IRMS
A very advanced analytical method for the authentification of vanilla extracts and vanilla flavors is stable isotope ratio analysis (SIRA). It was developed in the 1970s and became the most important tool for authenticity testing of nonchiral compounds. This analysis can be performed either by IRMS, mainly combined with a gas chromatographic separation, or by quantitative NMR measurement of the natural abundance at individual atomic sites (Schmidt et al., 2007).
In nature, the main bioelements such as hydrogen, carbon, oxygen, and nitrogen occur as mixtures of isotopes. The natural abundances of the stable isotopes have global average values. Owing to physical processes, (bio)chemical reactions such as photosynthesis, geographic parameters, and climate, their relative ratio can vary. Always the “light” isotopes (1H, 12C, and 16O) are by far dominant compared with the abundance of the “heavy” ones (2H, 13C, and 18O). The products formed in plants (or animals) and the ingredients of the extracts or the food prepared from them obtain a characteristic isotope ratio, which allows to correlate it to the photosynthetic pathways, and the climatic and geographic conditions.
Compounds with an aromatic ring—for example, vanillin—are generally synthesized in plants through the shikimic acid pathway from erythrose-4-phosphate and phosphoenol pyruvate. The formation of the products of this pathway is accompanied by a corresponding depletion of 13C relative to the primary plant products, the carbohydrates.
These carbohydrates are produced during photosynthesis of plants utilizing CO2 and water. The primary step is enzymatically catalyzed, with 13CO2 reacting somewhat more slowly than 12CO2. This phenomenon is named the “kinetic isotope effect.”
However, the 13C deficit is not identical for all plants.
Three major photosynthetic pathways for the CO2 fixation are known for plants: C3, C4, and CAM. The so-called C3 plants (e.g., wheat, barley, sugar beet, and most trees) use the ribulosebisphosphate-carboxylase reaction, the Calvin pathway, while C4 plants (e.g., sugarcane, maize, sorghum, and millet) use the phosphoenolpyruvate-carboxylase reaction, the Hatch–Slack pathway. The CAM plants such as succulents, orchids (e.g., V. planifolia), and some tropical grasses have the Crassulacean acid metabolism. Each group shows different values of the 13C/12C, 2H/1H, and 18O/16O ratios for their metabolites as can be shown by IRMS (e.g., carbohydrates, fatty acids, isoprenoids, amino acids, phenylpropanes, etc.). By analyzing these ratios, it is possible to distinguish between compounds produced by plants during the biochemical pathways and those produced by synthesis.
The changes in the isotope ratio caused by these effects are very small. So they are not indicated in the atom% scale. These minimal changes are compared with the values of international standards and expressed in per million (‰) as a variance from standard, the so-called delta value (Schmidt, 2003):
δ13C (‰) = (([ 13Csample ] / [ 12Csample ]) / ([ 13Cstandard ] / [ 12Cstandard ]) − 1) × 1000
The standard for the 13C/12C ratio is δ13C: V-PDB (Vienna-PeeDee Belemnite). The analysis of δ13C values for C3 plants (the so-called “light plants”) give values between −30‰ and −24‰, for C4 plants (the so-called “heavy plants”) between −16‰ and −10‰. The CAM plants have δ13C values from −10‰ to −30‰, making a differentiation from the other two types very difficult.
The authenticity proof of flavor substances by determination of their average δ13C value implies the combustion at about 1000°C of the purified organic samples in order to convert them into CO2. An online coupling of gas chromatography and IRMS via a combustion interface reduced the sample amount and made measurements easier. Later on, an online coupling of gas chromatography and IRMS via a pyrolysis interface (about 1300°C) was developed, primarily for 18O analysis, allowing the simultaneous online measurement of δ18O and δ13C values. This interface also affords the determination of δ2H when applying the pyrolysis at about 1450°C. Hener et al. (1998) compared the δ13C values of different vanillin samples measured via CO2 and CO and showed that the values are, in most cases, in good agreement. Nowadays, mainly the online coupling of gas chromatography with combustion or reductive pyrolysis to isotope ratio mass spectrometry (GC-C-IRMS or GC-P-IRMS) is used for the determination of the δ-values of carbon 13C, hydrogen 2H, and oxygen 18O.
V. planifolia belongs to the CAM plants, and vanillin ex-beans shows δ13C values in the range from −16.8‰ (V. tahitiensis) to −22.0‰ (V. planifolia) (see Table 15.3).
| Origin of Vanillin | δ13CV-PDB (‰) | δ2HV-SMOW (‰) | δ18OV-SMOW (‰) |
|---|---|---|---|
| Ex-beans (Bourbon) V. planifolia | −21.5 to −19.2a | 6.7–12.4b | |
| Ex-beans (Tahiti) V. tahitiensis | −19.7 to −15.9a | ||
| Ex-beans | −20.4 to −20.2c >−21.5e −21.5 to −16.8d −22.0 to −19.0g | −115 bis −52d | 12.2–14.0c 8.1–10.7f |
| Ex-guaiacol | −36.2 to −29.0h −26.1 to −24.9i | −23 bis −17i | −3.1 to −2.5f |
| Ex-eugenol | −31.7 to −29.9h | −87j | 11.8–13.3c 0.3k |
| Ex-lignin | −28.7 to −26.5d | −204 bis −170i −195 to −178b | 6.1–6.8f 6.0–9.8l |
| Ex-ferulic acid Ex-rice bran (biotechnology) | −37 to −36m −36.4 to −33.5n | −168 to −165n | 12.4–13.2c 10.7–11.2n |