phenmetrazine, phentermine, pholedrine, phenylephrine, pipradol, prolintane, propylhexedrine, pseudoephedrine, selegiline, strychnine Narcotics + Cannabinoids buprenorphine, methadone, morphine, pentazocine, pethidine, THC Beta-blockers acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bunolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol Anti-oestrogens aminoglutethimide, anastrozole*, clomiphene, cyclofenyl, exemestane*, formestane*, fulvestrant*, letrozole*, raloxifene*,tamoxifen, testolactone*, toremifene* Glucocorticoids beclomethasone, betamethasone, budesonide, desonide, dexamethasone, fludrocortisone, flumethasone, flunisolide, fluocortolone, fluticasone, methylprednisolone, prednisolone, triamcinolone Alpha-reductase inhibitors Finasteride, dutasteride

20 2002 (IOC MC) Anabolic Agents: Anabolic Androgenic Steroids (AAS): exogenous boldenone, clostebol, danazol, dehydrochlormethyltestosterone, drostanolone, fluoxymesterone, formebolone, mesterolone, metandienone, metenolone, methyltestosterone, nandrolone, norethandrolone, oxymesterone, oxandrolone, oxymetholone, stanazolol, trenbolone Anabolic Agents: Anabolic Androgenic Steroids (AAS): endogenous dihydrotestosterone, epitestosterone, testosterone Other Anabolic Agents clenbuterol Peptide hormones choriogonadotropin (hcg) Beta-2-agonists fenoterol, salbutamol, terbutaline Diuretics & Masking agents acetazolamide, bendroflumethiazide, bumetanide, chlortalidone, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, spironolactone, triamterene, canrenone Stimulants amfepramone, amphetamine, bromantan, caffeine, carphedon, cathine, cocaine, cropropamide, crotethamide, ephedrine, etamivan, etilefrine, fencamfamin, fenetylline, fenfluramine, heptaminol, mefenorex, mesocarb, methamphetamine, methoxyphenamine, MDA, methylephedrine, methylphenidate, nikethamide, pemoline, phendimetrazine, phentermine, pholedrine, phenylephrine, pipradol, prolintane, pseudoephedrine, selegiline, strychnine Narcotics + Cannabinoids buprenorphine, methadone, morphine, pentazocine, pethidine, THC Beta-blockers acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol

21 2005 (WADA) Anabolic Agents: Anabolic Androgenic Steroids (AAS): exogenous bolasterone, boldenone, boldione, clostebol, danazol, delta1-androstene-3,17-dione* dehydrochlormethyltestosterone, drostanolone, ethylestrenol*, fluoxymesterone, formebolone, mestanolone, mesterolone, metandienone, metenolone, methyldienolone*, methyltrienolone*, methyltestosterone, mibolerone, nandrolone, norbolethone, norclostebol*, norethandrolone, oxymesterone, oxandrolone, oxymetholone, quinbolone*, stanazolol, stenbolone*, THG, trenbolone, 4-hydroxytestosterone*, 18a-homo-17b-hydroxyestr-4-en-3-one*, 4-hydroxy-19- nortestosterone Anabolic Agents: Anabolic Androgenic Steroids (AAS): endogenous androstenediol, androstenedione, DHEA, dihydrotestosterone, testosterone Other Anabolic Agents clenbuterol, zeranol Peptide hormones choriogonadotropin (hcg), erythropoietin (EPO) + RSR13 Beta-2-agonists bambuterol, fenoterol, formoterol, salbutamol, salmeterol, terbutaline Diuretics & Masking agents acetazolamide, amiloride, bendroflumethiazide, bumetanide, chlorothiazide, chlortalidone, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, spironolactone, triamterene, canrenone, HES Stimulants amfepramone, amiphenazole, amphetamine, amphetaminil, benzphetamine, bromantan, carphedon, cathine, clobenzorex, cocaine, cropropamide, crotethamide, DMA, ephedrine, etamivan, ethylamphetamine, etilefrine, fampropazone, fencamfamin, fenetylline, fenfluramine, fenproporex, furfenorex, heptaminol, mefenorex, mesocarb, methamphetamine, methoxyphenamine, MDA, MDMA, mephentermine, methylephedrine, methylphenidate, modafinil, nikethamide, pemoline, phendimetrazine, phenmetrazine, phentermine, pholedrine, pipradol, prolintane, propylhexedrine, selegiline, strychnine Narcotics + Cannabinoids buprenorphine, methadone, morphine, pentazocine, pethidine, THC Beta-blockers acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bunolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol Anti-oestrogens aminoglutethimide, anastrozole*, clomiphene, cyclofenyl, exemestane*, formestane*, fulvestrant*, letrozole*, raloxifene*,tamoxifen, testolactone*, toremifene* Glucocorticoids beclomethasone, betamethasone, budesonide, desonide, dexamethasone, fludrocortisone, flumethasone, flunisolide, fluocortolone, fluticasone, methylprednisolone, prednisolone, triamcinolone Alpha-reductase inhibitors Finasteride, dutasteride

22 Antidoping analysis: 3 main areas Pre-instrumental stage: sample pretreatment and preliminary measurements (V, colour, sediments, ph, sg) Screening (GC-NPD, GC-MS, HPLC, LC- MS, immunological techniques ) Confirmation (GC-MS, GC-HRMS, GC-MS- MS, GC-HRMS-MS, GC-C-IRMS, LC-MS- MS ) + EPO/NESP Increasing complexity ( cost and time per sample)

23 Complexiy Confirmation analysis Screening analysis Hot area Reliability

24 Total number of targets for screening (minimum requested to pass the reaccreditation test or the PT) 92 in in in in 2005 over 200 in 2006?

25

26 2002 (6500 A urines) Stim (free) Stim (conj), bbloc Hormones (free) Hormones (total) LCAA Diur&Mask hcg, DOA GC-NPD GC-MS-NCI GC-MS GC-MS HRMS GC-MS IA

27 2002 (6500 A urines) 2003 (7000 A urines) Stim (free) GC-NPD Stim (free) GC-NPD Stim (conj), bbloc GC-MS-NCI Stim (conj), bbloc GC-MS-NCI Hormones (free) GC-MS Hormones (free) GC-MS Hormones (total) GC-MS Hormones (total) GC-MS LCAA HRMS LCAA HRMS Diur&Mask GC-MS Diur&Mask GC-MS hcg, DOA IA hcg, DOA IA HES GC-MS

28 2002 (6500 A urines) 2003 (7000 A urines) Stim (free) GC-NPD Stim (free) GC-NPD Stim (conj), bbloc GC-MS-NCI Stim (conj), bbloc GC-MS-NCI Hormones (free) GC-MS Hormones (free) GC-MS Hormones (total) GC-MS Hormones (total) GC-MS LCAA HRMS LCAA HRMS Diur&Mask GC-MS Diur&Mask GC-MS hcg, DOA IA hcg, DOA IA HES GC-MS 2004 (7800 A urines EPO + blood) Stim (free) Stim (conj), bbloc Hormones (free) Hormones (total) LCAA Diur&Mask hcg, DOA HES Glucocort, THG EPO GC-NPD GC-MS GC-MS GC-MS HRMS GC-MS IA GC-MS LC-MS Flaminia

29 2002 (6500 A urines) 2003 (7000 A urines) Stim (free) GC-NPD Stim (free) GC-NPD Stim (conj), bbloc GC-MS-NCI Stim (conj), bbloc GC-MS-NCI Hormones (free) GC-MS Hormones (free) GC-MS Hormones (total) GC-MS Hormones (total) GC-MS LCAA HRMS LCAA HRMS Diur&Mask GC-MS Diur&Mask GC-MS hcg, DOA IA hcg, DOA IA HES GC-MS 2004 (7800 A urines EPO + blood) 2005 (>8000 with 10% EPO planned) Stim (free) GC-NPD Stim (free) Stim (conj), bbloc GC-MS Stim (conj), bbloc Hormones (free) GC-MS Hormones (free) Hormones (total) GC-MS Hormones (total) LCAA Diur&Mask HRMS GC-MS LCAA Diur&Mask? hcg, DOA IA hcg, DOA HES GC-MS HES Glucocort, THG LC-MS Glucocort, THG EPO Flaminia EPO Blood

30 Screening analysis of a real sample (diclophenamide?)

31 Confirmation analysis (sample vs. positive reference urine)

32 Screening V Urine volume 5mL Internal standard Indomethacine, 200 ng/ml Extraction Sep-pak C18 cartridge Evaporation N 2 stream, 40 C Derivatization Acetone/CH 3 I= 1/9, 200 µl Anhydrous K 2 CO 3, 50 mg 70 C C 180 min Injection volume 1 µl (split 1:10) Analysis GC-MS EI SIM

33 Screening V Urine volume 5mL Internal standard Indomethacine, 200 ng/ml Extraction Sep-pak C18 cartridge Evaporation N 2 stream, 40 C Derivatization Acetone/CH 3 I= 1/9, 200 µl Anhydrous K 2 CO 3, 50 mg MW 900W 10 min Injection volume 1 µl (split 1:10) Analysis GC-MS EI SIM

34 Screening V 2004 Urine volume 5mL Internal standard Indomethacine/mefruside 1st Extraction No cartridge! ph corrected to 10 (800 µl carbonate/bicarbonate 4M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C 2nd Extraction No cartridge! ph corrected to 4 (100 µl formate/formic acid 5M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C Derivatization Acetone/CH 3 I= 1/9, 200 µl Anhydrous K 2 CO 3, 50 mg MW 1200W 6 min Injection volume 1 µl (split 1:10) Analysis GC-MS EI SIM

35 Screening V present Urine volume 6 ml Internal standard Indomethacine/mefruside 1st Extraction No cartridge! ph corrected to 10 (800 µl carbonate/bicarbonate 4M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C 2nd Extraction ph corrected to 4 (100 µl formate/formic acid 5M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C Derivatization Acetone/CH 3 I= 1/9, 200 µl Anhydrous K 2 CO 3, 50 mg MW 1200W 6 min Injection volume 1 µl (split 1:10) Analysis GC-MS EI SIM

36 Screening V present Urine volume 6 ml Internal standard Indomethacine/mefruside 1st Extraction No cartridge! ph corrected to 10 (800 µl carbonate/bicarbonate 4M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C 2nd Extraction ph corrected to 4 (100 µl formate/formic acid 5M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C Derivatization Acetone/CH 3 I= 1/9, 200 µl Anhydrous K 2 CO 3, 50 mg MW 1200W 6 min Injection volume 1 µl (split 1:10) Analysis GC-MS EI SIM

37 Screening V present Urine volume 6 ml Internal standard Indomethacine/mefruside 1st Extraction No cartridge! ph corrected to 10 (800 µl carbonate/bicarbonate 4M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C 2nd Extraction ph corrected to 4 (100 µl formate/formic acid 5M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C Derivatization Acetone/CH 3 I= 1/9, 200 µl Anhydrous K 2 CO 3, 50 mg MW 1200W 6 min Injection volume 1 µl (split 1:10) Analysis GC-MS EI SIM

38 Screening V present Urine volume 6 ml Internal standard Indomethacine/mefruside 1st Extraction No cartridge! ph corrected to 10 (800 µl carbonate/bicarbonate 4M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C 2nd Extraction ph corrected to 4 (100 µl formate/formic acid 5M) extraction by 12 ml CHCl 3 :isopropanol:tbme (80:10:10) Evaporation N 2 stream, 40 C Derivatization Acetone/CH 3 I= 1/9, 200 µl Anhydrous K 2 CO 3, 50 mg MW 1200W 6 min Injection volume 1 µl (split 1:10) Analysis GC-MS EI SIM 1/2 goes to IVa

39

40 Recombinant EPO Endogenous EPO Darbepoetin (NESP)

41

42 Microwaves, orientation of the magnetic field and rotational energy

43 Microwaves cook from the inside Text > Tint Text < Tint

44 Ideal derivatization reaction Microwaves diuretics, stimulants glucocorticoids

45 The tradition: Select an appropriate derivatization reagent Test everything you use (tubes, racks, septa) Work in segmented steps (i.e. 30 = ) Ensure thermostatic control (simplest way: put the reaction chamber(s) into a water bath inside the oven) Use technical ovens Keep a fire estinguisher at hand

46 7,00E+04 6,00E+04 5,00E+04 4,00E+04 3,00E+04 2,00E+04 1,00E+04 0,00E+00 time mw T Unspecific (methylation) 3,E+05 3,E+05 2,E+05 2,E+05 1,E+05 5,E+04 0,E+00 time mw T Specific (silylation)

47 Application of microwaves in derivatization reactions

48

49 OSSIMORO Una figura retorica in cui si accostano due termini in forte antitesi fra loro in modo da creare un originale contrasto, talvolta con sorprendenti effetti linguistici chiaramente ambiguo urlo silenzioso, silenzio assordante affollata solitudine insostenibile leggerezza definitively maybe

50 L ossimoro più famoso della lingua italiana ghiaccio bollente

51

52

53 On the boiling T of water (at 1 atm) conventional heating: C microwave irradiation, 1200 W: C microwave irradiation, 1200 W, + boiling chips: C This superheating effect has been attributed to retardation of nucleation during microwave heating The effect is eliminated in well stirred mixtures and using low microwave power (< 800 W) In theory, a sufficiently strong magnetic field could cause the solvent to freeze In practice, the effect is minimal, but detectable

54 The tested racks & water bath

55 The tested racks & water bath

56

57 Preliminary results (waiting for the oven )! " # $ %&'! ( ) *

58 Preliminary results (waiting for the oven ) %FLUOCORTOLONE-3TMS usp Time (min) Thermal 100 C MW in water MW dry 15 24%BUDESONIDE-3TMS 18 usp 95 30Time 45 (min) Time (min) Time 30 (min) Time (min) Thermal MW C in 35 water 40 %MPREDNISOLONE-4TMS usp Thermal 15 9MW 100 C in water %PREDNISONE-3TMS usp Thermal MW C in water %PREDNISOLONE-4TMS usp 19 Thermal 28 MW C 10in water 81 MW 97 dry MW dry MW dry MW dry

59 Preliminary results (waiting for the oven )!!!" # $!! % & #! #!"'()*%+ (!!,! #!!-.-!! #,!!! $!$ / #0! # #"! %

60 From urine to blood: key issues +,-!! "... / 0 10( * 2 3 (+* 4 1!".! 5! )

61 ! HBOCs hgh Robust and reliable procedure (visual screening + different confirmation) Lack of reference materials The procedure is reliable, but not robust Reagents not available in sufficient amounts Presently going from lab to industrial scale Blood Transfusions The procedure is reliable, but not robust No interlaboratory study so far

62

63

64

65

66

67

68 Acknowledgements Cristiana Colamonici, Flaminia Garribba, Monica Mazzarino National Italian Antidoping Commission (supporting the research activity of the lab in its entirety) The Athens, Barcelona, Koeln, Lausanne, London and Seibersdorf Laboratories