Detection of Performance-Enhancing Drugs ("Doping") in Sports - ChemPRIME

Detection of Performance-Enhancing Drugs ("Doping") in Sports

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"Doping" has been part of sports for a long time. Disqualification of cyclist Floyd Landis from the Tour de France in 2008[1], doping trials in US baseball, and positive tests of olympic athletes are included in a long list of doping cases in cycling or list of doping cases in sports.

Banner at 2006 Tour de France[2]

Now, with more sophisticated laboratories developing drugs that are virtually undetectable because they are also naturally occurring, you may wonder how synthetic versions of the naturally occurring drugs can be detected. They each have the same formula and molecular structure!

The new analytical techniques were developed by Don Catlin who was named Sportsman of the Year for 2002 by the Chicago Tribune[3] among many awards for doping control [4][5].

Contents

GC/C/IRMS

The technique Catlin developed is Gas Chromatography/Combustion/Carbon Isotope Ratio Mass Spectrometry (GC/C/IRMS). Drugs (or drug metabolites) from a urine sample are combusted (burned), and the resulting carbon dioxide (CO2) is analyzed by mass spectrometry to determine the ratio of the stable isotopes of carbon in the CO2 (and also the drug).

Carbon has two naturally occurring stable istopes 136C and 126C (and several radioactive ones that do not concern us here):

Isotope Symbol Protons Neutrons  % on Earth Isotopic Mass
Carbon-12

126C

6 6 98.93% 12.0000000
Carbon-13

136C

6 7 1.07% 13.0033548

If the drug is synthetic, it is likely that the starting materials were of plant origin, and will be enriched in 126C and have a different "isotopic signature" than the natural steroid. Various biological, physical, and chemical processes change (or "fractionate") the stable isotope ratio, changing the typical isotopic abundances in the Table.

What Catlin typically finds is that "δ13C values" for urine samples obtained from 43 healthy males were -23.8‰ and non-doped athletes’ urine samples were similar. But the "δ13C values" value in a "doped" athlete's sample was -32.6‰, suggesting that epitestosterone was administered. The difference is great enough to detect synthetic testosterone in the presence of the natural steroid[6][7].

13C" Values

The analyis is based on "δ13C" values, which represent very small differences in isotopic ratios. Delta values are measured in parts per thousand (or "per mil", ‰). The more negative the value, the more of the lighter isotope is present, so positive values represent material with more of the heavy isotope.

We saw above that carbon is normally 98.93% 126C and 1.07% 136C, but the range is about 98.85 - 99.02% 126C. The δ13C value assigned a value of 0 for a standard with a very high 136C/ 126C ratio, so values are more negative for sources with less 13C (like synthetic steroids). The details of the delta value calculations are not important, but in case you're interested, the formula is

\delta ^{13}C = 
\Biggl( 
\frac{\bigl( 
\frac{^{13}C}{^{12}C} \bigr)_{sample}

-\bigl(
\frac{^{13}C}{^{12}C} \bigr)_{standard}}

{\bigl( \frac{^{13}C}{^{12}C} \bigr)_{standard}} 

\Biggr) * 1000\ ^{o}\!/\!_{oo}

But is there a "normal" isotopic abundance ratio for an element? If the abundance of oxygen isotopes can vary by ~20‰ (2%), how can we have a single "atomic weight" for the element?

The "Normal" Isotopic Ratio: Atomic Weights

All atoms of a given element do not necessarily have identical masses. But all elements combine in definite mass ratios, so they behave as if they had just one kind of atom. In order to solve this dilemma, we define the atomic weight as the weighted average mass of all naturally occurring (occasionally radioactive) isotopes of the element.

A weighted average is defined as

Atomic Weight =
\left(\tfrac{\%\text{ abundance isotope 1}}{100}\right)\times \left(\text{mass of isotope 1}\right)~ ~ ~  +


 \left(\tfrac{\%\text{ abundance isotope 2}}{100}\right)\times \left(\text{mass of isotope 2}\right)~ ~ ~ + ~ ~ ...

Similar terms would be added for all the isotopes. Since the abundances change from place to place, IUPAC has established "normal" abundances which are most likely to be encountered in the laboratory. This important document that reports these values can be found at the IUPAC site. The abundances are also usually listed on the Table of the Nuclides which lists all isotopes for all elements. Surprisingly, a good number of elements have isotopic abundances that vary quite widely, so that atomic weights based on them have only 3 or 4 digit precision.


The atomic weight calculation is analogous to the method used to calculate grade point averages in most colleges:


GPA =
\left(\tfrac{\text{Credit Hours Course 1}}{\text{total credit hours}}\right)\times \left(\text{Grade in Course 1}\right)~ ~ ~  +


 \left(\tfrac{\text{Credit Hours Course 2}}{\text{total credit hours}}\right)\times \left(\text{Grade in Course 2}\right)~ ~ ~ + ~ ~ ...


Example: The Atomic Weight of Carbon

Calculate the atomic weight of an average naturally occurring sample of carbon, given the typical abundances and masses of the isotopes in the table above.


Solution

\frac{\text{98}\text{.93}}{\text{100}\text{.00}}\text{ }\times \text{ 12}\text{.000 + }\frac{\text{1}\text{.07}}{\text{100}\text{.00}}\text{ }\times \text{ 13}\text{.0034}=\text{12}\text{.011}

The exact isotopic mass of 126C may be surprising. It is assigned the value 12.0000000 as a standard for the atomic weight scale. Other masses are determined by mass spectrometers calibrated with this arbitrary standard.


Don Catlin and the Isotope Signatures of Steroids

The chemical properties of the synthetic administered to athletes, and normal testosterone are virtually identical. The only difference is that a bigger proportion of the carbon atoms in synthetic testosterone are 126C We don't distinguish the two in any way when we write chemical equations.

Detection of natural steroids posed a difficult problem for the World Anti-Doping Agency (WADA). The UCLA Olympic Analytical Laboratory, founded by Caitlin in 1982, tested Olympic, professional and collegiate athleste[8]. In the 1990s, the lab was first to offer the carbon isotope ratio test, and first to detect blood booster EPO (erythropoietin) in 2002, and has developed tests for several other designer steroids.

Example 2

The same calculation can be extended to more than two isotopes. Naturally occurring lead is found to consist of four isotopes:


1.40% 20482Pb whose isotopic weight is 203.973.


24.10% 20682Pb whose isotopic weight is 205.974.


22.10% 20782Pb whose isotopic weight is 206.976.


52.40% 20882Pb whose isotopic weight is 207.977.


Calculate the atomic weight of an average naturally occurring sample of lead.

Atomic Weight = \left(\tfrac{\text{98.93}\%}{100}\right)\times \left(\text{203.973}\right)~ ~ ~  +

 \left(\tfrac{\text{24.10}\%}{100}\right)\times \left(\text{205.974}\right)~ ~ ~ + ~ ~ ...
 \left(\tfrac{\text{22.10}\%}{100}\right)\times \left(\text{206.976}\right)~ ~ ~ + ~ ~ ...
 \left(\tfrac{\text{52.40}\%}{100}\right)\times \left(\text{207.997}\right)~ ~ ~ + ~ ~ ...

= 207.22

Defining the Mole

The SI definition of the mole also depends on the isotope 126C and can now be stated. One mole is defined as the amount of substance of a system which contains as many elementary entities as there are atoms in exactly 0.012 kg of 126C. The elementary entities may be atoms, molecules, ions, electrons, or other microscopic particles. This official definition of the mole makes possible a more accurate determination of the Avogadro constant than was reported earlier. The currently accepted value is NA = 6.02214179 × 1023 mol–1. This is accurate to 0.00000001 percent and contains five more significant figures than 6.022 × 1023 mol–1, the number used to define the mole previously. It is very seldom, however, that more than four significant digits are needed in the Avogadro constant. The value 6.022× 1023 mol–1 will certainly suffice for most calculations needed.


References

  1. http://en.wikipedia.org/wiki/Tour_de_france#Doping
  2. http://en.wikipedia.org/wiki/Tour_de_france#Doping
  3. http://en.wikipedia.org/wiki/Don_Catlin
  4. http://www.selectscience.com/product-news/agilent-technologies/agilent-technologies-presents-2008-manfred-donike-award-for-sports-doping-control-excellence-to-german-researcher/?artID=14791
  5. http://www.laboratorytalk.com/news/agi/agi473.html
  6. Rodrigo Aguilera, Caroline K. Hatton and Don H. Catlin, "Detection of Epitestosterone Doping by Isotope Ratio Mass Spectrometry", Clinical Chemistry, 48: 629-636, 2002
  7. Rodrigue Aguilera, Michel Becchi, Hervé Casabianca, Caroline K. Hatton, Don H. Catlin, Borislav Starcevic, Harrison G. Pope Jr. Improved method of detection of testosterone abuse by gas chromatography/combustion/isotope ratio mass spectrometry analysis of urinary steroids Journal of Mass Spectrometry, Volume 31 Issue 2, Pages 169 - 176.
  8. http://en.wikipedia.org/wiki/Use_of_performance-enhancing_drugs_in_sport
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