Better Playing Through Chemistry … Still

Abraham Almonte was suspended for 80 games in 2016 after testing positive for Boldenone. (via Erik Drost)

Editor’s Note: This piece initially was given as a presentation at the marvelous 2017 Saberseminar.

The announcement of player suspensions resulting from the use of performance enhancing substances have become commonplace in the game of baseball. Inevitably, a suspension is issued in late April or May, resulting from a positive test during spring training. And each suspension is followed by another player demanding cheating players be caught and decrying the current system as ineffective. And the truth is, the system isn’t as effective as it could be, and the game’s financial structure continues to incentivize players to take advantage of that system.

However, a savvy baseball player will recognize that he wields a significant amount of power in changing the terms of the Joint Drug Agreement (JDA)–not through social media, but through the MLB Player’s Association. The JDA is a collaboration between the MLB Player’s Association and the Commissioner’s Office. The JDA not only sets forth the list of prohibited substances, but the manner in which urine and blood samples will be collected, the laboratories that will analyze said samples, and the detection limits for prohibited substances. Included in the testing procedure is the number of samples to be collected and the time frame for testing.

More recently, the JDA has established the Longitudinal Profile Program in an attempt to establish baseline biological data for each player. The astute reader will be familiar with this as a biological passport. However, the bulk of the JDA is directed towards the appeals process and disciplinary actions. A player is presumed innocent until proven otherwise, and his name is withheld from public scrutiny until a positive test is confirmed.

The significant lag time between an initial positive test and the suspension is a direct result of the caution surrounding the JDA, and rightly so: In theory, a confirmed positive test and suspension should be detrimental to a player’s career and reputation, which should deter a player from the use of a prohibited substance. (And this doesn’t even broach the subject of the long-term effects of prohibited substances on a human’s body and mind as a deterrent.)

To the casual eye, the list of prohibited substances is quite lengthy; a significant portion of these prohibited substances are labeled as “Performance Enhancing Drugs.” The phrase “performance-enhancing drug” is nebulous. While many stimulants are expressly prohibited under the JDA, caffeine is not, although many people would consider caffeine to be a performance-enhancing substance.

And while the public generally associates the word “steroids” with performance-enhancing drugs, the term encompasses a number of naturally-occurring and therapeutically-beneficial compounds. Due to their use as therapeutic agents, as well as the role of steroids in the BALCO scandal, the steroids that are explicitly listed as prohibited substances have been thoroughly studied. While the generic term “steroid” encompasses everything from testosterone to cholesterol, androstenedione to cortisol, when we use the term “steroid” in the banned substance discussion, we are simply referring to anabolic, androgenic steroids, which are associated with muscle building and strength.

Fig. 1: A snapshot of human steroid biosynthesis. All of the depicted structures are steroids, but not all of them are prohibited substances.

The list of anabolic, androgenic steroids is clearly set forth in the JDA. However, the detection of the use of said steroids goes beyond the list of compounds that are specifically recited. The skilled, analytical chemist recognizes that the human body is a chemistry lab unto itself, capable of converting molecules into other molecules for the purposes of metabolism and elimination from the body.

Fig. 2: Structures of testosterone metabolites (Choi et al., Drug Metab. Disp., 2005, 33(6), 714-718).

Fig. 3: Chromatograms separating mixtures of A) testosterone and its metabolites formed by the human liver, compared to B) reference standards (Choi et al., Drug Metab. Disp., 2005, 33(6), 714-718).

Metabolism is the first step in the human body’s waste treatment facility, converting molecules into metabolites to facilitate removal from the body. Thus, the analytical chemist is actually looking for several compounds–the prohibited substance and its metabolites–in a mixture. While this might seem to complicate detection of prohibited substances, this mixture provides a fingerprint by which an analytical chemist can identify the presence of a prohibited substance.

Thanks to extensive research, the metabolism of numerous prohibited substances is well known, and the metabolites of said substances have been identified and structurally characterized. This is ongoing work, but many older prohibited substances, such as testosterone, stanozolol, boldenone, and nandrolone have been thoroughly studied.

Metabolite studies are conducted in a manner akin to that of an early stage clinical trial of a drug: Healthy volunteers are dosed with a compound and monitored at various time points afterwards. At each time point, a urine sample is analyzed for the presence and amount of the compound, as well as its metabolites.

A Hardball Times Update
Goodbye for now.

The metabolites of the prohibited substances provide a wealth of information, as metabolites can be detected long after the prohibited substance is undetectable. While the prohibited substance itself may only be detected for a scant 24 hours post administration, a metabolite may be eliminated through urine for days after the prohibited substance is no longer detectable.

Fig. 4: Six different subjects received oral administrations of 30 mg 17β-boldenone and were monitored for 17β-boldenone and two metabolites; note the time frame over which the metabolite BM1 is still detected (Wu et al., J. Pharm. Biomed. Anal., 2015, 115, 570-575).

Now that we know what we are looking for–prohibited substances and the metabolites of the prohibited substances–we turn our attention to how to look. The analytical chemist has a number of tools at her disposal. Under the current agreement, the contracted analytical chemistry laboratories use mass spectrometry to determine the presence of prohibited substances and their metabolites.

Mass spectrometry, or “mass spec,” is a well-established analytical technique that can quickly determine the presence of compounds in a sample based on the mass of the compounds. Mass spectrometry is typically coupled with a chromatography step before the actual mass analysis, separating the compounds of the mixture. The compounds are then ionized, creating charged compounds. The ionization step also can cause some compounds to fragment, resulting in charged compounds and charged fragments, which then pass through a magnet. Because the compounds and fragments are charged, they will be separated by the magnet, and the readout will provide the mass of the compounds and the fragments based on their charge (m/z).

Fig. 5: A really crude illustration of the concepts behind mass spectrometry.

Although “mass spec” is by no means a new technology, the field of mass spec is hardly stagnant. Method development in chromatography, ionization, and analyte detection are not to be overlooked; derivatization of analytes has been of value in amplifying signals, leading to prolonged detection of previously-overlooked metabolites.

It is relatively easy to detect the presence of a foreign substance in human urine at the right concentration. But what happens to endogenous substances, those naturally occurring compounds that are also on the prohibited substance list?

For example, testosterone is produced in the body from cholesterol, as are the pro-testosterone prohibited substances, dehydroepiandrosterone (DHEA) and androstenedione (commonly referred to as andro). How do we distinguish these externally administered, exogenously produced substances, from those produced by the player? Again, we turn to mass spectrometry.

We use mass spectrometry to detect the mass of compounds. But there’s a small, random, naturally-occurring variation in the mass of elements, in the form of isotopes. Most carbon atoms have six protons and six neutrons, resulting in an atomic weight of 12 (denoted as ¹²C). But occasionally, a carbon atom will have an extra neutron, resulting in an atomic weight of 13 (¹³C).

Testosterone produced endogenously, i.e., naturally in the human body, is 98.9 percent ¹²C and 1.1 percent ¹³C. However, pharmaceutical testosterone is typically produced from plant sources and has a slightly different isotope signature; synthetic testosterone has been found to be 99.0 percent ¹²C and 1.0 percent ¹³C.

While this may not sound significant, mass spectrometry can detect these slight differences. By comparing the presence of different carbon isotopes in a player’s testosterone, we can determine whether testosterone is endogenous (synthesized by the player himself) or exogenous, synthesized externally. Carbon isotope ratio mass spectrometry (IRMS) is a new addition to the Joint Drug Agreement arsenal and was attributed as the reason for a slight uptick in suspensions in 2016 and 2017.

Regardless of the state of the art in analytical chemistry, and despite the ongoing progress made in detection techniques, the Joint Drug Agreement is not ironclad. An enterprising player will find a way around the prohibited substance list as it stands, as there is a multitude of ways to circumvent the current agreement. The detection levels do not account for microdosing, wherein a player may take small doses of a prohibited substance more frequently, evading detection, but maintaining a steady, constant level of a performance-enhancing substance.

Drug cocktails also would evade detection, as ingestion of multiple compounds in minimal dosages most would likely remain undetected while providing the same benefits as a typical dose of a single compound. And of course, current testing doesn’t account for players who may be administering performance-enhancing drugs that are not prohibited substances. While this would not explicitly violate the terms of the Joint Drug Agreement, one could argue that certain substances would violate the spirit of the agreement, if not the letter of the law.

Similarly, the JDA does not account for advances in genetic engineering, which are not yet prohibited, but will easily provide an advantage to the competitive athlete. Gene therapy is making rapid progress as a way to correct deficiencies in DNA in order to treat disease. It is not difficult to envision the abuse of gene editing to modify an athlete’s DNA to provide him with genes associated with strength, endurance, or agility. Thus, given the nature of genetic engineering and its ability to fundamentally change DNA, a more rigorous biological passport must be established for each player before such techniques become easily accessible.

Taking the proactive step of establishing a more thorough biological passport for each player would be a significant, decisive action, and it would send a strong signal to the players and the public going forward. However, a much simpler approach could be enacted immediately and would only require increased participation from the players–increasing the frequency of testing.

The Odds Are Ever in Their Favor

We can examine the probability of catching a “cheating” player with simple math and logic. The JDA covers all players on the Major League Clubs’ 40-man rosters. Over 30 organizations, this means the JDA applies to 1200 players. There are three mandatory, unannounced urine sample collections: one upon reporting to spring training, one during the season, and one during the offseason. Additionally, the JDA mandates 4,800 urine samples will be collected from randomly selected players at unannounced times.

At first glance, 4,800 tests over a season sounds significant. You might expect each player would be tested four times–4,800 tests over 1,200 players. However, we must consider the frequency, distribution, and time frame over which these 4,800 tests are administered. For example, one player may be tested eight times, while another player may not be tested at all.

In an extreme example, over the course of a six-month season, 27 players may be tested every day of the season, while the other 1,133 are not tested at all. The more reasonable assumption is that these 4,800 tests are truly randomly distributed over the regular season among all players. In other words, there are 4,800 tests administered to 1,200 players over a roughly six-month (180-day) season. This would result in a two percent chance of any random player being tested on any random day. And a player who may be considered a frequent user would not necessarily test positive every day.

As we discussed, metabolites may be detected long after the prohibited substance itself is undetectable. However, even a metabolite has a finite time frame for detection. A player taking a prohibited substance may only test positive for three days after administration. If he is only taking a prohibited substance once a week, there’s a 43 percent chance he will test positive. Couple that with the slim, two percent chance of being tested on any day, and it’s no surprise players are willing to gamble for the chance at landing a significant contract.

The question of whether performance-enhancing substances or techniques should be allowed or prohibited is an ethical question beyond the scope of this essay. Regardless, the current Joint Drug Agreement is ineffective in deterring professional baseball players from using prohibited substances. Despite the increased length of a suspension–as well as the threat of a permanent ban from MLB–the number of suspensions resulting from the use of prohibited substances by professional baseball players has not decreased.

As it stands, the current JDA is more bark than bite. While the Commissioner’s office and certain baseball players allege they want to maintain the integrity of the game, it is the Commissioner’s office and the Player’s Association that are responsible for the terms of the JDA, and thus it is the Commissioner’s office and the Player’s Association that bear the responsibility for its ineffectiveness. Absent a more proactive approach from the Player’s Association, Commissioner’s office, or the owners, we can only expect the use of prohibited substances and other performance-enhancing drugs to continue, as the odds remain ever in the doping player’s favor.

References & Resources

  • MLB Joint Drug Prevention and Treatment Program and the Prohibited Substances list (This sets forth the list of prohibited substances, the time frames for testing, the number of tests, the laboratories conducting the testing, the procedure for the collection of samples–down to the dress code for the sample collectors!)
  • Maury Brown has a good story for Forbes every offseason: see “List Of Every 2015 MLB And Minor League Drug Suspension, (It’s A Long One)”, Forbes, November 12, 2015, and “The Definitive Look At Every Drug Suspension In Baseball For 2016,” Forbes, January 6, 2017, (bearing in mind that he’s looking at both MLB and MiLB)
  • Werner et al., “Performance-Enhancing Drugs in Sports: How Chemists Catch Users”, Journal of Chemical Education, 2011, vol. 88, no. 1, pages 34-41 (A great article from a chemistry professor who taught a “Chemistry and Athletic Performance” course.)
  • Harris, “How Mass Spectrometry Works,” (Written with the scientifically literate baseball fan in mind)
  • Catlin et al., “Issues in detecting abuse of xenobiotic anabolic steroids and testosterone by analysis of athletes’ urine”, Clinical Chemistry, 1997, vol. 43, no. 7, pages 1280-1288 (Don Catlin was a major figure in exposing the BALCO scandal.)
  • Muccio et al., “Isotope ratio mass spectrometry”, Analyst, 2009, vol. 134, pages 213-222 (Good review which also talks about the variations in carbon isotopes.)
  • Choi et al., “Characterization of testosterone 11β-hydroxylation catalyzed by human liver microsomal cytochromes P450”, Drug Metabolism and Disposition, 2005, vol. 33, no. 6, pages 714-718
  • Wu et al., “Metabolism study of boldenone in human urine by gas chromatography–tandem mass spectrometry”, Journal of Pharmaceutical and Biomedical Analysis, 2015, vol. 115, pages 570-575

Stephanie Springer is an organic chemist turned patent examiner. Follow her on Twitter @stephaniekays.
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5 years ago

Very well done, I have one minor quibble since everything else was so detailed and correct (I’m an analytical chemist). In Figure 1, it’s true that vitamin D is not a steroid, but neither are cholesterol or cholic acid. Yes, they are part of the steroid biosynthesis pathways, but they’re both sterols, not steroids. Steroids need to have some aromatic or conjugated functionality, usually in the A ring.

5 years ago
Reply to  James

As pregnanes, they would seem to fall into the general class of ‘steroid’.

5 years ago

STEPHANIE- you may want to look into something…Adderall exemptions on MLB clubs is approaching 11% whereas the national average of ADHD sufferers prescribed this powerful stimulant is somewhere around 5%. Does MLB really have an ADHD problem or a drug problem…

Jake From State Farm
5 years ago

Athletes in other sports have been caught using someone else’s urine that they transferred into their own bladder using a catheter. It stands to reason that somebody or a lot of somebodies in major league baseball has already done this.

5 years ago

Excellent article, thank you. Baseball, a cooperative agreement between owners and the players assoc., continues to celebrate and applaud the larger and larger contracts signed by the various known drug users. I hate the resulting hypocrisy of players like Nelson Cruz and Melky Cabrerra signing mega deals after being busted for drugs. Get rid of the financial incentives and impose real penalties and the players will stop cheating on their own. Again, thanks for the info.