Handbook of Vanilla Science and Technology

Most recently, Culp (2009) at the CAIS presented a two decade review of isotopic data on flavors, including vanilla. He demonstrated the need to have an in-depth understanding of the changing atmospheric ¹⁴C radiocarbon levels, as these are changing because of the atmospheric nuclear testing in the 1950s and 1960s and its subsequent cessation. He discussed data gathered on over 3,800 flavor samples during these two decades. Data included levels of radiocarbon (¹⁴C) and the stable isotopes of ¹³C/¹²C and D/H. This data can be used to authenticate flavor materials. Also, due to the yearly predictable decay of atmospheric ¹⁴C, the year of origin of the material can possibly be established.

10.4.4 Nuclear Magnetic Resonance, NMR

Martin et al. (1993) described the NMR authentication technique that they had developed, designated site-specific natural isotope fractionation (SNIF™). A variety of applications for different products, including vanilla, were detailed. Nuclear magnetic resonance (NMR) determines the deuterium (²H) concentration at each of the five isotomers on the vanillin molecule. They also point out that unlike other analytical methods, SNIF-NMR can detect ¹³C enriched vanillin but it does require long spectrometer time. A review by Martin (1998) then described the use of deuterium SNIF-NMR for the authentication of a variety of flavors, including vanilla, coupled with additional application of both deuterium anδ¹³C NMR. This permitted greater classification of the vanillin origins than that allowed by SNIF-NMR alone.

Jamin et al. (2007) reported on a collaborative study of site-specific isotope ratios of deuterium/hydrogen (D/H) in vanillin analyzed by deuterium-NMR (2H-NMR) spectrometry. Nine laboratories participated and the results supported the recommendation that the method be adopted as a First Action Official Method by AOAC International.

10.4.5 Isotopic techniques summary

Reported here are just a few examples of these isotopic methods, which can be used to authenticate the bean-derived origin of vanillin. There are many other reports of the applications of these techniques. Those reported here demonstrate the extent to which researchers are going in order to protect this valued product. Also indicated are the continued extension and development of these isotopic methods.

10.4.6 Integrated and miscellaneous methodologies

German researchers (Kaunzinger et al. 1997) used gas chromatographic profiling with quantitation for vanillic acid, 4-hydroxybenzaldehyde, anisic alcohol, anisic acid, and 4-hydroxybenzoic acid with subsequent δ¹³C isotopic analysis using IRMS, to map both the quantity and isotopic ratios for these components to cross-reference the authenticity of several vanilla extracts. They proposed that a database of these joint measures would give a more accurate assessment of authentic vanilla and acknowledged the intensive work needed to complete this goal.

Another approach combining analytical technologies to ensure authentic natural vanilla was described in the work of John and Jamin (2004). Because the HPLC ratios appeared to be changing, these researchers compared the HPLC ratios, stable isotope ratio analyses (SIRA), and the SNIF-NMR data on Indian vanilla. They report that for these products, many of the HPLC ratios were inconsistent with the French regulation, especially the vanillin to p-hydroxybenzaldehyde ratio, which was consistently high. The SIRA values were also slightly different than stipulated by regulation and they suggested a change to a minimum limit of —21.5%c for vanillin and — 19.2%c for p-hydroxybenzaldehyde. With respect to SNIF-NMR, differences in D/H values were noted, and the discrimination power of this technique to detect adulterated products was demonstrated.

Calabretti et al. (2005) reported on investigating an explicit experimental design methodology in order to determine the best parameters to be used for solid phase micro-extraction (SPME) analysis of vanilla. These researchers used a patented “cogrounds”, solid state activation (SSA) mixed with vanilla extract. They focused the analyses on volatiles “characterizing vanilla”, myrcene, methoxybenzaldehyde, ethylbenzoate, ethylvanillin, vanillin, coumarin, p-hydroxybenzaldehyde, and vanillin. Confusingly, there was no explanation as to why ethylvanillin and coumarin, which do not occur naturally, were targeted. However, this very detailed and statistically based experiment identified the 2 cm length DVB/CARB/PDMS fiber as the optimum to be used for further investigation.