Dr. Mary Scollay | Horse Racing Free Tips

Your Questions About Split Sample Testing (And More), Answered

Over the past week we have received numerous emails asking questions about the split sample testing process, as the racing world waits for the next news in the Medina Spirit Kentucky Derby scandal. We got even more questions after Louisville Courier-Journal reporter Tim Sullivan revealed that the split sample taken after Medina Spirit's Kentucky Derby run hasn't yet been sent out for analysis.

We examined the process briefly last year when racing fans grew restless awaiting the split sample results from the 2020 Arkansas Derby card, but wanted to address a few of the questions that were specific to this case.

We spoke with Dr. Mary Scollay, executive director of the Racing Medication and Testing Consortium (RMTC) and former equine medical director for the Kentucky Horse Racing Commission, to learn more about how this regulatory process normally works. (Current staff at the commission are prohibited from discussing an ongoing case, particularly one like this where the split sample has not yet been tested.)

What's a split sample and how is it collected?

The split sample, or “B” sample, is collected at the same time as the primary sample, in the test barn after the race. Urine or blood is collected from the horse and subsequently divided into two containers. One of these is sent off for analysis by the laboratory contracted by the commission – in this case, Industrial Labs – for post-race testing. The other is stored under lock and key at the track at which it was collected.

If a lab finds and confirms a drug positive or overage in the primary sample, the trainer is notified and provided the opportunity to request to have the split tested. The trainer is allowed to select which lab will test the sample, and in many places is required to select a lab with a certain level of accreditation, like RMTC accreditation. All RMTC-accredited labs are capable of performing split sample testing.

How often are split samples negative, and what happens if they are?

If a split sample is negative, or if the split sample laboratory finds the substance in question at a level below the regulatory threshold, then there is no violation of the rules and therefore no ruling issued.

Scollay said that in her experience at the Kentucky commission, it was extremely rare for a split to come back negative.

“Maybe over 11 years, maybe there were four [cases where a split lab found a lower, legal level of a substance in question],” she said. “I can only think of one split in all those years where a laboratory reported a finding and the split laboratory did not detect it. And in that particular case it was a fairly obscure substance.”

Do split sample labs know whose sample they're testing?

They aren't supposed to. In normal circumstances, the lab would receive the sample from the appropriate jurisdiction and would be told which substance had been found, and in what concentration. They would not be told the identity of the horse, trainer, or race. All testing samples are assigned numbers at the time of sampling to keep them anonymous to the primary and split sample testing labs.

Scollay said the publicity around this split sample could lead to a reluctance from some laboratories to take the sample on – but she has also heard from at least one lab director who isn't convinced that would be a factor.

How is a split sample test different from the original test?

A split sample test would be the same as the confirmatory analysis run by the primary testing lab. When the primary testing lab gets a post-race sample, it will first screen the sample against its catalogue of substances to see if any of them are present. When it does identify one, it then must perform confirmatory analysis to decide how much of the drug is present in the sample.

The split sample lab will not screen the split against its catalogue, but will instead perform confirmatory analysis similar to what the primary lab did. The split sample lab is provided with the concentration from the original test only to help technicians choose the proper calibrators for an accurate reading.

“The estimated concentration, the reason that's provided is so they know what calibrators – known positive concentrations – they need to run in order to make an accurate determination about the concentration about the sample,” she said. “If you know the concentration is five, your calibrators might be one, two, five, seven, and ten. But if you don't know the concentration, your calibrators might be one, 10, 25, 50, and 100. Then you're not able to get a very accurate estimate of the concentration.

“You're trying to sandwich your unknown or your test sample in the middle of the range of known concentrations. Think of it of if you have eight or ten glasses of water and you put blue food coloring in one and keep diluting across glasses until it gets lighter and lighter. You've got a glass with blue in it and you're going to line that up to try and see which one of those tinted blues is closest to your color.”

It's not unusual for there to be as much as a 25 percent variability between labs when you get down to very small measures like picograms. For substances that have thresholds governing how much of a drug is considered legal, that can make a big difference. (In this case, however, there is no threshold for betamethasone so any amount would be considered a violation.)

Once the lab actually begins the analysis, it takes the same amount of time as the initial post-race testing – but the hold-up is usually scheduling. Dr. Scott Stanley told us last year that it's not unusual to wait three to four weeks in the non-busy season to get a split result back. Given the constraints of the COVID-19 pandemic on staffing levels, he expected the Charlatan split sample in 2020 to take six to eight weeks; in the end, stewards in Arkansas issued a ruling on July 15 for races run the first week in May.

How is the split sample lab chosen, and how long does that take?

The process of choosing a lab depends on a few factors. Labs can reject a split sample request for various reasons. As we explained last year, contract work (like post-race and TCO2 testing) are the main priority for testing labs, because that's what pays their bills. Spring and summer are the busiest times for testing laboratories, as there is racing going on in more places than there is in the wintertime.

Scollay said in her role at the Kentucky commission, it wasn't uncommon for labs to tell her it might take weeks before they would be able to promise a result – or even months.

“When I worked for the horse racing commission, it was my goal to make sure a trainer always had a choice and sometimes that meant two laboratories,” she said. “Laboratories would respond, 'We're way behind' or 'We've got a bunch of confirmatory analyses lined up' or 'Our turnaround time would need to be 12 weeks.'

“I can't remember the last time I got a positive response from all the RMTC-accredited labs. I don't know that I ever did.”

If there's any kind of back-up for the lab's regular work – new equipment, staffing issues, etc. – that has a ripple effect on regular testing and hence, split sample timeframes.

It can also matter what substance is involved; if the primary lab found an unusual drug, or an uncommon drug at a particularly small concentration and the lab getting the split sample request already knows it can't reliably test for that substance to the same limit, it will reject the split sample request. Scollay said that in this case, that shouldn't be an issue because betamethasone is a controlled therapeutic drug which testing labs would encounter frequently. She expects all RMTC-accredited labs to have the same sensitivity of testing when looking for this particular substance.

Why can't we eliminate delays by having trainers choose split sample labs and send samples off before the first round of tests come back?

For one thing, it would be incredibly expensive for either commissions or horsemen to pay for double testing to be done on every sample. For another, split samples aren't obligatory – a trainer can decide to waive his or her right to the test and take the penalty if they feel it's not worthwhile to fight the positive.

Most importantly though, Scollay said the differences in testing capabilities for rare substances, and the variation in timelines for each lab through the year would make it meaningless to choose a lab before knowing what substance might be at play.

“It would be foolish for a commission to consent to that, because if the lab can't do the work and the sample gets sent to them, a positive test gets negated and that certainly doesn't support the integrity of the game,” she said.

So when will Baffert be banned?

Hang on a minute. The Kentucky regulations are very clear about the penalties stewards may hand to a trainer for medication violations. While they're given a range of potential suspension lengths and fines to work within, they don't have the latitude to throw those out the window and revoke a trainer's license. Under current regulations, betamethasone is a Class C medication in Kentucky, and Medina Spirit would be Baffert's second Class C in 365 days if the split is positive. (Merneith's overage for dextrorphan and the two lidocaine overages from last year fall in different drug classes by Kentucky standards. So, although this is Baffert's fifth therapeutic overage in a year, it's only his second of this type.) The penalty range for a second Class C violation in 365 days is a 10- to 30-day suspension and a $1,500 to $2,500 fine.

Scollay said there is a catch-all rule in Kentucky, as there is in many places, designed to deal with conduct “contrary to the best interests of racing,” but that's typically reserved for more extreme situations, not therapeutic overages.

What about the disqualification of the horse?

The same Kentucky regulation, KAR Title 810, Chapter 8, Section 030, states that a first Class C offense for an owner “shall” result in disqualification and loss of purse. Unlike trainer penalties, there is no language included allowing for stewards to consider mitigating circumstances. Medina Spirit owner Amr Zedan has engaged an attorney who's already preparing to argue that the stewards aren't bound to disqualify a horse.

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About That Connection Between SGF-1000 And Dexamethasone

When news broke last weekend that Medina Spirit had tested positive for the corticosteroid betamethasone, Paulick Report staff received several questions from readers asking about a phone conversation intercepted by federal agents. Court documents from the federal indictments of March 2020 recalled a conversation between trainer Jason Servis and veterinarian Dr. Kristian Rhein in which they were discussing a substance called SGF-1000, which prosecutors say was one of the misbranded or adulterated drugs at the heart of the case. Rhein told Servis that the substance could sometimes create a false positive for “dex,” widely believed to refer to dexamethasone, and Servis asked Rhein to alter his veterinary records to make it appear as though horses had been treated with dexamethasone in case of a positive test.

Read more about SGF-1000 in this Paulick Report feature.

Since both dexamethasone and betamethasone are corticosteroids, some readers wondered whether a positive test for betamethasone could actually be a guise for something more sinister.

According to Dr. Mary Scollay, executive director for the Racing Medication and Testing Consortium, the answer is no.

Scollay explained that the testing and confirmation process used in mass spectronomy makes it virtually impossible for one drug to be misidentified as another. She doesn't believe betamethasone is a false positive result, nor that SGF-1000 could actually have shown up as dexamethasone in post-race tests. (No one ever said the indicted individuals were always accurate in their intercepted conversations.)

Mass spectronomy works by identifying foreign molecules inside blood or urine and weighing them as part of a process called screening analysis. Those molecular weights are then checked against the lab's drug catalogue. The catalogue contains the molecular weights of known substances and is developed through rigorous testing of known drugs. If a molecular weight matches something in the catalogue, that's an initial finding.

Before the lab can actually call the test a positive for the substance though, it goes through a second process called confirmatory analysis. It's possible two substances could have the same weight but be made up of different components, so the lab must find out if their compositions are the same. In this process, the molecules of the substance are bombarded with energy until they split apart, and the ratios of the resulting pieces are measured against the catalogued substance.

“Each specific molecule has its own way of fragmenting,” said Scollay. “It's like a Hershey bar – it's scored in a certain way, it's going to break the same way every time if you apply force at certain points. When you go to identify the molecule, you look at the candidate ions, the ions that result from fragmenting it, and also the ratio of those ions to each other. They should be present in very specific proportions. If they're not, or if the candidate ions are not present, or even one of them is missing, you have not identified the substance.

“I would argue that if you identify the candidate ions in the right ratio, you've identified betamethasone.”

By the time a lab calls a positive using this testing method, it's justifiably confident that the substance at play has been correctly identified.

So what of the SGF-1000/dex connection?

“Maybe dexamethasone was in the SGF-1000, and that's why they said it would show as dexamethasone, but if a molecule has the same exact molecular weight as dexamethasone and you apply energy to it and it fragments, and the fragmented parts are the ions you would get from dexamethasone in the relative concentrations, I'm going to say you've identified dexamethasone,” she said.

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Hair Testing – What It’s Good For, What It’s Not Good For

After last weekend's revelation that Kentucky Derby winner Medina Spirit had tested positive for betamethasone post-race, trainer Bob Baffert outlined a few different methods for figuring out how the drug got into the horse's system, including hair testing the horse to look for the presence of the drug. On Tuesday, it seemed the need for investigative work was through, since Baffert admitted the horse had indeed been treated with a topical prescription that contained betamethasone.

Still, his suggestion raised questions about how hair testing can help in cases like that of Medina Spirit. Many have hoped hair testing would be the next great advance in racing's drug testing program, able to detect what blood tests cannot.

Hold your horses, experts say.

Using hair to detect the presence of drugs works because a stand of hair contains melanin, which gives it color and which carries a slight negative ionic charge. That means that when drugs go through a horse's system, those with a slightly positive ionic charge bind to the melanin of the hair at the base where it's growing out from the horse's skin. Laboratories can find the resulting band of the drug in question sitting crossways inside the hair shaft if they have a sample of the hair. Horses with black hair will bind drugs easily; those with less melanin in their hair, like grays and roans, do not retain drug remnants in that hair as readily.

Only the drugs with a slightly positive charge are going to bind to hair well enough to be detected. Dr. Rick Sams, equine drug testing expert and former lab director for HFL Sport Science, said this works well for certain types of drugs.

“Clenbuterol has a negative charge on it, it binds to melanin and it can be detected at a very low level because a lot of it binds to the hair,” said Sams. “Negatively-charged substances like flunixin, like phenylbutazone, are repelled by the negative charge on melanin and do not readily bind to the hair sample even though the blood concentration may be substantially higher than the concentration of substances like clenbuterol.”

Steroids – both anabolics and corticosteroids – are neutral, so they're not attracted to hair. Anabolic steroids are excreted through skin glands and may appear on the outside, rather than the inside, of the hair shaft, but that makes it difficult to say whether a horse was exposed to the steroid externally or internally.

A hair test would probably not detect a corticosteroid like betamethasone in a horse, because it wouldn't bind well to the melanin. If hair testing had been done on Medina Spirit, it wouldn't show the drug but that would be because it couldn't, not because it had never been given.

Then there's the question of gathering that hair sample.

“There are lots of challenges with hair testing that would need to be addressed and standardized,” said Scollay. “For example, I'm terrible at pulling manes, just terrible. I have a hard time with one pull or even two pulls getting a sufficient sample. I'm pulling and pulling and pulling and I finally get what I need. There are some people who just use scissors. If you look at those samples, they're not necessarily even cuts. If you're at the laboratory, you don't know how much hair remained on the neck between the site of the cut and the hair follicle itself.”

Without the root of the hair, Scollay pointed out there's also no way to conduct DNA testing on a sample, should there ever be a question about whether the sample came from the horse in question – and of course, with the majority of Thoroughbreds being bays, the color of the hair isn't going to be much help.

Hair testing also doesn't provide particularly specific information about drug administration, and that's why it's most commonly used to find prohibited substances that are never supposed to be given to horses. Finding a little band of drug in a hair shaft tells the tester that the drug was administered, but not how much was given, how it was given, or exactly when. A three- to four-inch length of hair represents about six months of growth. Most often, laboratories could give a range of time when the drug exposure might have happened but it's usually a range of days or weeks, not hours. Some drugs, like clenbuterol, require multiple exposures of a drug before it will show up in hair. Labs can cut the hair into sections to try to narrow the timeframe a drug was given, but that method isn't always a good one.

“The problem is hair sometimes stops growing before it falls out,” said Sams. “The hair shafts grow at basically the same rate but some of them stop growing, so if you do a sample even in sections, you're going to see a distribution of the drug probably through multiple sections just due to the fact some of those hairs stopped growing. It's an imprecise science.”

A hair test is only useful if enough time has elapsed since the administration of the drug for the hair to grow long enough that it can be taken in a sample. Sams said that in research settings, hair has been sampled using a set of clippers and revealed drug administrations from one or two days before – but that in the field, there's no standardized way to take a sample, and it's unlikely a test barn will be able to successfully cut that close to the skin. Scollay said she wouldn't use a hair sample as a basis to confirm a drug administration more recently than two weeks to a month after administration.

Clenbuterol was recently banned in racing Quarter Horses, and as a result, the American Quarter Horse Association conducts hair testing on horses ahead of major stakes races. There have been cases where a hair test has been negative for clenbuterol but a post-race sample has been positive, resulting in sanctions. Ironically, some of those cases were overturned by courts on appeal because trainers successfully argued that the post-race positive must be a mistake due to the negative pre-race hair test. In reality, Scollay said, it's possible two different labs could use different methodologies on the same horse's hair and come up with different results, neither of which should invalidate a post-race test on blood or urine.

Because of these inconsistencies, both experts agree it will be some time before hair testing becomes the go-to in the United States – if it ever does.

“You have to decide what your purpose is with hair testing; it's not going to replace blood and urine testing. It's not going to do it,” she said. “It's a regulatory tool. It's part of your arsenal, but relying on it solely – unless you're dealing with a prohibited substance – you're going to have a challenging time.”

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Report: Why Regulators Test For Picograms Of Betamethasone

On Sunday morning, trainer Bob Baffert shocked the racing world with his announcement that Kentucky Derby winner Medina Spirit's post-race test had returned a positive result for 21 picograms of betamethasone. During his press conference, Baffert went on to say that Medina Spirit has never been administered betamethasone.

During the ensuing social media storm, questions have arisen about what exactly betamethasone is, the legitimacy of testing for substances in concentrations as low as a picogram (one trillionth of a gram), and how it got into the horse's system in the first place.

Dr. Mary Scollay, executive director of the Lexington, Ky.-based Racing Medication and Testing Consortium, answered some of those questions in a series with Horse Racing Nation.

Betamethasone is a corticosteriod used to reduce inflammation. It can be utilized in four ways: direct injection into a horse's joint, injection into the bloodstream, subcutaneous injection near soft tissues that may be inflamed, or via topical applications.

Betamethasone “is a medication that has legitimate applications in the care of race horses,” Scollay told HRN. “It's not a heinous substance. But it is a substance that we want to control in proximity to a race, largely to protect the safety and welfare, of course, because anti-inflammatories have the ability to mask inflammation, signs of inflammation, that can be warning signs either to the horse's connections or the horse itself that there is an injury present that could escalate into something far worse if pressured.”

Read more about corticosteroids in the Paulick Report archives here and here.

The recommended withdrawal period in Kentucky for a betamethasone joint injection is 14 days, so no closer than two weeks before a race. The allowable threshold for betamethasone in a post-race test used to be 10 picograms, but that was changed last fall. Now, no trace amount is allowed.

When used as a joint injection, a typical dose of betamethasone would be nine milligrams, Scollay said.

“But then that drug leaves the joint, enters the bloodstream and is distributed throughout the body,” she told HRN. “And remember that a racehorse has upwards of 50,000 mls (milliliters) of blood. So you're not talking about 21 picograms in that entire horse's body. You're talking about 21 picograms in one ml of blood. And there's 49,999 other mls of blood, not to mention all the other tissues, the muscles, the organs, the brain, the skin, all the other tissues of the body. That drug distributes throughout the entire body. So 21 picograms, you know, you can be a little overly reductive and say that's nothing. But when you can contemplate the total sum of medication that may be in the body at that time point? It's a different story.”

If 21 picograms (remember, 21 trillionths of a gram) were found in a single milliliter of blood, that means upwards of 1,050,000 picograms of betamethasone was circulating through the horse's bloodstream at the time of the test. (That translates to 1.05 micrograms, or 0.00105 milligrams.)

Again, that doesn't include the amount of the medication remaining in the horse's tissues.

All of the above leads to the following question: if Medina Spirit was never administered this medication, how did it get into his system?

Scollay doubts that intentional sabotage is a factor in this case for two reasons. First, horses are under 24-hour security beginning on Tuesday of Kentucky Derby week. Second, the choice of a therapeutic medication to sabotage a horse just doesn't make sense.

Read more at Horse Racing Nation here and here

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