Handbook of Vanilla Science and Technology

The structure and flavoring properties of vanillin are the same, whether derived from vanilla beans or prepared synthetically from lignin, eugenol, and guaiacol or petroleum by-products. The ¹³C/¹²C ratio for natural vanillin from vanilla beans, however, is different from vanillin produced synthetically from lignin, eugenol, or guaiacol, all of which are also plant materials. In plants, the isotopic fractionation of carbon takes place during photosynthesis via CO₂ fixation. Plants fix CO₂ by one of three metabolic pathways: Calvin synthesis (C₃ plants), Hatch-Slack pathway (C₄ plants), or Crassulacean acid metabolic (CAM) pathway, each of which produce natural products with different ¹³C/¹²C ratios. In the CAM plants, both the Calvin cycle as well as Hatch-Slack pathway modes are operational. In these plants, CO₂ is absorbed at night and combines with phosphoenolpyruvate to form 4-carbon organic acids (malic and isocitric acids). During daylight, C₄-acid breaks down releasing CO₂ to be utilized by the Calvin cycle. The CAM pathway operates in vanilla and enriches vanillin with the ¹³C isotope. Bricout (1974), Bricout et al. (1981), and Hoffman and Salb (1979) applied the radio carbon technique to determine the δ¹³C values (generally reported as the stable isotope ratio analysis, or SIRA) of vanillin from different sources and used the information to establish the origin of vanillin in vanilla extracts. The δ¹³C results are reported in parts per thousand relative to the fossil Pee Dee (South Carolina) Belemnite Standard (PDB). They are computed as follows:

Fig. 9.6 Specific sites on vanillin molecule for natural isotope ratios. Vanillin from vanilla beans of many origins (49 samples) yielded the following values: D/H ratios: 1-130.8 SD ± 3.1, —157.3 ± 3, 4-196.4 SD ± 2.5, 5-126.6 SD ± 1.7.

The δ¹³C values for vanillin from different sources are given in Table 9.9.

Table 9.9 Carbon SIRA of vanillin from various sources (from Hoffman and Salb 1979)

Source of Vanillin | δ¹³C

Vanilla (Madagaxscar) | — 20.4

Vanilla (Indonesia) | — 18.7

Vanilla (Mexico) | — 20.3

Vanilla (Tahiti) | — 16.8

Lignin | — 27.0

Eugenol (clove oil) | — 30.8

Guaiacol | — 32.7

It is apparent that the addition of synthetic vanillin to vanilla extract can be detected by SIRA. However, a means of circumventing SIRA of vanillin was discovered, which involved addition of ¹³C-enriched vanillin to lignin-derived vanillin. This was done by replacing the methyl group of lignin-derived vanillin with methyl-¹³C, which made the synthetic vanillin appear natural to routine SIRA. Bricout et al. (1981) and Krueger and Krueger (1983) developed new tests to detect this addition of [methyl-¹³C]-vanillin to lignin-derived vanillin.

Their technique involves removal of methyl carbon from the vanillin molecule prior to performing SIRA on the resulting dihydrobenzaldehyde (DHB) or CH₃I. This analysis reveals whether lignin vanillin is altered with methyl-¹³C and makes it even more difficult to adulterate pure vanilla extracts with synthetic vanillin. This is illustrated in Table 9.10.

Table 9.10 Analysis of [methyl -¹³C]-Vanillin (from Krueger and Krueger 1985)

Sample | δ¹³C_total | δ¹³C_DHB | δ¹³C_methyl

Lignin vanillin | —27.0 | —26.7 | —

Altered lignin vanillin | —20.0 | —26.0 | —

Lignin vanillin | —27.3 | — | —28.4

Altered lignin vanillin | —20.6 | — | + 25.8

Besides the methyl group, there are seven other molecular positions in vanillin, in which the ¹³C label could be placed without detection by any of the above mentioned tests. Out of concern that some day an economical method could be developed for producing one of these, [carbonyl-¹³C]-vanillin, Krueger and Krueger (1985) have developed a method for detecting such an adulteration. Their method involves oxidation of vanillin to vanillic acid in the first step. This is followed by decarboxylation of vanillic acid with bromine, and performing SIRA on the resulting CO₂ from the carbonyl carbon. Vanillin carbonyl SIRA values for lignin and natural vanillin are given in Table 9.11

Table 9.11 Vanillin carbonyl SIRA on various vanillin sources (from Krueger and Krueger, 1985)

Sample | δ¹³C_carbonyl | δ¹³C_total | Remarks

Lignin | —37.7 ± 1.4 | —27.22 | Known lignin vanillin

Altered lignin | +17.1 | —19.94 | Lignin vanillin with added [carbonyl ¹³C]-vanillin

Bourbon | —25.7 | —21.4 | Known natural vanillin

Bourbon | —27.7 | — | Known natural vanillin

Bourbon | —23.2 | — | Known natural vanillin

Bourbon | —29.9 | — | Known natural vanillin

Bourbon | —24.7 | — | Known natural vanillin

Bourbon | —24.8 | — | Known natural vanillin

Commercial | —24.5 | — | Probably natural

Commercial | + 67.1 | —15.6 | Adulterated carbonyl-¹³C

9.4.2.2 SNIF-NMR technique