Isotope Ratio Mass Spectrometry

Mass Spectrometry represents one of the most effective techniques to characterize various molecular structures and to determine elementary composition and the dosage of molecules in complex matrices. A less frequent but very useful application is the precise determination of isotope ratios. Isotope Ratio Mass Spectrometry (IRMS) measures the abundance of a particular isotope of a given atom (C, N, O, H).  When carbon is of interest, the analysis is based on the fact that the proportion of 13C is not the same if the source of the carbon is organic or inorganic.

Studying isotope variations caused by natural phenomena  has proved very informative in a number of domains such as earth sciences, geochemistry, climatology, oceanography and biomedical sciences. A typical application in the latter case is the authentification of the origin of an organic substance. Indeed, the analysis of carbon isotopes makes it possible to differentiate an organic compound of natural origin from the same compound of synthetic origin. The obvious application of this technique is repression of fraud.

The principle of IRMS consists in measuring the relative abundance of stable isotopes of certain atoms (13C/12C, 15N/14N, 18O/16O, 2H/1H) found in the molecule under study. Once the ratio has been determined it is possible to differentiate two molecules  that have identical structures but different origins. All the classical mass spectrometry analyzers (quadrupole, ion trap, time-of-flight, etc) are capable of measuring isotope abundance. What sets IRMS aside is the very high precision and exactitude of the ratios that are obtained.

Isotopic fractioning

Natural carbon isotope variations are relatively rare  (approx. 0.03% in plants) and IRMS employs a specific notation when analyzing the measured data. The results are expressed as a per thousand difference (eg. 13Co/oo) relative to a reference substance (PDB).  Because most carbon sources contain less 13C than the reference, the obtained 13Co/oo values are usually negative.

Isotopic fractioning can be observed in nature.  Indeed, because humans eat plants and animals which themselves eat plants, the isotopic ratios in molecules synthesized endogenously may vary. Plants, like all other living organisms, exchange matter with their environment. As far as atmospheric carbon fixation is concerned, plants rely on three different photosynthetic modes to produce organic matter:

• The Calvin-Benson C3 cycle (wheat, sunflower, etc.)
• The Hatch et Slack C4 cycle  (maize, sugar cane, etc.)
• The Cam (Crassulacean Acid Metabolism) cycle that takes place in succulents, exotic plants and plants growing in desert-like environments.

The natural molecules found in plants exhibit a specific isotope signature that reflects their environment and the type of photosynthesis used.

Instrumentation

The first step in IRMS is to separate the volatile constituents in the sample using gas chromatography (GC). Each molecule is then oxidized during a combustion step at 940°C at the exit point of the GC column. The products generated by this combustion are essentially CO2 and H2O, with each carbon atom in the original molecule generating one CO2 molecule. Thus, the produced CO2  has the same 13C/12C isotope ratio as the original compound. The analyte then enters a reduction reactor, an oven at a temperature of 600°C where nitrogen oxides are reduced into dinitrogen (N2) and excess oxygen and residual water are eliminated. The CO2, produced earlier, moves into the mass spectrometer electron impact source which causes the ionization of molecules into ions 12C16O2+ and 13C16O2+  preserving  the isotope ratio of the original compound. The instrument is calibrated by introducing into the source a known amount of a standard CO2 sample.

Application in the fight against doping

In the fight against doping, IRMS is used to detect testosterone doping. According to the World Anti-Doping Agency (WADA), this type of analysis should be conducted when at least one of the following criteria is met:

• The testosterone/epitestosterone (T/E)  ratio is higher than 4.
• The concentration of testosterone or epitestosterone (glucuronide equivalents) is higher than 200 ng/ml.
• The concentration of androsterone or etiocholanolone (glucuronide equivalents) is higher than 10'000 ng/ml.
• The concentration of DHEA (glucuronide equivalents) is higher than 100 ng/ml.

The highest C13 values are obtained with specific populations, such as Americans, that consume foods rich in C4 plants. In order to take into account variations between individuals caused by different diets, it is recommended to measure the isotopic ratio in a reference compound produced endogenously. To truly reflect endogenous levels in an individual having a specific diet, the isotopic ratio in the reference compound should not be affected by the use of exogenous testosterone or any of its precursors. Currently, both 5-pregnandiol and 16(5alpha)androsten-3alpha-ol are use as references. Their concentrations are not affected by the use of testosterone or DHEA. Doping is confirmed when the delta13C value of the steroids in the sample differs significantly (by at least 3o/oo) from that of the endogenous reference compound. If the concentration of the reference is too low to be measured, the result is said to be «non-conclusive» unless the isotope ratio of the steroids present in the sample  is lower by more than –28o/oo compared to that of the natural steroids.

<sup>Delta 13Co/oo values compared to the PDB reference















GC-C-IRMS instrument</sup>

Last Update on 07.10.2008