Application of analytical methods in doping control

Application of analytical methods in doping control Supervisor: Dr. Y. Assadi By: A. Pajand Birjandi Nov 2006 Exogenous AAS Anabolic androgenic steroids (AAS) Endogenous AAS Other anabolic agents: (clenbuterol,zeranol,zilpaterol) Erythropoietin(EPO) Growth hormone (hGH) S1. anabolic agents S2.hormones and Related substances S3. β2-agonists Gonadotrophins (LH, hCG) Insulin Corticotrophins S4. agents with anti-estrogenic activity S5. diuretics and other masking agents S6. SIMULANTS S7. NARCOTICS S8. CANNABINOIDS (e.g. hashish, marijuana) S9. GLUCOCORTICOSTROIDS PROHIBITED METHODS M1. ENHANCMENT OF OXYGEN TRANSFER M2. CHEMICAL AND PHYSICAL MANIPULATION M3. GENE DOPING Blood Transfusion (homologous/heterologous) Erythropoietin (EPO) SUBSTANCES PROHIBITED IN PARTICULAR SPORT P1. ALCOHOL P2. BETA-BLOCKERS HISTORY  The first recorded attempt to enhance performance occurred as early as the 8th century BC, when Ancient Greek Olympians ate sheep's testicles; today we would recognize these as a source of testosterone. As early as the late 19th century professional cyclists were using substances like caffeine, cocaine and ether-coated sugar cubes to improve performance, reduce pain and delay fatigue. In the 1904 Olympics, Thomas Hicks (USA) won the marathon at St. Louis and collapsed. It took hours to revive him; he had taken brandy mixed with strychnine to help him win his gold medal. Nazi Germany athletes were rumored to use the first rudimentary testosterone preparations in the 1936 Summe Olympics... The first idea to create a Doping Control Laboratory Detection of alkaloid compounds in horse’s in 1900 First testing for doping in 1910 First scinificant control doping in 1960 For anabolic steroids Different Doping Controls 1. In-Competition Controls 2. Out-of-Competition Controls Unannounced Controls Announced Controls - Usually the controls are performed as unannounced controls in connection with competitions and training sessions - An athlete can be called to a doping control whenever and wherever, home or abroad 5. Dividing the sample into the ”A” and ”B” bottles Sample division: Pour a minimum of 50 ml (lower part of label) into the A bottle (orange label) and a minimum of 25 ml (lower part of label) into the B bottle (blue label) Checking pH and concentration of sample The doping control officer will check the pH and concentration of your urine sample which is left in the sample collection vessel to ensure that the sample is suitable for analysis. Sample B -Sample B will only be analyzed in case the sample A has been found positive .Sample B will be analyzed after a request of the athlete, and on the expense of the athlete or the sports federation. -The athlete or his/her representative has the right to be present when the sample B is analyzed in the laboratory. -In case the test result of the sample B does not confirm the positive test result of the sample A, the temporary suspension will immediately expire. 350 300 250 200 150 100 50 0 Athletics Icehockey Skiing Powerlifting Football 324 281 204 159 104 control/year What happen in a doping control laboratory? Screening confirmation Analytical procedures: 1. Sampling 2. Pre-treatment: (homogenisation, centrifugation, filtration) 3. Extraction/purification: (sample preparation methods) 4. Derivatization 5. Instrumental analysis Sample preparation methods 1-1) Liquid-liquid extraction 2-1) Solid phase extraction: 1-2-1) adsorption 2-2-1) apolar 3-2-1) polar 4-2-1) Ionic exchange 5-2-1) Mixed phase 3-1) Matrix solid phase dispersion 4-1) Immunoaffinity chromatography 5-1) Supercritical fluid extraction 6-1) dialysis General criteria (1) The substances measured in the analytical systems should cover the total range and be evenly distributed over that range(i.e., for GC–MS or LC–MS, the retention time (RT) of the standard substances ranges from 3 to 30min). (2) The standard deviations of measurements should be as small as possible and the values (i.e., RT) must be standardized in such a way that good reproducibility is obtained on an inter-laboratory scale. (3) When more than one system or technique is used, there should be low correlation between these systems or techniques. The general criteria for any procedure distinguish between the analyte and all known interfering materials that may possibly occur in the relevant matrix. Detailed information about the molecular structure of the analyteis essential. Each analytical technique possessesan identification power of its own and a definite number of identification points (IPs). 4.1. How to combine several analytical procedures (1) Screening urine samples with immunoassays is quite common and laboratories may use more than 15 different drug immunoassays. These are always used in combination with at least one other chromatographic technique for confirmation. (2) Most laboratories perform 1–5 (average 2) different extraction procedures on the biosamples, mainly urine and blood, before to the use of one or more chromatographic techniques to detect various groups of drugs and metabolites. These procedures comprise mainly liquid–liquid and solid-phase extraction (SPE) with various combinations of pH, organic solvents and SPE ready to use columns depending on the aims of the analysis. Derivatization of the extracts is made mostly with acetic anhydride, trifluoroacetic acid (TFA), tetramethylsilane (TMS) or methylation reagents. (3) Over the years, it appears that there is a decrease in the use of TLC and an increase in the use of diode array detection and GC–MS. LC–MS screening is still in its infancy. (4) Few laboratories make use of GC with nitrogen– phosphorus detection (NPD), mostly on basic extracts with no derivatization. The analyses are performed on capillary columns with temperature programming . (5) Automated GC–MS screening is made possible with rather simple and economic instruments equipped with the suitable automatic injection devices and comparison with adequate reference mass spectra databases . Selecting different chromatography and mass spectrometric techniques Choosing between GC–MS and LC–MS : Thermal stability of the extracted substances, their polarity detector sensitivity Impurities detected by one method may not be seen by another when based on a completely different physico-chemical detection approach ; (i.e.,LC with UV or MS detection and GC with flame ionization detection (FID), NPD or electron-capture detection (ECD)). Instrumental technologies The following analytical technologies were applied: (i) Gas chromatography with nitrogen detectors (GCNPD) for volatile stimulants and narcotics. (ii) Gas chromatography with quadrupole mass spectrometry for heavy volatile molecules like stimulants, narcotics, steroids, diuretics, etc. (iii) Gas chromatography with combustion isotope ratio magnetic sector mass spectrometry (GC-C-IRMS) for the measurement of 12C/13C isotope ratio, to discriminate endogenous steroids from exogenouslyapplied steroids, like testosterone. (iv) Gas chromatography high resolution mass spectrometry (GC-HRMS) to detect low to very low levels of anabolic agents. (v) Liquid chromatography tandem ion trap mass spectrometry (LC-MS-MS) for polar compounds incompatible to GC, like corticosteroids, proteins like haemoglobin based oxygen carriers (HBOCs) and compounds to be quantified with direct urine injection, like ephedrines, morphine and salbutamol,saving the sample preparation time and lowering the uncertainty. (vi) Size-exclusion high pressure liquid chromatography (HPLC) with ultraviolet diode array detector for HBOCs. (vii) Immunoassays: enzyme linked immunosorbent assay (ELISA) for recombinant human erythropoietin and chemiluminescence detection for measuring human chorionic gonadotropin (hCG),fluorescence detection for recombinant human growth hormone (rhGH) in serum. (viii) Electrophoresis with chemiluminescence detection for recombinant human Erythropoetin and HBOCs. (ix) Biochemical analyser for measurement of glucose levels in urine. (x) Flow cytometry with laser detection for blood analysis and homologous blood cell doping. a/a 1 2 3 4 Screening procedures Procedure I: Volatile nitrogencontaining compounds (stimulants, narcotics( Procedure II: Heavy volatile Nitrogen containing compounds (Stimulants, narcotics) instrumentation GC/NPD GC/MS Biochemical analyzer Plasma expanders Procedure IV: Combined free and conjugated anabolic agents, beta-blockers and beta2-agonists Combined free and conjugated Anabolic agents and beta2-agonists Glucocorticosteroids and combined free and conjugated anabolic agents Procedure V: Diuretics Procedure VI: Human chorionic gonadotropin (hCG) Protein hormone recombinant human erythropoietin (rEPO) GC/MS GC/HRMS LC/MS (ion trap) GC/MS Biochemical analyzer IEF and double blotting appliances 5 6 7 8 9 Β2-adrenoceptor agonists are used for the treatment pulmonary disorders, particularly in the treatment of asthma and exercise-induced asthma, which are the most common exercise pulmonary syndromes observed in competitive athletes. In addition to the stimulatory activity on respiration and on the central nervous system, the administration of B2-agonists promotes certain anabolic effects, depending on the does and the route of administration . In sport, the use of B2-agonists is prohibited by the international Olympic committee (IOC) and international sport federations and only salbutamol , salmeterol and terbutaline are permitted by inhalation and, even then , must be declared in writing ,prior to the competition, to the relevant medical authority. B2-agonists, according to their polarity , are excreted in urine as phase I (low polarity) or phase II (high polarity) metabolites. Owing to their potency , must of these drugs are employed in very small quantities and their determination in biological fluids requires the detection of nanogram or sub-nanogram per milliliter levels . The must usual analytical method for B2-agonists is gas chromatography/mass spectrometry (GC/MS) ,using liquidliquid extraction or solid phase extraction to isolate from biological samples unchanged compounds and metabolites and to eliminate matrix interferences. Derivatization has an important role in the detection of B2-agonists by GC/MS, making the molecules feasible for GC/MS analysis. Trimethylsilylation, acylation, combined Trimethylsilylation and acylation and formation of cyclic derivatives are the most commonly employed derivatization procedures. Multidimensional separation Multidimensional separation methods involve two different separation dimensions, for a range of analytical reasons including removal of potentially interfering matrix components, or to improve the resolution of specific analytes. for example, LC–LC has been Used extensively for the Analysis of drugs In matrices such as plasma, Serum or urine. Comprehensive 2D GC The first column is normally a non-polar or low polarity column of normal capillary column length and the separation mechanism is via solute boiling point. The second column is a fast elution column whit a more polar phase, hence the aim of the coupled system is to resolve closely eluting peaks of similar boiling point but of different polarity Various prolinate metabulites GC–FID; GC–MS GC×GC–FID. “In the name of all competitors, I promise that we shall take part in the Olympic games, respecting and abiding by the rules which govern them, committing ourselves to a sport without doping and without drugs, in the spirit of true sportsmanship, for the glory of sport and the honor of our teams”. references [1] J.F. Ferna´ndez-Sa´nchez, A. Segura Carretero *,Journal of Pharmaceutical and Biomedical Analysis 31 (2003) 859/865 [2] Frans T. Delbeke∗, Peter Van Eenoo, Journal of Steroid Biochemistry & Molecular Biology 83 (2003) 245–251 [3] Z. Hassankhani-Majd, V. Ghoulipour, ACTA CHROMATOGRAPHICA, NO. 16, 2006 [4] Chad R. Borges ,∗, James Taccogno International Journal of Mass Spectrometry 247 (2005) 48–54 [5] A.G. Fragkaki, I.-P. Leontiou, N. KioukiaFougia, Technovation 26 (2006) 1162–1169 [6] J. Marcos a,b, X. de la Torre a,b, J.C. GonzalezAnalytica Chimica Acta 522 (2004) 79– 88 [7] M.-H. Spyridaki a, P. Kiousi a, A. Vonaparti Analytica Chimica Acta 573–574 (2006) 242–249 [8] Laurent Rivier Forensic Science International 107 (2000) 309–323 [9] M. Gratac´os-Cubars´ı, M. Castellari Journal of Chromatography B, 834 (2006) 14–25 [10] M. Tsivou, N. Kioukia-Fougia, E. Lyris, Analytica Chimica Acta 555 (2006) 1–13 [11] Koichiro Teshima a,*, Takahiro Kondo Journal of Pharmaceutical and Biomedical Analysis 30 (2002) 299–305 [12] P. Kintza ,*, V. Cirimelea, H. Sachsb, T. Jeanneaua, B. Ludesa Forensic Science International 101 (1999) 209–216 [13] Laurent Rivier Analytica Chimica Acta 492 (2003) 69–82 [15] DOPING AND PROHIBITED DRUGS IN SPORT by Dr. Abazar Habibinia [16] DOPING WITH ANABOLIC STROIDS by Majid Gorgyaragh Thanks for your attention