Carbon isotope ratio mass spectrometry (CIR-MS) is an advanced analytical technique used to detect the use of synthetic anabolic steroids in sports by differentiating between naturally occurring and synthetic forms of the hormones based on their carbon isotope ratios (13C/12C). CIR-MS is highly sensitive and specific, making it a valuable tool in anti-doping laboratories for identifying the illicit use of performance-enhancing drugs.

The principle underlying CIR-MS is the natural variation in carbon isotope ratios found in different carbon sources. The biosynthesis of endogenous (naturally produced) steroids in the human body is derived from dietary sources, which have a specific carbon isotope ratio. Synthetic steroids, on the other hand, are usually derived from petroleum-based precursors, which have a distinctly different carbon isotope ratio compared to endogenous steroids.
The concept of the difference revolves around the differences in carbon isotope ratios between endogenous steroids (naturally produced by the body) and synthetic steroids (manufactured externally). To understand this, we need to dive deeper into the biosynthesis of endogenous steroids and the production of synthetic steroids.

1.Biosynthesis of endogenous steroids:

Endogenous steroids are synthesized within the body from cholesterol, which in turn is derived from dietary sources. The main dietary sources of cholesterol include animal-based products like meat, eggs, and dairy. Plants also incorporate carbon into their structures during photosynthesis, which ultimately makes its way into the food chain.
Carbon isotopes, primarily 12C and 13C, are found in different ratios in various carbon sources, depending on factors like the type of photosynthesis pathway (C3, C4, or CAM) and environmental conditions. When animals consume plants or other animals, they incorporate these carbon isotopes into their tissues, including cholesterol. Consequently, the carbon isotope ratio (13C/12C) of endogenous steroids reflects the isotope ratios of the dietary sources, which are relatively consistent within a population.

2. Production of synthetic steroids:
Synthetic steroids are manufactured externally, typically using chemical synthesis methods. The starting materials for the production of synthetic steroids are often derived from petroleum-based precursors. Petroleum, originating from ancient organic matter (mainly marine microorganisms), has a different carbon isotope ratio compared to contemporary dietary sources.
During the synthesis of synthetic steroids, the carbon isotope ratios of the petroleum-based precursors are carried over into the final product. As a result, synthetic steroids exhibit a different carbon isotope ratio compared to endogenous steroids.
By analyzing the carbon isotope ratios of steroids in an athlete’s sample using carbon isotope ratio mass spectrometry (CIR-MS), it is possible to differentiate between endogenous and synthetic steroids. If the carbon isotope ratio of the target steroid metabolite falls outside the expected range for endogenous steroids, it may indicate the presence of synthetic steroids, suggesting doping.

To further illustrate the concept that synthetic steroids have different carbon isotope ratios than endogenous steroids, let’s consider a specific example: the synthesis of synthetic testosterone, a widely used anabolic steroid.

1. Synthesis of synthetic testosterone:

Testosterone is an endogenous steroid hormone that plays a crucial role in the development of male reproductive tissues and the maintenance of secondary sexual characteristics. Synthetic testosterone, often used for doping purposes, is typically synthesized from petroleum-derived precursors, such as stigmasterol, a phytosterol obtained from soybean oil.

Stigmasterol is converted into testosterone through a series of chemical reactions, which include the following steps:

• Conversion of stigmasterol to progesterone
• Conversion of progesterone to androstenedione
• Conversion of androstenedione to testosterone

During these reactions, the carbon atoms in the precursor molecule (stigmasterol) are rearranged and/or modified but retain their original carbon isotope composition (13C/12C ratio).

2. Carbon isotope ratios in synthetic testosterone:

As mentioned earlier, petroleum-derived precursors like stigmasterol have a different carbon isotope ratio compared to the carbon sources in endogenous steroids. The distinct carbon isotope ratios in petroleum-based compounds are primarily due to the fact that they originate from ancient organic matter, which was subjected to different environmental conditions and photosynthetic processes than modern-day plants.

When synthetic testosterone is synthesized using petroleum-derived precursors, the resulting product inherits the carbon isotope ratio of the starting material. Consequently, synthetic testosterone exhibits a different carbon isotope ratio compared to endogenous testosterone produced by the body.

3. Detection using CIR-MS:
Carbon isotope ratio mass spectrometry (CIR-MS) can be employed to differentiate between endogenous and synthetic testosterone based on their carbon isotope ratios. By analyzing the 13C/12C ratio in the target steroid metabolites, anti-doping laboratories can determine if an athlete’s sample contains synthetic testosterone. If the carbon isotope ratio falls outside the expected range for endogenous testosterone, it suggests the presence of synthetic testosterone and potential doping.

The CIR-MS technique involves the following steps:

1. 1. Sample preparation: First, the athlete’s urine sample is processed to isolate the target steroid metabolites. This typically involves solid-phase extraction (SPE), liquid-liquid extraction (LLE), and derivatization procedures to concentrate and purify the compounds of interest.
   2. Combustion: The isolated steroid metabolites are then combusted at high temperatures, converting them into their constituent gases, primarily carbon dioxide (CO2), water (H2O), and nitrogen (N2).
   3. Gas purification: The generated CO2 and other combustion gases are separated and purified using gas chromatography (GC) to remove any residual impurities.
      Isotope ratio determination: The purified CO2 is introduced into the mass spectrometer, which measures the carbon isotope ratios (13C/12C) by comparing the relative intensities of the 13C and 12C ions. The mass spectrometer typically used for CIR-MS is an isotope ratio mass spectrometer (IRMS), specifically designed to measure the precise isotopic composition of small samples.
   4. Isotope ratio determination: The purified CO2 is introduced into the mass spectrometer, which measures the carbon isotope ratios (13C/12C) by comparing the relative intensities of the 13C and 12C ions. The mass spectrometer typically used for CIR-MS is an isotope ratio mass spectrometer (IRMS), specifically designed to measure the precise isotopic composition of small samples.
   5. Data analysis: The carbon isotope ratios obtained from the athlete’s sample are compared to established reference values for endogenous steroids. If the carbon isotope ratio of the sample falls outside the expected range, it may indicate the presence of synthetic steroids, suggesting doping.

CIR-MS has been instrumental in enhancing the detection capabilities of anti-doping laboratories and providing robust evidence for the use of synthetic anabolic steroids. The technique’s high specificity and sensitivity make it a valuable tool in the fight against doping in sports.