Handbook of Vanilla Science and Technology

A coupled technique, gas chromatography-isotope ratio mass spectrometry (GC-IRMS) was described by Fayet et al. (1996). The technique allows direct analyses of constituents, such as vanillin, in complex mixtures by the application of gas chromatographic separation and direct injection into the isotope ratio mass spectrometer. In addition, a German group (Kaunzinger et al. 1997) used coupled GC-C-IRMS as well as the gas chromatographic quantification of vanilla constituents to develop an integrated authenticity evaluation. The developed profile could be used for vanilla quality assurance. As they stated, “Nevertheless, our investigations clearly define the 4-hydroxybenzaldehyde/vanillin concentration ratios in connection with their δ¹³C values as characteristic parameters of genuine vanilla.”

In the mid-1990s, further efforts at the CAIS involved the investigation of tritium (³H) analysis (Neary et al. 1997). While it was demonstrated that vanilla bean vanillin and petroleum-derived vanillin could be differentiated with a gas proportional counter (GPC), the technique combusts vanillin and then electrolytically concentrates the tritium, but required inordinate amounts of material, 5-10 grams or more of vanillin.

Culp et al. (1998) at CAIS further investigated the application of isotopic analysis for the detection of vanilla adulteration. They applied accelerator mass spectrometry (AMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in order to authenticate flavors, with vanilla as one example. The vanilla extract was derivatized with bis (trimethylsilyl) triflouroacetamide (BSTFA). They were able to obtain the δ¹³C values of the major as well as ancillary compounds in the extract, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillin, vanillic acid, o-vanillin, and syringaldehyde. They proposed the use of these δ¹³C values in order to generate a fingerprint pattern that could be used to authenticate these flavors. Also, because this GC/IRMS analysis typically measures in nanograms to picograms, flavors in finished products could be analyzed for the detection of adulterated products. This demonstrated the application of AMS for ¹⁴C analysis and they hoped to reduce the amount of vanillin required so as to interface the AMS directly to the gas chromatography.

The French regulatory group, at Laboratoire DGCCRF (Fayet et al. 1999), used GC/IRMS to investigate the isotopic deviation of vanillin, vanillic acid, p-hydroxybenzaldehyde, and p-hydroxybenzoic acid. Twenty-two samples of vanilla beans from Madagascar, Indonesia, and Reunion were analyzed. The analytical data for the first three constituents were consistent with that previously reported. The stable isotope ratio analysis (SIRA) for p-hydroxybenzoic acid was first reported here and was suggested as a criterion for authenticity of vanilla.

Expanding on the work of Fayet et al. (1999), German researchers (Scharrer and Mosandl 2002) optimized the use of gas chromatography combustion-isotope ratio mass spectrometry (GC-C-IRMS) on the difficult-to-analyze phenolics, vanillin, and p-hydroxybenzaldehyde. In addition, analyte extraction efficiency was also investigated. They determined the optimum combustion temperature by moving the tip of the capillary column inside the heater and demonstrated that complete component extraction was not necessary in order to obtain a consistent δ¹³C/¹²C SIRA. They analyzed 15 samples of V. planifolia and 4 of V. tahitensis. The δ¹³C_v__pdb was determined on vanillin and p-hydroxybenzaldehyde and demonstrated its reliability in differentiating vanillin, ex-vanilla, from vanillin of synthetic or biotechnological origin.

Over the years, adulteration techniques that could circumvent isotopic detection have been developed, one being syntheses of isotopically altered ingredients. Researchers at Eurofins, a company specializing in isotopic analyses, demonstrated a method to detect this form of adulteration (Bensaid et al. 2002). They described a technique in which vanillin is deformylated and the product, guaiacol, analyzed by IRMS. This ¹³C information can be used in order to improve the authentication potential of C-IRMS. In addition, the ¹⁸O of the guaiacol is a better authentication tool then ¹⁸O of vanillin, which has exchangeable oxygen in the formyl group. Further collaborative studies were suggested in order to confirm that consistent results could be obtained between laboratories.