Mass Spectrometry in Sports Drug Testing

Characterization of Prohibited Substances and Doping Control Analytical Assays

Mario Thevis

• Publisher: Wiley
• Year: 2010
• ISBN:
  9781118035146
• Edition: 1
• Topic: Naturwissenschaften
• Subtopic: Spektroskopie & Spektralanalyse

Enables you to detect, identify, and characterize hundreds of drugs that may be used by athletes

Mass spectrometry has become essential to sports drug testing. This book examines both the principles of sports drug testing and the use of mass spectrometry techniques and mass spectral data to detect, identify, and characterize hundreds of known and unknown drugs that athletes may use to enhance their performance. The author provides a detailed overview of the mass spectrometry of numerous classes of therapeutics and agents, various analyzers to detect low- and high-molecular weight drugs, as well as techniques to discriminate between endogenously produced and synthetically derived compounds.

Mass Spectrometry in Sports Drug Testing begins with a full chapter dedicated to the history of sports drug testing. Next, the book provides the principles and techniques needed to maximize the specificity and sensitivity of mass spectrometric assays, including:

• Detailed, step-by-step assays with sample preparation
• Discussion of both chromatographic separation and mass spectrometric analysis
• Characterization of analytes in order to unequivocally identify banned substances
• Mass spectrometric behavior of low- and high-molecular weight analytes

Throughout the book, descriptive examples illustrate the principles, advantages, and limitations of different assays.

Mass Spectrometry in Sports Drug Testing not only sets forth the role mass spectrometry plays in detecting drug use among athletes, it also adds new insights into the health and ethical issues of doping in sports.

1

History of Sports Drug Testing

1.1 HISTORICAL ATTEMPTS OF ARTIFICIAL PERFORMANCE ENHANCEMENT

Sports competition is initiated by many different motivations, which include, but are not limited to, mankind’s desire for excellence and perfection, the enjoyment of contests, and financial as well as social benefits associated with victory and success at sporting events.¹ In particular, the latter facts are frequently mentioned as the major reasons for attempts of sportsmen to artificially increase their physical performance and that this modern and contemporary issue was not present between 776 B.C. and 393 A.D. at the ancient Olympic Games. However, the belief that athletes participating at the ancient Panhellenic Games, which included the Olympic Games as the most prestigious sporting festival, were motivated only by the glory and appreciation that they might receive, is weakened even by the interpretation of the term “athlete.” The origin of this expression, the Greek noun athlon, means “prize” or “reward;” its verbal form athleuein means “to compete for a prize.” Consequently, the athlete is a person who competes for a prize, which was reportedly a great value even in ancient times,² and sport for sport’s sake was not an ancient concept.³ In fact, even thousands of years ago, athletes were seeking competitive advantage over their rivals in many different ways, which included manipulation of equipment and corruption of judges. Moreover, the consumption of certain mushrooms as psychogenic aids was reported,⁴ and Philostratus (3rd–2nd century B.C.) and Pliny the younger (1st century A.D.) wrote notes about athletes consuming bread prepared with juice of the poppy plant (opium) and the use of a decoction of the hippuris plant, respectively.⁵ All was done with considerable support by the doctors and the goal to enhance the athletes’ performance, which would possibly be referred to as “doping” in a modern context. Physicians acted as coaches and doctors simultaneously, and one of the most famous medical attendants was Herodicus, the indoctrinator of Hippocrates, who was particularly interested in athletes’ nutrition and rehabilitation.⁶ In addition to the human competitors, horses were also the subject of treatment to increase their endurance and stamina as reported in the ancient Rome where hydromel, a mixture of honey and water, was administered to horses in chariot races.⁷ With the growing medical and pharmaceutical knowledge in the 19th and 20th century, more and more attempts of artificial performance enhancements were assumed and reported, which were conducted with humans and animals likewise. Consequently, various initiatives were started to counteract the misuse of drugs and methods to surreptitiously increase power, strength and athletic capabilities. Such approaches were first conducted with horse saliva in the early 20th century, and, approximately 50 years later, applied also to human urine and blood specimens.

1.2 BACKGROUND AND RATIONALE OF DOPING CONTROLS

Numerous reasons for banning drugs and methods of doping and manipulation from sports were defined by anti-doping authorities such as the medical commission of the International Olympic Committee (IOC) or the World Anti-Doping Agency (WADA) and first necessitated the recognition of the issue. In 1988, the IOC medical commission drafted a charter stating that, “The use of drugs and other substances and banned methods to enhance or accentuate athletic performance is a tragic reality that must be eliminated from modern sport.”⁸ These fundamental words still reflect the principles that are still seminal to anti-doping programs, which have been coined to preserve the “spirit of sport.” According to the World Anti-Doping Code, the spirit of sport is characterized by various values including ethics, fair play and honesty, health, dedication and commitment, and respect for rules, laws, self, and other participants, etc.⁹ Doping, however, contravenes to all of these aspects and, thus, modern doping controls are focused on substances and methods of doping that meet at least two of three criteria as defined by WADA: (1) A substance or method has the potential to enhance or enhances sport performance as evidenced by medical or scientific data, pharmacological effects, or experience; (2) a substance or method represents an actual or potential health risk to the athlete as evidenced by medical or scientific data, pharmacological effects, or experience; and (3) a substance or method violates the spirit of sport as defined in the World Anti-Doping Code.⁹

1.2.1 Cheating

Sportsmanship implements the idea of fair play and the integrity of all members of the sporting community. The ideology that only “eligible” persons should be allowed to compete was present also during the ancient Olympic Games and outlined in the facts that only athletes who were never convicted of a crime should participate. Moreover, sportsmen were requested to swear that they had trained for 10 months prior to coming to Olympia, and another 4 weeks on-site being supervised by the Helenedonakai—the judges.⁴ Doping contravenes the most basic principles of fair play and results in beguilement of competitors and spectators. Both are hoodwinked, and in particular the deceived athlete might suffer from financial, social, and probably occupational disadvantages in addition to a personal disappointment, if he/she loses a competition against an athlete who artificially increased his/her performance. Consequently, doping must be regarded as cheating in numerous regards, and the rights of those athletes, who are devoted to clean and fair sports, must be protected.

1.2.2 Health Issues

Doping practices can compromise the short- and long-term physical and mental health of athletes; hence, health and safety concerns have been a major aspect of the fight against doping. Numerous articles were published dealing either with case reports about serious or even fatal consequences of drug abuse in professional and amateur sport as well as general undesirable effects observed and associated with doping,^10–20 which were supposedly the final trigger for international sport federations to establish anti-doping rules and test their athletes for drug abuse.²¹

1.2.3 Ethical Issues

According to the World Anti-Doping Code⁹ and the common understanding of the intrinsic value of sport, doping categorically contradicts the spirit of sport. This issue has also ethically been evaluated and all values attributed to the spirit of sport have been subject of ethical considerations.²² Fair play and honesty, character and education, and the virtue of athletes, are an integral part of sport pedagogy and pedagogic ethics, which is complemented by numerous additional aspects of ethics in sports concerning the health and the exploitation of the human (or animal) body as well as the respect for rules, laws, self, and other participants. The violation of ethical principles is not acceptable in sport, and noncompliance is regarded as a doping offence.

1.3 EARLY DETECTION METHODS: POSSIBILITIES AND LIMITATIONS OF ASSAYS WITHOUT MASS SPECTROMETRY

Doping of animals, primarily horse and hound, has been considered a major pacemaker of doping practices in modern human sports but also as a driving force of anti-doping activities. In 1666, the first decree was enacted in England, which prohibited the administration of substances to horses aiming to improve their performance in races at Worksop,¹² and severe consequences up to the death penalty were announced and executed as reported in the late 18th century in Cambridge (Great Britain) when horses were poisoned at Newmarket.²³ In light of such regulations and their strict enforcement, it was a logical consequence that the first successful attempts to detect doping agents using bioassays and analytical chemistry were introduced in horse racing rather than in human sports.

1.3.1 First Applications Using Chemical and Biological Approaches in Horse Doping Control

In 1910, the Austrian Jockey Club hired a Polish pharmacist named Alfons Bukowski²⁴ (who has, at some occasions, been referred to as a Russian scientist) to establish a method that allows the detection of alkaloids such as morphine and heroin in equine saliva. He reportedly succeeded in developing such a method but never disclosed any details and returned to his home country, which prompted the Austrian Jockey Club to call in Professor Sigmund Fränkel from the University of Vienna to install a new procedure enabling saliva drug testing. Although never published by Fränkel himself, the principle procedure was later described by G. Lander, the chief chemist for the Jockey Club of England,²⁵ who published a method that included various consecutive extraction and concentration steps followed by chemical reactions forcing precipitation and/or color reaction of alkaloids for visual inspection. In general, the employed approach was mainly a miniaturized application of the Stas-Otto process,²⁶ which gained public recognition as early as 1850 when its use helped reveal the murder of Gustave Fougnies and strongly influenced the newly born arena of forensic sciences.²⁷

First, a comparably large volume of saliva was required, which was preferably obtained by washing the horse’s mouth using a 0.16 M acetic acid solution over a period of up to 5 minutes. The obtained material was extracted using 90% pure ethanol and diluted acetic acid, followed by filtration and concentration of the extract in vacuo to an aqueous residue of approximately 5 mL. The solution was purified by ether extraction and its volume further reduced by evaporation to yield a viscous remainder, which was again extracted with small amounts of ethanol that was finally concentrated to dryness. The dry residue was extracted using 0.3 M aqueous hydrochloric acid, the aqueous layer was adjusted to alkaline pH followed by extraction using chloroform and benzene, the combined organic layers were concentrated to dryness, and the remaining residue reconstituted in 0.16 M acetic acid. The presence of an alkaloid (including cocaine, strychnine, quinine, morphine, and heroin) was visualized by formation of a precipitate or opalescence when placing approximately 5–10 µL of the saliva extract in a capillary test tube and adding different reagents as listed in Table 1.1.²⁵ Depending on the target analyte and the employed chemical, estimated detection limits between 0.05 and 20 µg were accomplished using diluted reference compounds. The applicability to authentic saliva specimens and, as such, the proof-of-principle, was provided using blank saliva samples of reportedly untreated animals as well as specimens derived from administration studies to outline the specificity of the method and the ability to “unambiguously” differentiate between positive and negative results.

TABLE 1.1: Estimated Detection Limits (µg) for Alkaloids Using Colorimetric Test Methods According to Lander (1930)^a

^a Ref. 25.

Chemical saliva tests underwent further developments that aimed for optimized extraction conditions of target analytes and more sensitive assays.²⁸ Using a defined array of tests, opiates were the first to screen for preferably by the Marquis reagent (formaldehyde and sulphuric acid), which yields a dark red-to-purple color in the presence of opiates, particularly morphine and heroin. Subsequently, analyses for strychnine (vanadic acid/sulphur-chromate test), quinine (bromine-ammonia test), cocaine, nikethamide, atropine, etc. (crystalline methods) were conducted. These and other compounds were used as mixtures, e.g. 1.5 g of heroin, 2.5 g of strychnine, 2 minims of nitroglycerine (accounting for 1/250 U.S. fluid ounces or 0.12 mL), 5 minims of tinctura digitalis, and 2 ounces of cola nut, and applied to race horses approximately 1 hour before a race.²³ Numerous additional concoctions made from stimulants, narcotics, herbal extracts, and organic as well as inorganic poisons were employed for horse doping purposes in the early 20th century,²⁹ and astonishing estimations about the prevalence of doping in equine sports were published mentioning more than 50% of doped horses in the United States in the early 1930s.³⁰ However, the numbers dropped in the following years possibly due to improved analytical procedure...