Source丨Science Compound(ID:kexuedayuan)
Author 丨 Tan Xiaotian
The schedule of the Beijing Winter Olympics is nearing its end, and in addition to the wonderful competition, there are also some discordant news:
As an onlooker, you may often hear words such as "blood test" and "urine test" in the news related to doping, but how exactly is the stimulant detected? In this Winter Olympic Games, what new means of testing doping has the Beijing Winter Olympic Organizing Committee used?
Stimulant detection: chromatography + mass spectrometry
After decades of struggle with doping, the detection and screening technology of doping molecules in the world has become more and more sound. Although the Beijing Winter Olympic Organizing Committee has not yet announced the specific method of doping detection in this Olympic Games, we can know the principle of doping testing from the conventional testing methods of previous large-scale sports events.
Conventionally speaking, most of the doping tests in sports events are first extracted and isolated from various molecules in the biological samples of athletes, and then the "identity" of these molecules is determined in turn. Although the samples taken from athletes are generally liquid samples, such as blood, urine, sweat, etc., in the process of detection, it is actually divided into two tracks: gas and liquid.
Urine is the most commonly used test specimen, while blood samples are mainly used to detect molecules that are generally not easy to enter the urine, such as proteins/glycoprotein hormones such as growth hormone and erythropoietin, as well as exogenous red blood cells and other active biological products that can improve their performance in the competition. Some of the more volatile molecules in the urine (such as some small molecule steroid stimulants) will participate in the gas track test, while the remaining molecules in the blood and urine that are larger and more "honest" will appear in the liquid test track.
The current general process of doping analysis
(Image source: the author made it himself)
Detect step 1 – Separation of different molecules by chromatography
Although the tracks are not the same, the detection process for different molecules in the gas and liquid tracks is the same. The first step in both testing methods is to separate the various molecules contained in the complex sample and let them stand in line.
Liquid chromatography/gas chromatography techniques
(A) Schematic diagram of a typical liquid chromatography structure, where the transverse column is an LC separation column. (B) Gas chromatography structure diagram, of which the shape of the coil is a GC separation column. (C) A miniature GC separation column integrated into the surface of a silicon wafer in the form of a chip.
(Image source: Wikipedia (A), Reference 1 (B+C))
Through the difference in the molecular mass, charge, hydrophilicity and other physical characteristics of different molecules in the sample, different molecules can be separated by certain laws, this method is called "Chromatography". It's like letting these molecules run a long marathon and then distinguish the difference between their "endurance" over a long distance.
The name of the chromatography actually comes from the Russian botanist Mikhail Zvet (Михаил Семённович Цвет) a hundred years ago. I believe that this experiment, many friends have learned and experienced in the middle and high school biology class (using filter paper to separate chlorophyll A, chlorophyll B, lutein and β carotene from the leaf juice). But today, the physical characteristics of molecules that use chromatography to separate molecules rarely include optical features such as molecular color.
The original molecule used in modern chromatographs to separate molecules is called a separation column, which is now generally called a column by "biochemical ring material experimental dogs". Generally speaking, a slender pipe is filled with fillers that can interact with molecules. The columns used in Liquid Chromatography (LC) appear to be short and thick (generally no more than one meter in length), while in Gas Chromatography (GC), the filler distance required to separate different molecules often takes 5-10 meters or more due to the weak interaction between gas molecules and fillers. For this reason, the separation column of gas chromatography actually looks more like a coil.
A typical mass spectrometry analyzer
( Image source: Wikipedia)
Test step 2 – Mass spectrometer to verify the body
Whether it is a liquid molecule or a gas molecule in a sample, after they have separated different molecules through a chromatograph, the next hurdle they face is a mass spectrometer (MS) that can "verify their body". Mass spectrometers can separate and purify molecules by chromatography for ionization, and then analyze their "characteristic fingerprint" - mass spectrometry , to determine the true identity of this molecule.
It is worth noting that there are currently many different and common mass spectrometry techniques in the field of doping testing, and they all have their own unique skills. For example, DFS high-resolution dual-focus magnetic mass spectrometry, which is mainly used to detect trace stimulants, and DELTA V isotopic mass spectrometry, which is mainly used to distinguish endogenous and exogenous testosterone (synthetic testosterone tends to have higher carbon isotope consistency, while the carbon atoms in the testosterone molecules produced by the body itself will contain a portion of C13 or C14).
Speaking of testosterone, in fact, there is a very thrilling old thing, which happened to liu Guoliang, a "fat man who doesn't understand the ball" that we are very familiar with. At the 1999 World Table Tennis Championships, Liu Guoliang was found to be high in serum episterone (a variant of testosterone), suspected of taking exogenous stimulants, and was almost banned. But in reality, his abnormal serum testosterone levels were caused by his own physiological circumstances (perhaps born with a clear skeleton?). ), not taking stimulants. At that time, it was precisely by relying on the new technology of isotopic quality spectrum and the multiple flight sampling inspections of the International Table Tennis Federation that Liu Guoliang was able to clear his grievances.
While the current combination of chromatography + mass spectrometry (LC-MS/GC-MS) is still the gold standard for doping testing, other technologies have also emerged over the years. For example, the rapid detection technology of special molecules in sweat (drugs, stimulants, etc.) based on microfluidic immunoassay (testing can be completed within 20-30 molecules), ELISA immunoassay kit for some common stimulants (such as ephedrine), stimulant detection technology that takes the technical route of electrochemistry, capillary electrophoresis, etc.
Beijing Winter Olympics:
The dried blood point technique was officially used for the first time
Not only detection technology, but also the transportation and preservation technology of athlete samples and sample extraction technology also have a great impact on the effective detection of illegal stimulants.
As mentioned earlier, the biological test materials of traditional athletes generally include blood/serum as well as urine. In order to prevent stimulant molecules in the urine/blood sample from being hydrolyzed or inactivated under the influence of enzymes, the storage and transportation conditions of the sample are quite harsh, for example, the sample needs to be sent for testing as soon as possible in a refrigerated (4 C) environment, and the sample that needs to be stored for a long time must be maintained below -20 C.
In 2020, the International Doping Testing Machine ITA also set up a special preservation facility for long-term storage of samples, which can preserve all kinds of specimens collected by athletes for more than ten years. In addition, after the sample is transported to the laboratory, it also needs to go through a more complex extraction process, and after eliminating the interference of cells and proteins, it can enter the process of chromatography/mass spectrometry detection.
Dry blood spots on three different filter papers. After the blood is dripped on the test strip, it will gradually dry out over a period of about 30 minutes. However, for the sake of safety, the air drying time is at least 2 hours (Image source: Reference 2)
After the Tokyo Olympics trials, a new sample collection/transportation technology, Dry Blood Spot (DBS), was officially incorporated into the doping testing process at the Beijing Winter Olympics. In fact, this technology is very easy to understand. It is made by dripping a drop of the athlete's blood (which can be venous blood/fingertip blood) on the filter paper (or nitrocellulose membrane) and allowing it to dry naturally for two or three hours. The dried blood point technique is not a new technology, it has played a great role in the fight against infectious diseases such as AIDS, hepatitis B/C, malaria and so on, especially in the sample collection activities of patients in underdeveloped areas.
For doping testing alone, in the process of drying the dried blood point, not only the concentration of the small molecules of stimulants in the sample can be concentrated, but also the cells contained in the blood will lose water and die. Without water and enzyme interference, dried blood point samples are very stable and can be stored at room temperature for days or even weeks. That is to say, in the future Olympic Games, it is even possible to transport samples without carrying thermal insulation equipment, and only through the simple encapsulation method of envelopes.
At the same time, since there is almost no water in the dry blood point sample, it is more conducive to the extraction of some hydrophobic organic compounds when using special organic solvents such as methanol. These technical advantages all help to improve the detection ability of stimulants in subsequent gas chromatography/liquid chromatography/mass spectrometry tests.
epilogue
As the saying goes, even with so many anti-doping advanced technologies and equipment, there are still a few athletes who will take the risk of doping. Ensuring that the competition is completely fair remains a very difficult task, and anti-doping also requires the joint efforts of all parties.
bibliography
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[2] Denniff, Philip, and Neil Spooner. "Effect of storage conditions on the weight and appearance of dried blood spot samples on various cellulose-based substrates." Bioanalysis 2.11 (2010): 1817-1822.
[3] Tretzel, Laura, et al. "Dried blood spots (DBS) in doping controls: a complementary matrix for improved in-and out-of-competition sports drug testing strategies." Analytical Methods 7.18 (2015): 7596-7605.
[4] Ramesh, Bokka, et al. "LC–HRMS determination of piperine on rat dried blood spots: A pharmacokinetic study." Journal of Pharmaceutical Analysis 6.1 (2016): 18-23.
[5]http://www.xinhuanet.com/politics/2016-02/27/c_128756864.htm
[6] http://wsjkw.hebei.gov.cn/html/zwyw/20220214/386104.html
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Author Affilications:Beijing-Tianjin-Hebei National Technology Innovation Center