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RonnyT
07-08-2010, 05:11 PM
Tetrahydrogestrinone (THG)
Before mid-October, few people had heard of the synthetic steroid tetrahydrogestrinone (THG). That changed with the official announcement of the identification of THG and the development of a test method to screen urine samples for it, which altogether is being hailed by sports-governing authorities as a milestone in the 40-year history of international antidoping efforts. Meanwhile, a federal investigation is ongoing to determine if there was a conspiracy to synthesize and distribute THG or other unapproved or controlled substances.
The fallout from these events is having an unprecedented impact on the sports world. On the surface, it would seem that the uncovering of THG indicates that enhanced antidoping efforts during the past few years are starting to make a difference. Yet experts on performance-enhancing substances and methods are quick to point out that the serendipitous discovery of THG, which had the potential to quietly become widely used by athletes, illustrates the undiminished severity of the doping problem in sports. Critics assert that sports-governing authorities may never come up with an adequate solution to quash the problem.
The THG chemical detective story began in June at about the time of the U.S. Outdoor Track & Field Championships. A coach tipped off the U.S. Anti-Doping Agency that several athletes from the U.S. and other countries were using a new steroid. The coach backed up the tip by supplying USADA officials with a syringe that had contained the steroid. The agency in turn rinsed the syringe with a few milliliters of methanol and sent the sample to the Olympic Analytical Laboratory at the University of California, Los Angeles.
The UCLA lab is one of about 30 testing labs worldwide accredited by the International Olympic Committee (IOC) and the only one in the U.S. accredited by IOC and the World Anti-Doping Agency (WADA). USADA and WADA are independent nonprofit organizations created in 2000 that govern drug testing and provide drug education for athletes.
The lab processes some 25,000 samples per year for U.S. and international sports-governing authorities, the National Collegiate Athletic Association, and the National Football League, among other clients. It is led by Don Catlin, a professor in the department of molecular and medical pharmacology at the university.
Athletes in Olympic sports and most professional sports are tested during competitions or in random out-of-competition visits. They are required to provide a urine sample of at least 75 mL, which is divided into an A sample and a B sample.
The basic testing procedure starts with an aliquot of the A sample, Catlin tells C&EN. A few hours of wet chemistry using column chromatography are required to remove water, ions, and proteins from the sample, leaving a small volume of residue for the analysis. A separate aliquot of the urine sample is used when testing for each class of compounds--for example, stimulants, diuretics, and steroids. The chemical workup is optimized for each class of compounds.
IN THE CASE of steroids, functional groups of the compounds in the isolated residue are next derivatized to render the molecules more volatile for standard gas chromatography-mass spectrometry (GC-MS) analysis, Catlin explains. Trimethylsilyl groups typically are added to the keto or hydroxyl oxygen atoms in the 3-position and 17-position of the four-ring steroid system.
A microliter or so of the derivatized sample is then injected into a benchtop GC-MS for the initial screen, Catlin continues. An algorithm is used to search for 30 to 40 banned steroids based on GC retention time and two or three ions per molecule in the mass spectrum. The amount of steroid in a positive urine sample will be in the nanogram-per-milliliter range, he says. For comparison, testosterone in men is typically about 40 ng per mL, while in women it is about 10 ng per mL or less.
If there appears to be a hit, then a new aliquot of the A sample is used in a confirmation procedure. This involves tailoring the chemical workup to make better derivatives of the drug or metabolite that was observed. The GC-MS analysis is then repeated.
In some cases, liquid chromatography with tandem MS (LC-MS/MS) or high-resolution MS is used to detect or conclusively identify a compound, Catlin notes. Polar compounds are more amenable to LC-MS/MS, he says, and sometimes the steroids don't need to be derivatized. The tandem MS allows the researchers to home in on a particular molecular ion or fragment and monitor its fragmentation to a specific subfragment. Regardless of which methods are used, in routine screening the MS results usually are not confirmed by other analytical methods.
If the confirmation test is positive, the result is reported back to the sports-governing authority that submitted the sample. The sports authority informs the athlete or coach, and then the B sample, which has remained sealed, is opened and tested as well. The athlete and his or her representative--for example, a lawyer or chemist--are allowed to be present during the opening and testing of the B sample.
Once the THG syringe sample had been sent to UCLA, Catlin and a team of seven chemists set about trying to identify the unknown steroid. When a new steroid such as THG is in a urine sample, it's not readily detectable using the standard algorithm, Catlin notes. However, there will be a telltale prominent peak in a region of the chromatogram where steroids generally appear.
"You see evidence that it is steroidal, but you can't necessarily say it is a steroid," he says. "There are hundreds of steroids in urine, so it takes considerable time to work out. We will stop and try to identify it. But if we don't quickly recognize it from our extensive database, or if it's not in our sphere of knowledge, we have to move on. But we save it for later when we have time to look at it more closely."
As the UCLA researchers found out, THG is unstable under the conditions of the routine assay. "That's why we have been blind to it in previous testing," Catlin says.
"In the syringe material we received, we tried to make the TMS derivative, but we saw 20 to 30 peaks in the gas chromatogram," Catlin relates. "We couldn't tell if these were breakdown products from the steroid or if these were by-products or impurities from whomever had synthesized it." At that point, the team knew it would have to try a different approach.
Switching to an LC-MS/MS system made the process less dependent on the derivatization, Catlin notes. "We were actually able to get the retention time and mass spectrum of underivatized THG," he says. "It was difficult, but we got it."
That process turned out to be a good screen. It also gave the researchers a leg up once they found a suitable way to derivatize the material from the syringe, allowing them to determine the molecular ion of the steroid and begin to piece together what the molecule might be.
In early July, after a couple of weeks of work making different types of derivatives and obtaining the spectra, the researchers had identified enough different MS peaks to allow them to draw the structure of THG on paper, Catlin says.
THG is thought to be derived from gestrinone, a synthetic steroid used to treat gynecological disorders. The two steroids differ by only four hydrogen atoms: THG has an ethyl group at C-17, whereas gestrinone has an ethynyl group there. THG also is structurally similar to trenbolone, a synthetic steroid that is used by bodybuilders and by U.S. ranchers in combination with estradiol to bulk up cattle.
Both gestrinone and trenbolone are listed as anabolic steroids in the IOC/ WADA Olympic Movement Anti-Doping Code, which is also generally adhered to by professional sports leagues. A new steroid such as THG also is inherently banned, owing to the catchall phrase "and related substances" that is added to the end of the list for each class of substances in the code.
While the UCLA chemists were still at the "pencil and paper stage," they were able to synthesize THG by simple hydrogenation of gestrinone, Catlin says. "We were able to show a match between the spectra of the synthesized THG and the THG from the syringe."
The synthesis set off a series of other investigations, he adds. As a confirmation, USADA had another testing lab synthesize THG. Catlin's lab also got help from Jane M. Strouse of the UCLA chemistry and biochemistry department's nuclear magnetic resonance imaging facility to characterize THG by NMR spectrometry. USADA further arranged to have an independent NMR expert do the same. USADA also arranged for THG to be given to a baboon. Urine samples collected from the baboon over a few days provided a close approximation of human urine to test the detection procedure and check for characteristic THG metabolites.

RonnyT
07-08-2010, 05:15 PM
THE ADDITIONAL WORK allowed Catlin's group to develop a rapid screen for THG using the LC-MS/MS system. The best derivative has a methyloxime group (5NOCH3) at the 3-position and a trimethylsiloxy group at the 17-position. The confirmation test is carried out using high-resolution MS.
By August, the screening and confirmation techniques were sufficiently refined to begin routine testing. At that point, at the request of USADA, the UCLA lab reprocessed approximately 550 A samples that had been collected at the U.S. Outdoor Track & Field Championships and in out-of-competition testing this past summer. Some of the out-of-competition samples came from the International Association of Athletics Federations (IAAF), track-and-field's world governing body, which had athletes suspected of using THG tested.
From these samples, four U.S. athletes and one athlete from the U.K. had positive THG tests for both their A and B samples. At C&EN press time, the names of the four U.S. athletes had not been officially released, although three of them have been identified in news media reports. The U.K. athlete, sprinter Dwain Chambers, has been officially named.
The THG test methods have been distributed to all IOC/WADA-accredited laboratories for implementation, Catlin says. WADA has encouraged all sports-governing authorities to retest currently stored samples, which means that more positive tests could be in the offing.
IAAF has requested that A samples from the track-and-field world championships held in Paris in August be retested by the Paris lab. The International Federation of Swimming has announced that it will retest some 300 samples taken during its world championships held in July. Some professional sports leagues around the world also are considering retesting some samples.
Each of the athletes who have tested positive for THG will next face an adjudication hearing to determine their fate. For Olympic sports, this is typically a two-year competition ban. However, a positive test doesn't necessarily mean an athlete will be punished.
The athletes or their lawyers can argue that they were unaware of ingesting a banned substance, and that the positive test was inadvertent. This is a routine occurrence, even though each elite athlete is made well aware that he or she is personally responsible for any banned substances in their body. "Ignorance is never an excuse," sports-authority documents stipulate. In addition, a chemist or other expert hired by the athlete can try to punch holes in the testing procedure. With these modes of defense, many athletes have avoided suspensions or had suspensions overturned on appeal.
On Oct. 16, USADA issued a statement officially announcing many of the details of this series of recent events. "What we have uncovered appears to be intentional doping of the worst sort," USADA CEO Terry Madden said. "This is a far cry from athletes accidentally testing positive as a result of taking contaminated nutritional supplements. Rather, this is a conspiracy involving chemists, coaches, and certain athletes using what they developed to be 'undetectable' designer steroids to defraud their fellow competitors and the American and world public who pay to attend sports events."
According to USADA, the coach who turned in the syringe sample identified the source of THG as Victor Conte, the founder and owner of the Bay Area Laboratory Cooperative (BALCO). The Burlingame, Calif., company conducts blood and urine analyses and provides nutritional counseling and dietary supplements based on the results. BALCO has become famous in recent years by catering to a clientele of professional athletes and Olympic stars, which the company has used to promote products on its website.
In early September, the Internal Revenue Service searched BALCO headquarters for evidence. A federal grand jury has been convened in San Francisco to determine if charges will be brought against Conte or others for tax evasion and for the sale and distribution of THG or other controlled substances. One of those substances is the stimulant modafinil, which is used to treat the sleeping disorder narcolepsy. In an additional blow to track and field in recent months, there has been a separate set of positive drug tests for modafinil. Sanctions against those athletes are pending.
As many as 100 professional football and baseball players, well-known track-and-field athletes, and others have been subpoenaed to testify before the grand jury. It's unclear if the athletes have tested positive for the steroid or if they have been summoned only as witnesses, but many were known to have been BALCO customers.
For BALCO's part, the company denies supplying the steroid and claims that THG may not have anabolic properties. Former employees and friends of Conte's have commented to the press that they have never known Conte to be involved with steroids.
THE MYSTERIOUS SYRINGE is the first time in Catlin's 20-year career as director of the UCLA lab that a drug sample has turned up out of the blue. He suspects that a few underground labs are always attempting to design steroids and other drugs that can't be detected. "Our lab has been interested in designer steroids for some time, and we know the theory behind how one can make them to be undetectable. We have been doing some research trying to find them, but the vast number of possibilities you are looking for makes this a difficult project."
About a year ago, Catlin's group did find a previously undetected steroid in a single urine sample from a cyclist, he notes. The steroid, norbolethone, is not exactly a designer steroid like THG. Rather, it was developed by a drug company 40 years ago, but it never made it to market. "Someone out there was reading the old literature, which basically says norbolethone is very potent," Catlin says. "They must have made it. Then we found it in a urine sample." Norbolethone was added to the IOC/WADA list of prohibited substances and methods earlier this year.
Catlin believes it's likely that THG was designed specifically to avoid detection, but he acknowledges that it could just be a coincidence that the steroid's chemical properties made it undetectable. His group has gone back over spectra of previous samples looking for signs of THG, but they have not seen it, suggesting that the steroid has been used for only a short period of time.
"The underlying issue of the THG story is that we have found a steroid that heretofore we did not know about," Catlin says. "That points to some sort of industry--of one or more people--who are involved in developing, synthesizing, and distributing steroids. Getting on top of that is something that sports authorities are going to have to deal with proactively rather than after the fact."
The THG saga suggests that doping in sports will continue to be a cat-and-mouse game. As scientists attempt to develop better testing procedures, unscrupulous athletes will seek out other ways to cheat and, if caught, will continue to insist that their infractions don't violate the "spirit of the rules." Sports authorities probably will continue to be slow-footed in reacting because of the billions of dollars of revenues generated worldwide by professional and Olympic sports. Heading into the 2004 Summer Olympic Games next year in Athens, Greece, those involved on all sides of the doping issue are going to remain on edge.

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