STEROIDS IN WASTE WATERS AS MARKERS OF ABUSE Renzo BAGNATI Mario Negri Institute of Pharmacological Research- IRCCS Milano - Italy
A first attempttoapplythe sewage epidemiologyapproachin doping Compare urban waste water samples with samples taken in sites during sport events. Major methodological problems: very small populations (hundreds ), only spot sampling available, presence of mixed population. This study tried to obtain a first results about the presence of significative chemicals in waste waters from amateur sport sites.
Sampling site 1 Sampling well of sewage waters in the University Sport Center (CUS) - Università della Calabria - Arcavacata di Rende (Cs) South of Italy. Practised sports: basket, volley, football, rugby, martial arts etc. People: about 150 athletes/day; age: 18-25, mostly registered to the University, with some external addition. 2 samplings: june and october 2011
Sampling site 2 Sampling well of sewage waters, School Camp (FIDAL), Via degli Stadi, Cosenza - Italy. Samplings: a) Regional Athletics Masters (june 2011); about 200 participants, age 35-75 b) Italian Athletics Masters (october 2011); about 2500 participants, age 35-75 Praticed sports: shot put, discus, javelin, hammer throw, high and long jump, all running specialities (short and long distances).
Compounds analyzed Doping substances: anabolic steroids, aromatase inhibitors Pharmaceuticals: anti-acids, antibiotics, anti-convulsive, analgesics, anti-hyperlipidemics, β-blockers, diuretics Illicit drugs: amphetamines, cocaine, morphine, codeine, cannabinoids
Doping substances Anabolic steroids α- and β-testosterone androstenedione α- and β-boldenone α- and β-19-nortestosterone β-trenbolone 17-α-methyltestosterone 4-chloro-1,2-dehydromethyltestosterone mesterolone methandrostenolone oxymetolone Aromatase inhibitors anastrozole exemestane letrozole
Some steroids and aromatase inhibitors structures
Sample preparation 1. Double filtration (1.2 e 0.45 μm) 2. Internal standars spiking 3. Acidification (HCl 37%), purification and pre-concentration with SPE (Oasis)
Instrumental analysis HPLC-MS Quantitative analysis Triple quadrupole API-5500 Qualitative analysis High resolution LTQ-Orbitrap XL
Quantitation by MRM using a triple quadrupole
Isotopic dilution Addition of known amounts of isotope labelled substances
MS/MS instrumental conditions Substance declustering potential (DP) (V) Precursor ion (m/z) Product ion I (m/z) and collision energy (ev) Product ion II (m/z) and collision energy (ev) β-trenbolone 60 271.2 199.1 (34) 107.1 (44) α-19-nortestosterone β-19-nortestosterone α-boldenone β-boldenone α-testosterone β-testosterone β-testosterone-d3 (IS) 60 60 60 60 60 60 60 275.2 275.2 287.2 287.2 289.2 289.2 292.2 109.1 (32) 109.1 (32) 121.1 (32) 121.1 (32) 97.0 (28) 97.0 (28) 97.0 (28) 79.0 (62) 79.0 (62) 135.1 (20) 135.1 (20) 109.1 (32) 109.1 (32) 109.1 (32) Methandrostenolone 60 301.1 121.1 (40) 149.1 (22) 17α-methyltestosterone 17α-methylestosterone-d3 (IS) 60 60 303.2 306.2 97.0 (32) 97.0 (32) 109.1 (34) 109.1 (34) Mesterolone 60 305.2 91.0 (66) 229.2 (30) Oxymetolone 60 333.2 99.0 (40) 43.0 (120) 4-chloro-1-dehydro-17αmethyltestosterone 60 335.2 155.1 (44) 157.1 (44) Anastrozole 60 294.2 225.1 (32) 115.1 (76) Exemestane 60 297.2 121.1 (26) 149.1 (22) Letrozole 60 286.2 217.1 (20) 190.2 (44)
HPLC conditions for quantitative analysis Column: Xterra MS C18, 100x1 mm, 3.5 μm Eluent A: acetic acid (0.1%) in water Eluent B: acetonitrile Agilent 1200 - API 5500 ESI source triple quadrupole
Substance Retention time (min.) Method characteristics LOQ in samples (ng/l) Relative Standard Deviation % "intraday" Relative Standard Deviation % "interday" Recovery tests from water samples Reccovery % SD % 17-α-methyltestosterone 18.27 2 6.5 6.2 76.3 5.4 4-chloro-1,2-dehydrometiltestosterone 19.24 1 10.9 4.1 71.5 6.8 α-19-nortestosterone 17.51 5 9.1 6.6 80.8 6.8 α-boldenone 17.25 1 7.4 8.4 88.9 8.9 α-testosterone 18.46 1 10.6 8.9 66.4 7.4 anastrozolo 15.47 1 5.2 5.6 92.7 9.6 androstenedione 18.37 1 5.1 8.9 83.1 3.5 β-19-nortestosterone 16.61 5 7.4 10.9 78.8 4.6 β-boldenone 16.24 1 10.6 4.4 93.9 11.5 β-testosterone 17.49 1 8.9 7.4 72.6 4.3 exemestane 18.66 2 5.2 4.9 83.9 8.2 letrozole 15.63 1 6.8 3.3 85.4 8.6 mesterolone 20.19 1 7.4 3.8 64.1 6.5 methandrostenolone 16.94 5 7.4 7.6 91.3 9.2 trenbolone 15.91 5 11.2 8.6 84.3 4.1 oxymetolone 22.69 1 10.8 14.7 62.4 5.9
HPLC-MRM chromatograms of a standard mix of steroids [API 5500 positive ions] XIC of +MRM (38 pairs): 287.200/97.000 Da ID: Androstenedione 1 from Sample 1 (06 St_1000-1000pg) of 06 St_1000-1000pg.wiff (... Max. 1.9e5 cps. Intensity, cps 18.15 1.9e5 1.5e5 1.0e5 androstenedione 5.0e4 6.07 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Time, min XIC of +MRM (38 pairs): 287.200/121.100 Da ID: Boldenone 1 from Sample 1 (06 St_1000-1000pg) of 06 St_1000-1000pg.wiff (Turb... Max. 2.4e5 cps. Intensity, cps Intensity, cps Intensity, cps 2.0e5 1.0e5 β - boldenone 16.22 17.18 α - boldenone 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Time, min XIC of +MRM (38 pairs): 289.200/97.100 Da ID: Testosterone 1 from Sample 1 (06 St_1000-1000pg) of 06 St_1000-1000pg.wiff (Tur... Max. 2.9e5 cps. 2.9e5 2.0e5 1.0e5 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Time, min XIC of +MRM (38 pairs): 292.200/97.100 Da ID: Testosterone-D3 1 from Sample 1 (06 St_1000-1000pg) of 06 St_1000-1000pg.wiff (... Max. 2.2e5 cps. 2.0e5 1.0e5 β - testosterone 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Time, min 17.38 17.32 18.31 α - testosterone testosterone D3
HPLC-MRM chromatograms of a waste water sample [API 5500 positive ions] I n t e n s i t y, I n c t p e s n s i t y, I c n p t e s n s i t y, c p s XIC of +MRM (38 pairs): 287.200/121.100 Da ID: Boldenone 1 from Sample 1 (18 Mi_Nosedo_10) of 18 Mi_Nosedo_10.wiff (Turbo S... 1.4e6 1.0e6 5.0e5 β - boldenone α - boldenone Max. 1.4e6 cps. 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Time, min XIC of +MRM (38 pairs): 287.200/97.000 Da ID: Androstenedione 1 from Sample 1 (18 Mi_Nosedo_10) of 18 Mi_Nosedo_10.wiff (Tur... Max. 8.4e5 cps. 8.0e5 6.0e5 4.0e5 2.0e5 6.61 7.04 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Time, min XIC of +MRM (38 pairs): 289.200/97.100 Da ID: Testosterone 1 from Sample 1 (18 Mi_Nosedo_10) of 18 Mi_Nosedo_10.wiff (Turbo... Max. 2.5e5 cps. 2.5e5 2.0e5 1.5e5 β - testosterone 1.0e5 18.25 20.26 5.0e4 14.34 12.77 13.51 15.37 16.11 19.47 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Time, min 17.15 androstenedione α - testosterone
Resultsforthe analysisofdoping substances α- and β-testosterone, androstenedione and α- and β-boldenone were found in similar concentrations in all samples coming from sport sampling sites. α-testosterone and androstenedione are metabolic products of β-testosterone α- and β-boldenone are not normally present in natural conditions. None of the other artificial anabolic steroids and aromatase inhibitors were found.
Result comparisons boldenone and testosterone Urban and sport sites waste waters (ng/l) 90.0 80.0 70.0 60.0 50.0 40.0 β-boldenone α-boldenone β-testosterone α-testosterone 30.0 20.0 10.0 0.0 CUS event Calabria (Site 1) Master events Calabria (Site 2) Milano waste Bologna waste Cagliari waste water plant (mean) water plant (mean) water plant (mean)
Boldenone and testosterone concentrations in urine and waste waters Human urine (non diluted (ng/ml) Human urine (non diluted) incubated with β-glucoronidase /arylsulfatase(ng/ml) Human urine diluted 5 timeswithwaste water and incubated (ng/ml) Wastewater used for dilution (ng/ml) β-boldenone < 0.4 1.97 14.4 0.18 α-boldenone < 0.4 0.64 2.42 0.046 β-testosterone 0.071 46.2 81.1 0.033 α-testosterone 0.068 107 80.3 < 0.4
Finalconsiderationsabout anaboliccompoundsin wastewaters The only anabolic compounds identified in waste waters are β-testosterone and some of its metabolic and/or bacterial products (α-testosterone, α- boldenone and β-boldenone). A comparison with data from urban waste waters and a sample of normal clean urine showed that boldenone is most probably derived from testosterone excretion of normal individuals and subsequent bacterial transformation in waste waters, not from its use as anabolic agent.
Pharmaceuticals in urban and sport sites waste waters (ng/l) 4500.0 4000.0 3500.0 3000.0 2500.0 2000.0 1500.0 CUS event Calabria (Site 1) Master events Calabria (Site 2) Milano waste water plant 1000.0 500.0 0.0
Considerationsaboutpharmaceuticals in wastewatersduringsport events Most of the pharmaceuticals found in waste waters of general population are also present in samples from sport dedicated areas There is a trend toward (to be confirmed) of a higher levels of consumption of analgesic/anti-inflammatory drugs, despite a prevalence of a young healthy population in one of the sampling sites Also the presence of high concentrations of β-blockers and diuretics should be confirmed Anti hyperlipidemics are less used among sport practising people.
Results - Illicit drugs in waste waters (ng/l) 700 600 500 400 300 200 CUS event Calabria (Site 1) Master events Calabria (Site 2) Milano waste water plant 100 0
Considerationsaboutillicitdrugsin wastewatersduringsport events Measurable levels of illicit drugs residues were found in this study (cocaine, morphine, cannabis) In contrast with levels found in large urban settings (Milano) cannabis use looks higher than cocaine Illicit drug consumption is higher in younger amateur athletes.
Qualitative analysis with LTQ Orbitrap XL - nano-esi source Column: Zorbax SB C18, 50x0.5 mm, 5 μm Eluente A: formic acid (0.05%) in water Eluente B: acetonitrile
LTQ- Orbitrap scheme
Quadrupoleand Orbitrapresolutions Quadrupole Orbitrap
LTQ-Orbitrap analysis TIC - FTMS TIC MS2 β-boldenone α-boldenone cocaina
Boldenone confirmation by high resolution HPLC-MS β-boldenone FT-MS MS2 spectrum
LTQ ORBITRAP XL Mario Negri