Just Breathe: Finding New Ways to Test for Banned Substances

Earlier this month, the Partnership for Clean Competition (PCC), in conjunction with Major League Baseball, announced they had begun trials of “exhaled breath testing technology” to detect the use of prohibited substances by athletes. PCC touts breath testing as inexpensive, efficient, and less invasive than urine and blood testing, while acknowledging that breath testing is intended to complement, rather than supplant, current testing methods. PCC hopes to enroll 500 athletes in the trial, which will validate the use of breath testing by comparing the breath testing results to current testing methods.

Developing and testing a new method for testing for prohibited substances requires the same basic considerations, whether you’re testing urine, blood, or breath: collecting the sample, maintaining the sample integrity so it reflects the amount of substance present in the sample at the time sample was taken, and analyzing the sample to detect the presence of prohibited substances. You may be thinking that we’ve already tackled these problems when it comes to breath testing – what about the breathalyzer tests used for roadside sobriety checks?

If you’re thinking that “exhaled breath testing technology” is a fancy way of saying “breathalyzer”, you’re right – to a point. The breathalyzer that one associates with roadside alcohol testing won’t be used for anti-doping tests, although the principles are the same. In general, for breath testing, the subject simply breathes into a device, which collects the sample (the breath) and analyzes it to detect the presence of particular analytes, such as ethanol or a drug.

The alcohol breathalyzer test only detects and quantifies a single compound, and benefits from having a relatively simple task. In the case of the alcohol breathalyzer, the analyte is the volatile organic compound ethanol. Ethanol is relatively easy to detect using methods such as infrared spectroscopy; a device that is capable of detecting multiple prohibited substances would require additional considerations. In this case, we’re collecting and detecting multiple compounds, and these compounds have very different chemical structures. Most drugs of abuse and other prohibited substances are non-volatile organic compounds, so the breath testing technology that the PCC is field testing for anti-doping purposes requires technology beyond what is present in the breathalyzers used for roadside sobriety checks.

The “breath testing technology” in this case is a device manufactured by a company called SensaBues, and is primarily based on the work of pharmacologist Dr. Olof Beck of the Karolinska Institute in Sweden. While SensaBues has some experience in using exhaled breath to detect prohibited substances, PCC disclosed that the the trial is also being conducted with Dr. Mario Thevis of the Center for Preventive Doping Research at the Institute of Biochemistry, German Sport University Cologne. Dr. Thevis’s research is directed specifically towards detecting substances that are prohibited by many sports organizations, such as anti-doping agencies and professional leagues like MLB.

Breath test sampling provides a number of advantages over traditional urine and blood sampling, particularly when it comes to taking, storing and transporting the sample. When it comes to collection, a blood sample is best taken by a trained phlebotomist – it is better for the person relinquishing the sample (no one wants a hematoma!) and ensures the appropriate quality and volume of sample is collected. But even when the blood draw goes smoothly, there are storage and transportation concerns. Blood samples need to be kept at the right temperature, or you risk degradation; the substances you’re looking for may degrade even if the sample is handled properly.

And transportation of the sample isn’t trivial – you can’t just walk onto a plane with vials of blood, nevermind trying to ship liquids. This is why dried blood spot samples are preferred – a drop of blood from a simple finger prick provides the sample, which is dried onto a card. When it’s time for analysis, this dried blood sample can then be removed from the card by a solvent. This is certainly much easier than obtaining an entire vial of blood, but it’s still invasive. Taking a sample of someone’s breath doesn’t require a needle. The subject simply breathes into a small collection device for two to three minutes; the device has a small polymeric membrane that collects microparticles from the subject’s exhaled breath.

Another advantage a breath sample provides: it can be done in the open, on the spot. Obviously, there are privacy and personal space concerns with collecting a urine sample. There’s also the issue of collecting a urine sample in an unsecured bathroom, as someone might try to pass an older, clean sample of urine off as a current sample. That’s part of the reason why you’ll see temperature strips on a urine sample cup, to confirm that the urine sample was taken recently. Additionally, when you can easily visually monitor the collection of a breath sample, it minimizes the chances of a sample being swapped out for a sample from someone else.

For these reasons, a breath-based detection system would be ideal – you could collect the sample on the spot, out in the open. The SensaBues device is small, and easily fits into a pocket – there are no worries about glass vials or needles. And the collection membrane found inside the device is relatively easy to handle, so testers don’t need to worry about spilling subjects’ samples.

For all its advantages, breath testing for drug testing purposes is not without its faults. While the ethanol breathalyzer is notable for its quick response, analysis using the SensaBues device is not trivial. Detecting a single, small, volatile compound is a little simpler than trying to detect many different compounds having more complex chemical structures. An ethanol breathalyzer can provide a reading on the spot. But for anti-doping purposes, the SensaBues device is more properly thought of as a device for the collection, storage, and transportation of a sample. Analysis still requires ultra high performance liquid chromatography (UHPLC) coupled with a mass spectrometer (MS). UHPLC-MS devices are still quite large — it’s not practical to have one on site in a clubhouse, for example.

Although the use of liquid chromatography paired with mass spectrometry is well known at this point, having a means by which to detect something is only so useful if you don’t know exactly what you’re trying to detect. While we have many studies showing how different known drugs are metabolised by the human body, and how to detect these drugs and their metabolites, those studies were primarily detecting drugs and their metabolites in urine and blood.

A breath detection device will be able to collect analytes from the mucous membranes and airways, notably from the bronchiolar and alveolar fluids from the lungs. We’ll need to consider the metabolism and pharmacokinetics of drugs in exhaled breath – how drugs are processed and transported throughout the body will play a role in what drugs we will be able to detect in exhaled breath. The profile of drugs and the corresponding metabolites found in lung fluids may vary from what we observe in blood and urine.

Analysis of exhaled breath may require looking for other analytes, as well as considering the time frame over which the parent drug and its metabolites are present in the breath. In order to validate the test, researchers have compared the breathalyzer results with the typical urine and blood samples. By comparing various samples taken from the same patients, researchers can confirm the various body fluids, collection methods, and analytical testing provide the same results.

Until now, the vast majority of these validation studies have been conducted on outpatients in treatment programs for drug abuse. In one study, 122 patients were enrolled from outpatient treatment programs, and urine and breath samples were collected simultaneously from each patient. The samples were analyzed for amphetamine, methamphetamine, the benzodiazepines diazepam and oxazepam, tetrahydrocannabinol, cocaine, buprenorphine, methadone, morphine, and codeine, using mass spectrometry. Of the positive urine samples in this cohort, 58 percent of the subjects also had positive breath tests. In patients on methadone treatment, all patients were positive for methadone in urine, but only 83 percent of those were positive in breath. And in patients on buprenorphine, 92 percent were positive in urine, while 80 percent were positive in breath.

You might wonder why the breath test resulted in a lower positive test rate than the urine test, but it’s important to note that this study relied on self-reporting to determine when the patient last administered the drug. Breath testing is likely to have a shorter detection time; in the situations where a urine test was positive but the breath test was negative, it’s possible a significant amount of time had elapsed since the drug was administered.

Naturally, as the use of exhaled breath for drug testing has been explored, it has gained interest from the anti-doping world. More recent studies have expanded the detection of drugs in exhaled breath to include substances that are of interest to the athletic community. It has now been demonstrated that chlorodehydromethyltestosterone (Turinabol), meldonium, pseudoephedrine, methylphenidate (Ritalin), the beta blocker bisoprolol, and the stimulant methylhexaneamine can be detected in exhaled breath. While there is evidence that a variety of stimulants, narcotics, and cannabinoids are detectable in exhaled breath as well, at this stage testers wouldn’t be able to detect anabolic agents, hormone and metabolic modulators, or diuretics and masking agents, among other compounds on the prohibited substances list.

Additional studies are ongoing, as there are still many areas that require further investigation. For example, there may be a need for additional qualitative rather than quantitative measurements – a positive test is a positive test, but if there is an issue of a threshold level or amount that is acceptable in a sample, that needs to be considered. And the volume of breath collected needs to be more accurately measured in order to determine a concentration. More importantly for anti-doping purposes, it is important to remember the drugs that can be detected by the breathalyzer are very limited – it’s a very small subset of the entire list recited in the CBA. This testing method will never completely replace blood or urine tests. It’s clearly meant to be a complementary detection method, providing another window of insight. But the use of breath testing technology may be a useful tool to have in the ongoing fight against the use of prohibited substances.

In one sense, the addition of exhaled breath sampling to the typical urine sampling will provide a more accurate look at the use of prohibited substances, particularly because it will fill in some gaps in terms of timing. Let’s say someone were to take a sublingual pill, just before a urine test. That pill wouldn’t show up in their urine at that point in time. But if the test administrator were to also ask for a breath sample in conjunction with a urine sample, the window of detection widens. In other words, using breath testing technology with the typical urinalysis could allow for prohibited substance detection immediately after the drug was ingested, all the way until the substance and its metabolites are completely excreted through urine.

Further study is warranted, but as with many trials, recruiting trial subjects can be challenging. This is where MLB and MiLB come in – here we have a group of athletes who are representative of the people who will eventually be subject to breath testing if it is approved for testing for banned substances. Although a rigorous study will anonymize the samples so as to protect patient privacy, the collection of any kind of biological sample will raise alarms among athletes, and rightly so.

Interestingly, the patent filings related to the device, and the methods of collection using the SensAbues device, also refer to biomarker detection. This could be used to reveal additional information about a player’s health or propensity for injury. Given that the company manufacturing these breath collection devices is clearly thinking of expanding the use of the devices beyond drug detection, there may be talk of using these breath collecting devices for other purposes down the road. This again brings us to the thorny issue of consent and privacy concerns.

Any professional athlete willing to voluntarily enroll in this study will have much to consider prior to enrollment, given the privacy concerns and the potential for the misappropriation of samples and data. It’s worth noting that the 2017-2021 Collective Bargaining Agreement only discusses the collection of urine and blood samples, and the Joint Drug Agreement only provides procedures for the collection or urine and blood samples.Regardless of the trial results, before breath testing could be implemented across the league, the league and the Player’s Association would have to negotiate it into CBA.

Minor league players are not covered by the protections of the CBA. They also have more to lose – remember, marijuana is not a prohibited substance in the CBA, but it’s use is prohibited for minor league players. Although the press release doesn’t explicitly state that minor league baseball is involved, a tweet from PCC mentions MiLB as a partner. Given the timing of the study, it’s possible minor league players at instructional leagues or in the Arizona Fall League are being recruited as well.

Exhaled breath testing is a promising tool for monitoring illicit substance use, and future research could provide insight into the use of exhaled breath to monitor a patient’s overall health. However, there is still much work to be done before it will be ready to meet MLB requirements, and there is ambiguity regarding the blinding process as it pertains to further study with MLB and MiLB players. As with any study, players would be well advised to do their research and ask plenty of questions before enrolling in PCC’s exhaled breath testing technology trial.

References and Resources

• Olof Beck et al., “Detection of drugs of abuse in exhaled breath using a device for rapid collection: comparison with plasma, urine and self-reporting in 47 drug users,” Journal of Breath Research, 2013, vol. 7, no. 2
• Himes, S.K. et al., “Cannabinoids in exhaled breath following controlled administration of smoked cannabis,” Clinical Chemistry, 2013, vol. 59, no. 12, pages 1780-1789
• Joseph Stromberg, “Cops Could Soon Use Breathalyzers to Test for Illegal Drugs,” Smithsonian.com, April 26, 2013
• Darren Sugrue, “Hold your breath — drug testing just got more advanced,” Elsevier Connect, March 2015
• Mario Thevis et al., “Sports drug testing using complementary matrices: Advantages and limitations,” Journal of Pharmaceutical and Biomedical Analysis, 2016, vol. 130, pages 220-230
• Mario Thevis et al., “Expanding analytical options in sports drug testing: Mass spectrometric detection of prohibited substances in exhaled breath,” Rapid Commun. Mass Spectrom., 2017, vol. 31, pages 1290-1296