History of mass spectrometry at the Olympic Games

Transcription

1 JURNAL F MASS SPECTRMETRY J. Mass Spectrom. 2008; 43: Published online 20 June 2008 in Wiley InterScience ( SPECIAL FEATURE: HISTRICAL History of mass spectrometry at the lympic Peter Hemmersbach 1,2 1 Aker University Hospital, slo, Norway 2 School of Pharmacy, University of slo, slo, Norway Received 11 April 2008; Accepted 9 May 2008 Mass spectrometry has played a decisive role in doping analysis and doping control in human sport for almost 40 years. The standard of qualitative and quantitative determinations in body fluids has always attracted maximum attention from scientists. With its unique sensitivity and selectivity properties, mass spectrometry provides state-of-the-art technology in analytical chemistry. Both anti-doping organizations and the athletes concerned expect the utmost endeavours to prevent false-positive and false-negative results of the analytical evidence. The lympic play an important role in international sport today and are milestones for technical development in doping analysis. This review of the part played by mass spectrometry in doping control from Munich 1972 to Beijing 2008 lympics gives an overview of how doping analysis has developed and where we are today. In recognizing the achievements made towards effective doping control, it is of the utmost importance to applaud the joint endeavours of the World Anti-Doping Agency, the Internationallympic Committee, the international federations and national anti-doping agencies to combat doping. Advances against the misuse of prohibited substances and methods, which are performance-enhancing, dangerous to health and violate the spirit of sport, can be achieved only if all the stakeholders work together. Copyright 2008 John Wiley & Sons, Ltd. KEYWRDS: doping control; doping analysis; mass spectrometry; lympic ; anti-doping INTRDUCTIN The modern lympic have been important for the development of competitive sport throughout the last century. The 4-year period of an is celebrated every second year with Summer and Winter, respectively. The next will be held in Beijing from 8 to 24 August 2008, celebrating the XXIX. Regrettably, the development of sporting performances has been accompanied by many attempts to use prohibited substances and methods, in other words, doping. 1 Various performance-enhancing plant extracts or drinks were even used during the ancient lympic, which were held from 776 B.C. until 393 A.D. 2,3. Doping control analyses using instrumental analytical tools have been performed since the 1960s, 4 when various measures were initiated to control the misuse of performance-enhancing drugs in sport. The analysis of stimulants began after gas chromatographic separation was introduced. 5 The International lympic Committee (IC) Ł Correspondence to: Peter Hemmersbach, Aker University Hospital, N-0514 slo, Norway. Peter.Hemmersbach@farmasi.uio.no Medical Commission was formed in following obvious indications of the misuse of stimulating and other possible performance-enhancing agents, 7 including the occurrence of fatal accidents in sport, in particular the death of two cyclists in 1960 and This commission, headed by Prince Alexandre de Merode, initiated the first doping controls at the lympic in Grenoble and Mexico City in The doping controls and doping analyses at these may be regarded as a pilot project, but systematic doping controls and analyses were performed in all sports at the lympic in Munich in It was at these of the XX that mass spectrometry (MS) was introduced as a tool to identify doping substances. From that time until today, doping testing has been an integrated part of the fight against doping by the lympic Movement 12,13 and MS has played a decisive role both for the effectiveness of the testing and the legal security of the athletes. The definition of doping and the regulations for doping controls at the lympic have been set forth by the IC. Until 2003, the IC was also responsible for the List of Prohibited Substances 14 and for laboratory quality control and accreditation. From 2004, this task has been taken over by the World Anti-Doping Agency (WADA), which now Copyright 2008 John Wiley & Sons, Ltd.

2 840 P. Hemmersbach Table 1. List of laboratories, either IC-(until 2003) or WADA-accredited, performing doping analyses with mass spectrometric detection at lympic lympic Year/place Date IC/WADA laboratory Head of laboratory Number of Number of adverse samples b findings c of the XX XII lympic Winter of the XXI XIII lympic Winter of the XXII XIV lympic Winter of the XXIII XV lympic Winter of the XXIV XVI lympic Winter of the XXV XVII lympic Winter of the XXVI XVIII lympic Winter of the XXVII XIX lympic Winter of the XXVIII XX lympic Winter of the XXIX 1972 Munich a 26 August 11 Cologne M. Donike September 1976 Innsbruck 4 15 February Innsbruck G. Machata Montreal 17 July 1 August Montreal R. Dugal Lake Placid February Montreal R. Dugal Moscow 19 July 3 August Moscow V. Semenov Sarajevo 8 19 February Sarajevo B. Nikolin Los Angeles 28 July 12 August Los Angeles D. H. Catlin Calgary February Calgary S. Chan Seoul 17 September 2 Seoul J. Park ctober 1992 Albertville a 8 23 February Paris J. P. Lafarge Barcelona 25 July 9 August Barcelona J. Segura Lillehammer February slo P. Hemmersbach 529; Atlanta a 19 July 4 August Indianapolis/Los B. Sample Angeles 1998 Nagano a 7 22 February Tokyo M. Ueki Sydney 15 September 1 ctober Sydney R. Kazlauskas 2769; Salt Lake 8 4 February Los Angeles D. H. Catlin 700; 77 8 City a 2004 Athens August Athens C. 2926; Georgakopoulos 2006 Turin a February Rome F. Botré 919; 300; Beijing 8 24 August Beijing M. Wu a At these an IC/WADA-accredited laboratory was temporarily established at the lympic site. b The figures for the two last rows are either from original publications cited in this review or from Clasing s book. 12 The figures indicate the numbers of urine and blood samples taken under the authority of the IC. c The number of adverse findings is related to laboratory reports that resulted in a sanction. issues the new Prohibited List 15 each year in ctober that comes into effect on 1 January the following year. This list has obviously been subject to many changes over the years, and this is also reflected in the variety of mass spectrometric instrumentation. Table 1 lists the IC/WADA-accredited laboratories that were involved in the doping analysis at the different lympic. Many of them are still in operation and are registered today as WADA-accredited laboratories. 16 Munich 1972 From 1969 onwards, the IC Medical Commission has regulated the analytical methodology for the detection of doping agents in urine samples. A list of classes of prohibited substances with examples was established and identification criteria were defined. 11 The list composed of stimulants, narcotics and analgesics, and the list of examples was rounded off with the expression and related substances to cover newly synthesized substances with

3 History of mass spectrometry at the lympic 841 Figure 1. Atlas MAT CH-5 sector mass spectrometer coupled to a gas chromatograph with packed column via a glass capillary interface. The instrument was installed in Munich similar pharmacological activities. The analytical strategy was as follows: an initial screening procedure, mainly based on gas chromatography (GC) and detection with a nitrogen phosphorus detector (NPD), was followed by a strictly regulated identification procedure for suspicious samples. MS had already been applied as a detection method at that time, but it had not yet become mandatory. 17 In all 2079 urine samples were forwarded to the laboratory and seven adverse findings were reported. They included the detection of amfetamine, ephedrine, phenmetrazine and nikethamide. Figure 1 shows the mass spectrometer used then to identify prohibited substances. The mono-sector instrument coupled to a gas chromatograph with a packed column was state-of-the-art technology at that time. Colleagues working with this kind of instrumentation remember well how difficult it was to handle the glass capillary interface between GC and MS. Figure 2 shows the mass spectrum from one of the ephedrine findings; the spectrum differs in no way from an electron impact (EI) spectrum of underivatised ephedrine today. Many elements of doping analysis that are still important requirements for an lympic laboratory were already introduced in The laboratory had to provide a report within 24 h of receiving the samples to guarantee a quick and effective management of the results, and the laboratory also had the challenge of analysing four control samples. These urine samples contained doping agents at relevant concentrations and were introduced into the normal sample flow without the laboratory being able to recognise them. The basis for the identification of stimulants and narcotics by GC separation and MS detection was laid at that time and modified later on. 18 Prof. Dr Manfred Donike, heading the Institute of Biochemistry at the German Sport University in Cologne, Germany, from 1977 until his death in 1995, was the main initiator for the introduction of MS in doping analysis. Already since 1968 he had been engaged in doping analysis with chromatography and MS as the key techniques. The establishment of the Cologne Workshop, which has been arranged yearly since 1983, has given all IC/WADAaccredited laboratories a scientific forum to exchange new ideas and results. Through his engagement in many international federations and the IC, Prof. Donike laid the basis for the development and quality control of doping control laboratories all around the world. Montreal 1976 The list of prohibited substances was expanded in 1975 with the class of anabolic-androgenic steroids (AAS), in good time to be effective at the lympic in Montreal. It was decided that the doping analyses during the Winter in Innsbruck 19 should be performed at the lympic site under the supervision of Prof. Donike. A MAT 112 mass spectrometer was used for substance identification. At these controls, AAS were not yet tested for. Although indications of the misuse of AAS in various sports had existed long before 1975, this class of substances was only put on the list when analytical methods for their detection became available. In Montreal, the screening procedure for AAS was based on an immunoassay, 20,21 while confirmation with GC-MS was applied after hydrolysis, purification and concentration and finally a trimethylsilyl derivatisation step This method has been substantially modified since then, 27,28 but the principles are still the same. 29,30 It became obvious that MS was the detection method of choice for the definite identification of AAS and their metabolites in urine. The view on the potential of mass spectrometric detection in the 1970s is well characterized by the following wording from that time: The ionisation and subsequent fragmentation of sample molecules introduced into the mass spectrometer produces a spectrum,... which is so characteristic of the compound concerned as to enable a suspect positive to be confirmed unequivocally. 4 Further quality assurance elementswere introduced into the regulations for doping analysis, such as the right of the athlete to be present at the re-analysis of his or her sample. Selected ion monitoring (SIM) and chemical ionization (CI)

4 842 P. Hemmersbach Abundance H CH 3 H CH CH N 58 CH m/z--> Ephedrin M+ 165; Aufnahme G.Sigmund 2000 Figure 2. Mass spectrum of underivatized ephedrine from an adverse finding during the lympic in Munich 1972 compared to a spectrum acquired more recently. were introduced to complement full-scan detection in the EI mode. As many as 1786 urine samples were analysed during the of the XXI in Montreal, and 275 of these were subjected to the new analysis for AAS. f these, 11 samples with an adverse finding led to sanctions, including 8 for AAS. Moscow 1980 It is difficult to retrieve data from the literature about the detection of doping agents during the Moscow lympic. No adverse analytical finding was mentioned after the analysis of 1645 samples in the reports, but even progress in mass spectrometric detection had not eradicated the misuse of prohibited substances during these. Whereas exogenous androgenic anabolic agents had been subjected to testing since Montreal, a new challenge arose through the administration of testosterone esters. The excretion of natural metabolites, such as testosterone glucuronide, could not disclose misuse before 1983 when the introduction of a testosterone/epitestosterone ratio threshold and the re-analysis of the anonymized urine samples collected during the Moscow gave clear indications of administration of testosterone in a considerable percentage of the samples. 31 Los Angeles 1984 Doping analysis at the lympic in Los Angeles marked a considerable change concerning the use of MS as a detection technique. 32 ne year earlier, in 1983, a doping control laboratory equipped with two GC-quadrupole MS instruments was set up for the Pan American in Caracas. Several cases of AAS misuse were disclosed, 33 but it was more striking that many athletes from various countries left the when it became obvious that doping controls were being performed. The Los Angeles in 1984 was the first time when all 1510 urine samples were submitted to a screening analysis using the hyphenated technique of GC-MS. Highperformance liquid chromatography (HPLC) and radioimmunoassay (RIA) were also applied. When the analytical work was initially being planned, it was anticipated that RIA would be used to screen for AAS, but the experience from the Moscow, the re-analysis of the Moscow samples with respect to testosterone doping and advances in detection methods 27,31 changed this plan. The ratio of testosterone to epitestosterone (T/E ratio) was only measurable with the help of GC-MS. This ratio was placed on the list of prohibited substances and a value exceeding 6 constituted an adverse finding. Later, it was discovered that certain athletes showed a natural increase in their T/E ratio 34 and the management of such results was modified. Testosterone and epitestosterone were not the only endogenous steroids playing an important role in the fight against doping. The interpretation of the urinary steroid profile became, and is still, an important tool in the fight against doping. 35 Additionally, another new class of substances, beta-blockers, was also included on the list. Prior to the, 10 test samples were used to assess the laboratory for IC accreditation from the IC Medical Commission Subcommission for Doping and Biochemistry of Sport (Secretary Prof. Dr M. Donike), which was founded in To perform the AAS screening, six gas chromatographs coupled to low-resolution quadrupole mass spectrometry (LRMS) of type HP 5996 were operated; a seventh instrument was used to confirm other doping agents. This enabled 100 samples to be handled each day, with a reporting time of 24 h. The GC-MS instruments were operated in SIM mode, and fused silica capillary columns were directly led into the ion source. The group of analytes for this screening procedure contained both endogenous and exogenous AAS. Eleven adverse findings were reported. Seoul 1988 The development of mass spectrometric detection after gas chromatographic separation was quite intense in the 1980s and for the Seoul new types of mass-selective

5 History of mass spectrometry at the lympic 843 Figure 3. Confirmation result for a sample containing stanozolol metabolites at the lympic in Seoul 1988 and press coverage after the case of Ben Johnson. detectors were installed in the lympic laboratory. Twelve GC-MS combinations (HP5890/5970B) were being used for screening and confirmation. Additionally two so-called MS- Engines (HP5988A) were installed, one with an interface to agcandonetoanlc. ne of the highlights of low-resolution quadrupole MS detection happened without doubt during the lympic in Seoul in After setting a new world record of 9.79 s in the 100 m race, Ben Johnson (Fig. 3) was stripped of the gold medal when he tested positive for the anabolic-androgenic steroid stanozolol (Fig. 3). At that time two metabolites of stanozolol, 3 0 H-stanozolol and 3 0 H-epistanozolol, were detected in the free fraction after liquid/liquid extraction as N-HFB-bis--TMS derivatives. This prohibited steroid was an excellent example of how growing knowledge of steroid metabolism, conjugation and excretion into urine, difficulties with derivatisation and chromatography contributed to a successful detection method in urine. 37 A total of 1601 urine samples were analysed at the laboratory in Seoul with a staff of about 50 scientists and technicians, and 10 positive cases documented. They included anabolic-androgenic steroids, beta-blockers and stimulants. ne finding was caffeine, where the threshold of 12 µg/ml was exceeded. Caffeine was in 2004 removed from the Prohibited List. However, caffeine, together with other substances, has been put on a Monitoring Program 38 and continued laboratory analysis gives WADA the opportunity to review this and other issues later on. In its preparation for the, the Seoul laboratory was quite active in developing GC-MS methodology for stimulants, narcotics and anabolic steroids Diuretics were included into the Prohibited List for the first time in Corticosteroids were substances subjected to certain restrictions in 1988, and the Seoul laboratory did research on the analysis of this class of substances with LC-MS technology. The ionization technique applied was thermospray. 44 The lympic in Seoul were the first in which horses also had to undergo doping control tests. The urine and blood samples were analysed in the same laboratory with analytical methods established and implemented by Prof. Donike. The rigid identification principles, requiring MS as the detecting technique, were also applied to those samples. Barcelona 1992 Between the in Seoul and Barcelona, a new era of doping developed through the occurrence of doping with protein hormones such as the glycoprotein erythropoietin (EP). A recombinant form of EP had entered the pharmaceutical market in 1987 and its oxygen-transportenhancing capacity contributed to a quickly growing doping misuse. Although MS can reveal many details about its structure, 45 other methods such as isoelectric focusing 46,47 have to be used to detect misuse in urine samples. EP was placed on the prohibited list in 1990, although no detection method was available at that time. This also applied to autologuous and non-autologuous blood transfusion. Planning and accomplishing doping analysis during lympic is a challenging and resource-demanding task. 48 Fig. 4 shows the staff involved in doping analysis at

6 844 P. Hemmersbach Figure 4. Laboratory team for doping analysis at the lympic in Barcelona the in Barcelona. This amount of human resources is also reflected in the instrumentation installed to perform the analyses. Thirteen GC-MS instruments (HP5970, HP5971) were used for the screening and confirmation procedures of AAS and non-volatile stimulants, beta-blockers and narcotics. 49 In addition, GC and LC were applied with a variety of other detectors. For the first time, two LC-MS systems with a particle beam interface and a quadrupole as the MS detector were used to confirm diuretics 50 and labile compounds. 51 ne of the adverse findings, the metabolite of the stimulant mesocarb, of Russian origin, could favourably be confirmed with this technique. 52 We are aware that this interface had limitations to fulfil the expectations for a suitable transfer of analytes in a liquid mobile phase to the ion source of a mass spectrometer as is now common with other soft ionization techniques, but at that time particle beam was used because the MS spectra were very comparable with the EI spectra and existing MS libraries. In all 1871 urine samples, 70% from males and 30% from females were submitted to six different screening procedures, with 192 samples as the daily maximum. Five adverse findings were recorded, containing strychnine, norephedrine, mesocarb and clenbuterol (n=2). Mesocarb (Sydnocarb), which was developed as a stimulant in Russia in the 1970s, 53 was and is almost unknown in Western countries. Sharing of knowledge between the doping control laboratories is required to disclose doping with such substances. 54 Further examples from the 1990s will be dealt with later. The other remarkable adverse finding was clenbuterol, a beta-2 agonist, which also shows anabolic properties, 55,56 detected at concentrations down to 1 ng/ml as its methyl boronate derivative. 57 This led to a controversy as to whether the substance should be regarded as prohibited in sport. 58,59 Clenbuterol has since been prohibited, and the former class of anabolic-androgenic steroids was renamed anabolic agents. Lillehammer 1994 In 1994 the Winter were organized between two summer games for the first time, 2 years after the start of the new. With respect to the number of athletes participating, Winter are only a quarter of the size of Summer, and this is also reflected in the number of doping tests (Sapporo 211, Innsbruck 390, Lake Placid 440, Sarajevo 424, Calgary 492, Albertville 522) and adverse findings. However, new mass spectrometric technologies are sometimes introduced in Winter, though perhaps not with the same attention as they would draw in Summer. Two major technical advances in the field of MS were introduced into doping analysis during the Winter in Lillehammer in 1994 and Nagano in Although doping control for stimulants and narcotics by in-competition testing is very efficient and the expected concentrations in urine are quite high, controlling for AAS misuse requires a totally different strategy. The Ben Johnson case in Seoul showed that only out-of-competition testing can be effective in this respect. Analytically, the limit of detection should be as low as possible to achieve a long detection period. Efforts to increase the analytical sensitivity were therefore made in the early 1990s. While the former use of MS for detection in doping analysis was based on a quadrupole mass filter with LRMS, high-resolution mass spectrometry (HRMS) seemed to be promising for two reasons. The increased mass resolution of 5000 to would decrease the biological background in the SIM mode, especially for certain analytes, and the double-focusing sector instruments would be more sensitive than the quadrupole mass filters. After optimization, detection limits for AAS metabolites down to pg/ml could be achieved During the Lillehammer, a subset of the 529 urine samples was analysed by GC coupled to high-resolution MS (GC-HRMS). A double-focusing sector instrument with reverse geometry (Finnigan MAT 95) was installed for that purpose. 63 The laboratory in slo, which served the Lillehammer, had eight GC-MS systems (HP5890-MSD 5970, MSD 5972, Finnigan SSQ 7000) in addition to the HRMS, and the daily number of samples reached a maximum of 60. The introduction of a more sensitive technique for anabolic-androgenic steroids also revealed new elements for the evaluation of analytical results. Low concentrations of norandrosterone in a female had to be evaluated using the scientific knowledge available. In addition to the possibility of endogenous production, 61,64,65 the occurrence of norandrosterone due to the administration of a nonprohibited substance, such as norethisterone, 66 had to be taken into account. This was done in an actual case in 1994, and a WADA Technical Document now provides the harmonized rules, 67 including explanatory notes that take into account new knowledge about the stability of norandrosterone in urine samples. 68

7 History of mass spectrometry at the lympic 845 The Lillehammer were also the first lympic in which blood samples (n=59) were taken and tested for the use of non-autologuous blood transfusions. 69 It is a curiosity that the samples had to be destroyed immediately after analysis, because no research was to be done on them afterwards. Atlanta 1996 During the lympic in Atlanta, urine samples were subjected to doping analysis, and two adverse findings of anabolic-androgenic steroid metabolites ended with sanctions. Increased sensitivity was achieved by using double-focusing sector mass spectrometers for all the samples in the initial testing step. However, decreasing the limit of detection always means that new knowledge about the biological background of the urinary matrix will be obtained. The development of isotope ratio MS in doping analysis had just started and a GC-combustion-isotope ratio mass spectrometer (GC-C-IRMS) was installed in Atlanta. Any introduction of new technology has to be discussed in detail before it is routinely applied. As this discussion was incomplete at the time of the, the IRMS instrument was not used for official analyses (see section Nagano 1998). In former, some of the adverse findings that created major scandals were achieved through new analytical technology or decreased limits of detection. In Atlanta, however, the big headlines were generated because of analytical signals in the mass spectrometers that were so large that the detector might have become overloaded. The Atlanta will be remembered because of the adverse findings identifying the stimulant bromantan, which following intense juridical evaluation ended with the athletes concerned being exonerated. Bromantan 71 is a stimulant that was totally unknown at that time in Western countries. It was developed in Russia and publications in Russian described it as a new immunostimulator and a substance exhibiting psychostimulant features. 72,73 Possible heat-protective effects 74 were also put forward during the hearings. The outcome showed both the benefits and the drawbacks of the wording that ends the list of examples for the various classes of prohibited substances:...and other substances with a similar chemical structure or similar biological effect. 15 We can describe bromantan as a designer doping agent, which was followed by carphedon (4-phenylpiracetam) shortly afterwards. This stimulant was also developed in Russia and both incidents also underline the need for continuous sharing of knowledge between all WADAaccredited laboratories. Carphedon can be regarded as phenyl-substituted piracetam, in which through this substitution a phenylalkylamine structure is generated. Analytically, these designer drugs were easily detected by GC and GC-MS 75,76 once the structures (Fig. 5) were known. The mass spectrometric properties soon found their way into most mass spectrometric libraries. 77 Nagano 1998 The other MS technology introduced at lympic Winter was the hyphenated technique of GC combined with H N Br (1) (2) N NH 2 Figure 5. Designer stimulants bromantan (1) and carphedon (2), disclosed in doping controls at the Atlanta and Turin, respectively. a combustion unit and an isotope ratio mass spectrometer (GC-C-IRMS) in Nagano The application of IRMS techniques was developed for doping control purposes after It was shown that administration of testosterone led to differences in the 13 C: 12 C ratio between testosterone metabolites and endogenous reference compounds that were not involved in the testosterone metabolism. 81 The same technological approach was extended to other endogenously produced steroids, such as dehydroepiandroesterone. 82,83 Steroid profiling and IRMS became the two analytical means to disclose doping with endogenous steroids. 84 lympic always provide opportunities to evaluate normal ranges of urinary constituents. The fact that no finding of norandrosterone above 2 ng/ml was detected in more than 1000 urine samples during the Lillehammer and Nagano provides support for the correctness of the threshold setting for that metabolite. 85 The laboratory in Tokyo had seven GC-MSD instruments, two GC-HRMS instruments and one GC-C-IRMS instrument. In all 621 urine samples were analysed and one muchdiscussed adverse finding was made, the detection of 11- nor-1 9 -tetrahydrocannabinol-9-carboxylic acid, a cannabis metabolite at a concentration exceeding the threshold of 15 ng/ml. At that time cannabis products were subject to certain restrictions; today they are prohibited during competition. Sydney 2000 Although the 1990s were characterized by the increasing appearance of doping with a protein hormone, the EP, including the scandals during the 1998 Tour de France, detection of this misuse was still not developed. This would change at the beginning of the new century, but regrettably not yet through mass spectrometric detection. Two approaches were followed to reveal EP misuse, a direct approach of identifying recombinant forms of EP in urine, 46,86 and an indirect approach of determining physiological effects on haematological parameters in blood During the of the XXVII in Sydney, focus was directed at revealing doping practices that increase the transport capacity of oxygen. At that time, analytical proof was based on a combination of the two approaches described above. A total of 625 blood samples were drawn. f the 2769 urine samples were taken out-ofcompetition because effective doping control requires outof-competition testing. Many classes of prohibited substances can also be effective as doping agents after the metabolites have left the athlete s body (e.g. AAS or EP).

8 846 P. Hemmersbach All previously implemented mass spectrometric techniques, such as GC-LRMS (20), GC-HRMS (4), GC-Ion Trap MS (1), GC-C-IRMS (2) and LC-MS (1), were applied during the. The greater number of samples required more instruments, and the number is shown in brackets. IRMS was fully implemented for the first time during these Summer and criteria for applying this technique included increased steroid concentrations in the initial testing procedure. The T/E ratio, androsterone, etiocholanolone and dehydroepiandrosterone concentrations and parameters indicating dihydrotestosterone misuse were evaluated. The WADA performed an Independent bserver Programme 93 for the first time during the Sydney. The Sydney lympics are also associated with a new trend, which can be regarded as a result of a legislative change in the United States of America in The Dietary Supplement Health and Education Act 94 came into effect at that time and initiated the production and distribution of nutritional supplements that caused grave concern in the fight against doping. Many of these products, sold over the counter in the US, contained prohormones, steroids which could be metabolised to doping agents in the human body and excreted as their markers and metabolites. These prohormones, 95,96 including androstenedione, 97 dehydroepiandrosterone, 98 norandrostenedione and norandrostenediol, caused adverse analytical findings and led to athletes being sanctioned. Some companies also put other nutritional supplements on the market, which were advertised with energy-boosting properties and contained substances such as pyruvate, ribose and creatine. These products were not prohibited, but sometimes turned out to be contaminated with prohormones In general, an increasing use of nutritional supplements was recorded during the Sydney 104 and some of the 11 adverse findings that were generated through an optimized sensitivity of mass spectrometric detection were probably related to such contaminations. The progress of the identification of doping agents by MS has been supported by a considerable work in synthesizing reference standards. While many of the anabolic steroids metabolites were first synthesized by the Cologne laboratory, 30 in the preparation for the Sydney a project for these tasks was initiated, which resulted in the establishment of a company still supplying the laboratories with certified reference standards. 105 Today many stakeholders in anti-doping are funding this important task and many laboratories are involved in it. Salt Lake City 2002 The 2002 Winter in Salt Lake City, along with those in Turin in 2006, are mainly remembered in the fight against doping for the discoveries of doping practices related to the enhancement of oxygen transfer. This comprises both the misuse of glycoprotein hormones, such as recombinant erythropoietins and its analogue darbepoetin or the novel erythropoiesis stimulating protein (NESP), and the practices of autologuous and non-autologuous blood transfusions. It was a considerable surprise for certain persons that the newly released preparation Aranesp, containing darbepoetin, also called NESP, was revealed during the Salt Lake City in three athletes, including in the Spanish cross-country skier, Johann Mühlegg. 106 Seven hundred urine samples, including 100 from outof-competition tests, were analysed, in addition to 77 tests in which blood and urine were collected. The mass spectrometric procedures were extended by a method for detecting hydroxyethyl starch (HES) misuse. This substance belongs to a new group of doping agents, plasma expanders, which was included in the group of diuretics and other masking agents in the Prohibited List. 15 The first adverse findings were made during the 2001 World Championships in Nordic Skiing in Lahti, Finland. The analytical methodology, also used in Salt Lake City, was based on hydrolysis of the macromolecule, with subsequent extraction, derivatisation and GC-MS detection. 107,108 The use of LC-MS 109,110 or matrix assisted laser desorption- time-offlight (MALDI-TF) MS 111 also reveals this misuse and that of other plasma expanders. Certain new pharmaceutical developments, such as efaproxiral, which is a synthetic allosteric modifier of the affinity of haemoglobin for oxygen, could be detected by GC-MS using existing methods. 112 Additional to the adverse findings of NESP, one analytical result generated by MS attracted considerable attention, a finding of levmetamfetamine (R-( )-methamphetamine). Both stereoisomers (R-( )- and S-(C)-methamphetamine) are prohibited, but have a very different potential of stimulating properties. 113 Therefore, today the level of a sanction might be different. In order to discriminate between the two optical isomers, chiral separation has to be performed either chromatographically or by transferring the enatiomers into diastereomers through derivatisation Such chiral separation has found other applications in doping control as, for example, for the differentiation of oral and inhaled salbutamol administration. 121 Themisfortunewiththiscase was the fact that the administration of levmetamfetamine might have been caused because a preparation with the same name contained the prohibited substance in the United States, while it did not in Great Britain. Athens 2004 The of the XXVIII were held in Athens between 13 and 29 August In all 27% of the participating athletes were selected for doping control. The total number of samples analysed was 3617, of which 691 were blood tests. 122 WADA again issued a detailed independent observer report, which also covered the laboratory analyses. 123 When preparations were being made for the Athens, possible new doping practices were identified, placed on the Prohibited List and included in the analytical repertoire of the lympic Laboratory. Agents with anti-estrogenic activity were included into the laboratory s repertoire, but doping control had also to meet new techniques such as enhancement of the uptake, transport or delivery of oxygen in the body through, for example, haemoglobin-based oxygen carriers (HBCs). The initial testing for these preparations is straightforward in blood samples, because administration will colour the

9 History of mass spectrometry at the lympic 847 serum red. For confirmation, LC-MS detection methods were developed in addition to the size-exclusion LC procedure. ne of the most striking incidences in doping control in the time before the Athens was the BALC Affair. BALC stands for Bay Area Laboratory Co-operative, a laboratory in California, USA, which was involved in the synthesis of designer steroid doping agents. 128 The most interesting substance was tetrahydrogestrinone (THG), a previously totally unknown steroid. After disclosure of the substance by analytical means, 129 its anabolicandrogenic properties could also be proven. 130 nly a few examples of true designer doping agents are known (e.g. bromantan and carphedon), but the skill, knowledge and creativity in the THG case is striking (see Fig. 6). Not only THG but also the improved detection of glucocorticoids made LC-MS-MS very important in the WADA laboratory in Athens, 131 where the total number of mass spectrometers included 23 GC-MSDs, 4 GC-HRMS, 6 LC-MSD Ion Traps, 2 GC-C-IRMS and 1 GC-TF-MS. 132 The mass spectrometric detector technology was subsequently combined with Norethisterone H Norgestrel Gestrinone H H Norethandrolone H H Norboletone H Tetrahydrogestrinone (THG) Figure Ethinyl-substituted steroids norethisterone, norgestrel and gestrinone with no or minor anabolic properties being reduced to the anabolicandrogenic steroids norethandrolone, norboletone and tetrahydrogestrinone (THG) and indication of year of first reporting. Cl TMS H M + : m/z = 420 N m/z = m/z = TMSHN m/z = Cl Figure 7. Ion chromatograms for a clenbuterol confirmation with GC-HRMS at the lympic in Athens. The suspicious sample (left) is compared to a negative control urine and a positive control sample at 0.2 ng/ml (right). The selected ion masses are indicated.

10 848 P. Hemmersbach ultra performance liquid chromatography (UPLC-TF-MS) and applied to the detection of corticosteroids. 133 When Yahoo! Sports drew up its world sports poll in 2004, the record-breaking 23 positive doping cases in Athens 2004 came high on the list, beaten only by Greece as European Champion in football, the lympic itself, Lance Armstrong as Tour de France winner and Michael Schumacher from Formula 1 racing. Most of the adverse findings were related to old-fashioned anabolic-androgenic steroids and other anabolic agents. This was partly because HRMS also made it possible to apply initial testing with a sensitivity of about 10% of the minimum required performance levels (MRPL) set by WADA. 134 Fig. 7 shows a confirmation result for clenbuterol as a bis-tms-derivative in the SIM mode at an estimated urinary concentration of 0.1 ng/ml. Many of the adverse findings were at low levels, possibly indicating that not all nations have an effective dayto-day and all-year-around doping control programme with out-of-competition tests at appropriate times. Doping analysis always has some surprises in stock. During the lympic in Athens, one adverse finding of the stimulant isometheptene by GC-MS created the need for a follow-up to study the metabolism and compare it with that of heptaminol. 135 When reviewing the doping cases of Athens, one has to realize that it was the first time that doping cases without an analytical result attracted the main attention, cases where the athletes denied to deliver a urine sample or where it not even was possible to get into contact with the athletes for conducting a doping test. Turin 2006 Recent developments in the LC-MS-MS technique continued in preparation for the Turin. 136 GC and LC were simultaneously applied to joint urinary extracts. 137 The urine samples were extracted and concentrated with the inclusion or exclusion of hydrolysation steps. After dividing the extracts, the samples were subjected to both GC-MS and LC-MS, depending on what was most suitable for the substances in question. The GC runs were normally preceded by a derivatisation step. In general, it has been shown that several classes of prohibited substances are more easily tested by LC-MS/MS and considerable efforts have been made for a method transfer of stimulants, narcotics, anabolic agents, diuretics, beta-blockers and other doping agents. Today, GC and LC supplement each other to achieve the necessary separation before mass spectrometric detection. When new doping agents appear, as in the case of sibutramine, the first approach will be to incorporate the new analyte into an existing procedure. 147,148 The mass spectrometric instrumentation in Turin differed from that in Athens only in that 3 LC triple-stage quadrupole MS instruments (Applied Biosystems API 4000/3200) were used in addition to 14 GC-MSD (Agilent 5975), 2 GC-HRMS (Waters Autospec Ultima) and 2 GC-C-IRMS (Thermo Delta Advantage Plus) instruments. Installing mass spectrometric instruments in a new lympic laboratory is not always very trivial as shown in Fig. 8. Figure 8. Transportation of the magnet of a high resolution mass spectrometer into the new building of the Turin lympic Laboratory. Not all adverse findings during the result in a sanction. Many of the 39 adverse findings related to beta-2-agonists and glucocorticosteroids, where athletes had received an abbreviated therapeutic use exemption (ATUE) from their respective anti-doping organization. Such findings were previously not reported, but only checked after the initial testing procedure. Thankfully, this practice is again in place from 1 January ther results reported relate to the disclosure of a prohibited substance, which might be the metabolite of a non-prohibited compound. This was the case with a couple of cathine determinations higher than the threshold of 5 µg/ml in urine. The concurrent identification of high amounts of pseudoephedrine indicates that the cathine amount was in agreement with the administration of pseudoephedrine. Pseudoephedrine is not prohibited. As a sanctioned adverse finding, the designer stimulant carphedon resurfaced after almost a decade (see also Fig. 5). As mentioned before, most focus was placed on blood doping and EP testing of 392 samples. An assessment of the anti-doping activities can be found in the report of WADA s Independent bserver Programme for these. 149 Beijing 2008 The of the XXIX will be held in Beijing from 8 to 24 August It is expected that 4500 samples will be taken from the participating athletes. The 2008 Prohibited List 15 will form the basis for judging potential violations of the World Anti-Doping Code. The analyses will be performed according to the International Standard for Laboratories, 150 MS will be applied for both initial testing and confirmation procedures.atthisstep, MSisthe only authorized confirmation method. MS may be applied in conjunction with GC or LC. This statement from the International lympic Charter of Doping in Sport in has been applied since 1972 in Munich and will be applied in Beijing in The only exceptions are made for the analysis of protein hormones. For certain proteins, such as insulins, which were prohibited after the lympic in Nagano 1998, mass spectrometric detection methods are in place today. 152

11 History of mass spectrometry at the lympic 849 Strict criteria will be applied 153 to identify prohibited substances, their metabolites and markers. The mass spectrometric instrumentation will include, but is not limited to, the hyphenated techniques of GC coupled with lowand high-resolution MS, a combustion unit and IRMS, as well as LC coupled to single- and triple-stage quadrupole mass spectrometers and ion-trap, TF or orbitrap technologies. The exact analytical repertoire will, as usual, not be announced before the. Any adverse finding from the WADA-accredited laboratory 16 will be assessed by members of the IC Medical Commission Group before the legal procedure starts immediately. Again an adhoc division of the International Court for Arbitration for Sport will be available in Beijing. 154 CNCLUSINS The use of MS in connection with doping analysis during the lympic gives a perspicuous picture of the analytical challenges and the solutions that the IC and WADAaccredited laboratories have developed and applied. MS is the only accepted technique when a prohibited substance, its metabolite or marker is being identified. Alternative detection is allowed only for substances and methods, for which mass spectrometric detection is impossible or currently impossible in the expected concentration range. In general, MS is now applied in WADA-accredited laboratories, following separation by either LC or GC, with ionization techniques such as EI, CI, electrospray ionization (ESI), atmospheric pressure ionization (APCI) and atmospheric pressure photoionization (APPI). The mass spectrometer configuration ranges from lowresolution quadrupole techniques, via ion-trap and sector instruments, to time-of-flight (TF) detection. MS n experiments are achieved either in triple-stage quadrupole or ion-trap instruments, including linear ion-trap and orbitrap technology with collision-induced dissociation (CID), electron transfer dissociation (ETD) and pulsed Q dissociation (PQD). Both target analysis and general approaches for unknown doping agents are applied. 158,159 Acknowledgements Special thanks are due to all my colleagues in IC/WADAaccredited laboratories and other laboratories, national anti-doping agencies and the International lympic Committee, which contributed through their friendly collaboration to the information brought forward in this article. REFERENCES 1. Andren-Sandberg A. Doping, 2nd ed. rion Pharma AB: Stockholm, Prokop L. The struggle against doping and its history. Journal of Sports Medicine and Physical Fitness 1970; 10: Burstin S. La Lucha contra el Dopage. Revista de Derecho Sportiva 1963; 3: Beckett AH, Cowan DA. Misuse of drugs in sport. British Journal of Sports Medicine 1979; 12: Beckett AH, Tucker GT, Moffat AC. Routine detection and identification in urine of stimulants and other drugs, some of which may be used to modify performance in sport. Journal of Pharmacy and Pharmacology 1967; 19: Dirix A, Sturbois X. The First Thirty Years of the International lympic Committee Medical Commission. International lympic Committee: Lausanne, 1998; Dirix A. The doping problem at the Tokyo and Mexico City lympic. Journal of Sports Medicine and Physical Fitness 1986; 6: Beckett AH. Use and abuse of drugs in sport. Journal of Biosocial Science-Supplement 1981; 7: Clasing D, Donike M, Klümper A. Dopingkontrollen bei den Spielen der XX. e München Leistungssport 1974; 4: Clasing D, Donike M, Klümper A. Dopingkontrollen bei den Spielen der XX. e München 1972 (III). Leistungssport 1974; 4: Donike M, Clasing D, Klümper A. Dopingkontrollen bei den Spielen der XX. e München 1972 Teil 2. Leistungssport (Berlin) 1974; 4: Clasing D. Doping und Seine Wirkstoffe Verbotene Arzneimittel im Sport. Spitta Verlag: Balingen, 2004; Todd J, Todd T. Significant events in the history of drug testing and the lympic movement: In Doping in Elite Sport The Politics of Drugs in the lympic Movement, Wilson W, Derse E (eds). Human Kinetics Publishers: Champaign, 2001; International lympic Committee. List of Prohibited Classes of Substances and Prohibited Methods of Doping, Lausanne, World Anti Doping Agency. The 2008 prohibited list, List En.pdf, accessed on 05 April World Anti Doping Agency. WADA-accredited laboratories, idd333, accessed on 05 April Donike M, Kaiser CH. Moderne methoden der dopinganalyse. Sportarzt und Sportmedizin 1971; 3: Hemmersbach P, de la Torre R. Stimulants, narcotics and ß- blockers: 25 years of development in analytical techniques for doping control. Journal of Chromatography B 1996; 687: de Mérode A. Doping tests at the lympic in Journal of Sports Medicine and Physical Fitness 1979; 19: Brooks RV, Firth RG, Sumner NA. Detection of anabolic steroids by radioimmunoassay. British Journal of Sports Medicine 1975; 9: Brooks RV, Jeremiah G, Webb WA, Wheeler M. Detection of anabolic steroid administration to athletes. Journal of Steroid Biochemistry 1979; 11: Bertrand M, Massé R, Dugal R. GC-MS approach for the detection and characterisation of anabolic steroids and their metabolites in biological fluids at major international sporting events. Farmaceutische Tijdschrift Voor Belgie 1978; 55: Donike M. N-Methyl-N-trimethylsilyl-trifluoracetamid, ein neues Silylierungsmittel aus der Reihe der silylierten Amide. Journal of Chromatography 1969; 42: Donike M. Zum problem des Nachweises der anabolen steroide: gas-chromatographische und massenspezifische Möglichkeiten. Sportarzt und Sportmedizin 1975; 26: Donike M, Zimmermann J. Zur Darstellung von Trimethylsilyl-, Triethylsilyl- und tert.-butyldimethylsilyl-enoläthern von Ketosteroiden für gas-chromatographische und massenspektrometrische Bestimmungen. Journal of Chromatography 1980; 202: Ward RJ, Shackleton CH, Lawson AM. Gas chromatographic mass spectrometric methods for the detection and identification of anabolic steroid drugs. British Journal of Sports Medicine 1975; 9: Donike M, Zimmermann J, Bärwald KR, Schänzer W, Christ V, Klostermann K, pfermann G. Routinebestimmung von Anabolika im Harn. Deutsche Zeitschrift für Sportmedizin 1984; 35: Masse R, Ayotte C, Dugal R. Studies on anabolic steroids I. Integrated methodological approach to the gas chromatographic-mass spectrometric analysis of anabolic