Fighting the doping cheats

Daniel Leigh explores the evolution of mass spectrometric approaches in sports drug testing At the Winter Olympics in Sochi in February, the biathlete Evi Sachenbacher-Stehle tested positive for the stimulant methylhexaneamine. Methylhexaneamine is found in some dietary supplements and was added to the World Anti Doping Agency (WADA) list of prohibited substances in 2010. Following this incident and others like it, experts speculated that many doping cases are still likely to remain undetected. Other high profile cases in recent years include that of the cyclist Lance Armstrong, who last year admitted to blood doping, and the use of growth hormone and steroids throughout his career¹. Sprinters Tyson Gay and Asafa Powell have also been in the news recently having returned to competitive sport after being banned from competing after failed drug tests. Doping has been a problem in competitive sports since ancient times and is ever evolving. New compounds are constantly being developed and new strategies are being used to avoid detection. The challenge is to identify the next doping agent and to develop a test able to detect it. This requires methods that can detect substances that are unknown and have not been chemically characterised. The accuracy and sensitivity of the method of detection is also important, as a false positive result could mean that an athlete was falsely banned from competing or a false negative result could mean an athlete who had taken a performance enhancing substance could escape detection. Numerous classes of drugs including anabolic androgenic steroids, ?2 agonists, hormone agonists, diuretics, peptide hormones and growth factors are used by doping athletes and are screened for in doping control programs². New active pharmaceutical ingredients and designer substances that are coming onto the market and are not yet approved as drugs also have the potential for abuse. As a result, the regulations and detection strategies for banned substances and methods of doping are frequently updated. The WADA updates the list of doping substances annually and each year more compounds are added to the prohibited list³. Historically anabolic compounds such as orally administered or injectable steroids were abused. Then there was a move towards endogenous steroids, which were more difficult to detect. However, improvements in analytical methods have meant that endogenous steroids as well as synthetic steroids can be detected and distinguished from one other⁴. Since then, mimetics and prohormones have become popular⁵. Today, some of the most potent stimulating agents (e.g. amphetamine) are less commonly used, because relevant doses of these substances are easily detected. However, less efficient stimulants such as caffeine, modafinil or methylhexaneamine have been used, with caffeine being added to the WADA monitoring program and modafinil and methylhexaneamine added to the prohibited list. Today, athletes have what is known as a ‘biological passport’ that can be used to track the athlete’s blood profile and selected biomarkers over time. The biological passport is important because it can help identify irregularities in an athlete’s profile. For example, haemoglobin concentration normally remains relatively constant throughout life but can be altered following use of drugs such as testosterone. Thus a large change in haemoglobin concentration would provoke further testing. Mass spectrometry methods are used for routine screening of samples for drugs of abuse. If peaks are identified in the spectra that indicate a banned substance, an athlete’s sample can be subjected to further analysis and investigation. At present mass spectrometry (MS) coupled with chromatographic separation is the dominant analytical technique used for the detection of doping agents. Over the years there has been a progressive shift from gas chromatography (GC)/MS to liquid chromatography (LC)/MS and LC/tandem MS (MS-MS) (see Table 1). [caption id="attachment_40681" align="alignleft" width="200"] Table 1: Changing trends in doping agents and methods of detection. Adapted from (6).[/caption] The advantage of LC/MS-MS is that it can be more accurate, more sensitive and higher throughput than alternative methods. Recent LC/MS-MS systems allow rapid screening, identification and quantification of hundreds of compounds in a single analysis. Conventional methods can involve specialised immunoassays, which may require multiple assays and repeated experiments and can be more expensive and time consuming. LC/MS-MS and ultra-high performance liquid chromatography (UHPLC)/MS-MS are tandem MS techniques that overcome the limitations of other more traditional technologies such as GC-MS and immunoassays. UHPLC/MS-MS and LC/MS-MS can easily and rapidly quantify multiple drugs simultaneously and in various matrices such as saliva, urine and serum. Urine is the simplest fluid to analyse but the analysis of blood samples is growing in popularity. Detectability of compounds in urine requires knowing how the compound is chemically transformed in the body and its kinetics of excretion. The time windows for detection can be determined by pharmacokinetics and the analysis of terminal metabolites. Modern LC/MS systems are available that can accurately identify drug metabolites such as the AB SCIEX QTRAP 5500 LC/MS-MS system. Blood (including serum, plasma or dried blood spots) can be used for the detection of blood doping and of erythropoiesis (EPO) stimulating agents. The traditional method for the detection of EPO is an isoelectric focusing test developed in 2000 by a team in France. The isoelectric focusing test has limitations in that the method takes a long time to perform and it can be very difficult to detect synthetic EPO. The development of a mass spectrometric method for the detection of EPO has been challenging. However, last year a paper was published in the journal of Drug Testing and Analysis, which presents the first analytical data on purified human urinary EPO generated with a high resolution high accuracy mass spectrometer (LTQ-Orbitrap)⁷. These results suggest that researchers are one step closer to developing a suitable mass spectrometric method. Advances have been made in the coupling of mass spectrometry with the most recent developments in chromatography and with novel sampling approaches such as solid-phase microextraction, which has been used in detection of the latest small molecule – doping agents. For example, Dr Ezel Boyaci and team have developed a new fully automated high-throughput method based on thin-film solid-phase microextraction and LC/MS for the simultaneous quantitative analysis of 110 doping compounds. The developed assay will allow the fast and reliable analysis of various prohibited substances and may be an attractive alternative to more traditional assays⁸. Other advancements in drug testing methods in sports include the paired ion electrospray ionisation (PIESI) method, which was developed by Dr Daniel Armstrong and his team at the University of Texas⁹. The method can be used on existing test instruments with the addition of a capillary tube. A high voltage is applied to the capillary tube where the fluid sample becomes a fine spray, the charged sample molecules then enter the mass spectrometer to be analysed. This method is useful for detection of steroids and amphetamine but not larger proteins such as EPO. A chemical agent is used to bind to traces of steroid or amphetamine making them more detectable and the method is thought to be 1000 fold more sensitive than other mass spectrometry techniques. Doping in sports is an ever-evolving problem and investment in research is needed to keep up with the latest trends in doping and to develop methods able to accurately detect banned substances. Mass spectrometry is likely to remain the dominant technology used for the detection of doping substances as it allows fast, sensitive, accurate quantification of many substances simultaneously. This can mean shorter analysis times and the potential for cost reduction making mass spectrometry a more efficient and attractive platform than alternative methods such as immunoassays. References

1. Carmody, T. 2013. Hacking your body: Lance Armstrong and the science of doping. The Verge. http://www.theverge.com/2013/1/17/3886424/programming-your-body-lance-armstrong-and-doping-technology
2. Ahrens, B.D., Starcevic, B., Butch, A.W. 2012. Detection of prohibited substances by liquid chromatography tandem mass spectrometry for sports doping control. Methods in Molecular Biology. 902:115-128.
3. WADA prohibited list: https://elb.wada-ama.org/en/what-we-do/prohibited-list
4. Thevis, M., Kuuranne, T., Geyer, H., Schänzer, W. 2012. Annual banned-substance review: analytical approaches in human sports drug testing. Drug Testing and Analysis. 4:2-6.
5. Ayotte, C. 2010. Detecting the administration of endogenous anabolic androgenic steroids. Handbook of Experimental Pharmacology. 195:77-98.
6. Hemmersbach, P. 2008. History of mass spectrometry at the Olympic Games. Journal of Mass Spectrometry. 43(7):839-853.
7. Reichel, C. 2013. Differences in sialic acid O-acetylation between human urinary and recombinant erythropoietins: a possible mass spectrometric marker for doping control. Drug Testing and Analysis. 5(11-12):877-89.
8. Boyac?, E., Gorynski, K., Rodriguez-Lafuente, A., Bojko, B., Pawliszyn, J. 2014. Introduction of solid-phase microextraction as a high-throughput sample preparation tool in laboratory analysis of prohibited substances. Analytica Chimica Acta. 809:69-81.
9. ACS press release. 2014. New method is a thousand times more sensitive to performance-enhancing drugs. http://www.acs.org/content/acs/en/pressroom/newsreleases/2014/march/new-method-is-a-thousand-times-more-sensitive-to-performance-enhancing-drugs.html