Doping: a search of banned drugs

Which compounds are doping, what technology to identify banned substances in athletes and what is needed biological passport?Dr. Doping has prepared for you material about techniques to identify doping in athletes.

Doping is a wide variety of methods of influence on the body that have the potential to enhance athletic performance, which makes their use by athletes unethical and contrary to the very concept of sport.

The doping consists mainly of drugs developed for the treatment of various diseases and used in a variety of medicinal purposes. Periodically sheet doping drugs varies, and there are additions. So, we know that in 2016 it was Meldonium - a metabolic drug. Some of the compounds are included in the list in groups - to the use of all steroids banned. This sheet is a separate section, saying that banned all so-called designer drugs, and drugs that are in clinical trials. That is, even if we do not clear whether the newborn medicine to improve the results of sportsmen, by default it is not allowed. The doping list also hit and noble gases - argon and xenon, which are now very actively used in medicine and cause a number of amazing physiological effects.

In order to identify the fact of use of many drugs included in the list of restrictions, conducted standard and relatively simple tests based on enzyme-linked immunosorbent assay and other routine biochemical methods, and in the same way as the blood testing in biochemical laboratories.

But the search for some kinds of drugs in blood and urine is a very complex technological procedure. We may think that doping analysis is simple, because we all go to the lab for blood tests and urine tests. In this technologically rigorous doping analysis is fundamentally more complicated process. Basically, in order to determine an athlete taking a doping or not, using a urine test, but now it is connected to, and a blood test. The most common test for doping occurs during a sporting event, but it is likely that competition between the officer World Anti Doping Agency (WADA) will come and take a sample of the athlete, to find out if he did not accept doping in the run-up. This is due to the fact that all medications are washed out of the body, so the closer to the time of dosing were analyzed, the more sensitive.

Methods for doping detection

Prohibited substances include steroids and many all "complex" organic molecule, which is used to detect a very advanced technology in high performance liquid chromatography with tandem mass spectrometry.

Various substances in the blood can be "drived", i.e. to allocate spectrum of column for liquid chromatography. After that we see a range of different compounds and we know what a strip of the spectrum are those or other compounds with similar chemical properties of the molecule can be collected in one strip. And to find out what the connections are included in each strip of the spectrum, we need to "weigh" them in microquantities and determine their precise quality composition. This is done by means of mass spectrometry.

For a more accurate determination of compounds within the range of separate fragments of a spectrum obtained by using liquid chromatography, is transferred to the mass spectrometer (in ionic form). In the mass spectrometer, we see the peaks of compounds having different molecular weight, and the more the compound in the urine, the higher peak. Very simplifying, we can say that the researcher conducting high performance liquid chromatography in tandem with mass spectrometry, there is a great "mountain range" of the various peaks and, until he became interested in a particular peak, he does not understand what a compound is hiding behind them.

At the moment, there is no methodology that would allow a high degree of confidence to analyze automatically and without significant effort of the operator as any of the compounds is in the urine. The amount of various lipids to which steroids are extremely difficult to install, despite the rapid development of biochemical techniques. NMR spectroscopy is capable of much faster method to identify and sort molecules than mass spectrometry, but it does have some limitations: the method of cost, availability and complexity of the installation service. This method is only suitable for the identification of molecules with low molecular weight, which is what the majority of illicit drugs and steroids.

There is a list that includes several dozen steroids, molecular weight and properties of ions which are well known to us, and we know what the peak corresponds to a particular steroid or its metabolite. If an athlete did not use, for example, steroid Methandienone, in the place where he could appear on a mountain ridge, is a failure. If he took it, then this place will be a small peak, and the longer he took it, the higher the peak.

Natural steroids contained in humans also form a peak, but if a person uses an excess amount of the natural steroid, this peak will be extremely high. This applies to those steroids that we know. But the real difficulty with tests is that athletes can make "designer" (made to order) steroids with a variety of molecular weights, which can hide behind the higher peaks of natural steroids or other compounds present in our urine or blood. To calculate this, you need to guess to isolate and analyze specific peak of the mountain range, which previously were sizing. And it should be noted that the method is able to some extent to simplify the procedure and streamline detection of illicit substances has been developed only in the 2005 year.

The biological passport

In order to make the sport fair, came up with the biological passport. Previously, they were introduced to stop the manipulation of the blood - the introduction of the blood itself, which was harvested before the competition, or the introduction of the hormone erythropoietin, which increases the number of red blood cells and enhances endurance athlete. Even if you can not find itself in the urine erythropoietin, you can see a radical increase in red blood cells.

A few years ago, we have been introduced and "steroid passport" that is, bringing the total number of fixed steroids in the urine of athletes. This means that is not so important, there are some peaks on a mass spectrometer, and high performance liquid chromatography. If the athlete number of test steroid greatly exceed that which was previously recorded, it is established that the use of steroids, even if a particular steroid was not determined.

The process of metabolism of various compounds in the body is extremely complex, so even if an athlete takes a steroid molecule, a synthetic hormone, which we all know, it can be modified in his body in a completely different connection. To date, we have models that could reliably predict that our liver specific make of a particular molecule. Some researchers, in order to more accurately predict the range of metabolites suggest using special mice that human liver was implanted. The method is to produce the compound WADA prohibited mice and analyzing metabolites in their urine, which will produce humanized liver. The information obtained will allow for a fresh look at the spectra of chromatography in tandem with mass spectrometry and pay attention to those areas that have not been of interest.

Laboratory of Sherlock Holmes

Thus, the search for illicit drugs is a very complex technological process, so in the world, only 34 laboratories accredited by the WADA, which can carry out such tests. The more serious competition, the more severe methods of identifying compounds used in them. In not the most major championships search doping is performed through the list with the help of not very sensitive method (without mass spectrometry), allowing in some cases hide steroids. If normal competition can conceal the fact of doping, on the major sporting events it becomes almost impossible.

It should be understood that the lipid-rigorous logic and mass spectrometry testing is done only in special cases, it was never widespread. And with routine mass spectrometry, even in ongoing WADA accredited laboratory, performed the definition of a fixed list of prohibited molecules and their metabolites, that is, search is carried out that it is well known how to look for. Advanced expert in mass spectrometry is similar to Sherlock Holmes - a single ultra-high-skilled people from connecting to the testing only in cases of a high likelihood of an athlete banned (including design) of drugs that have not managed to find in the usual way.

It seems that if you hold a mass in-depth analysis of urine and blood plasma of athletes from different countries, who have won the competition, using the latest generation of mass spectrometry and IR spectroscopy, it is very likely there may show very interesting substance present only in the urine and the blood of elite athletes.