Handbook of Vanilla Science and Technology

Chinese researchers investigated supercritical CO₂ (SCO₂) flavor component isolation from vanilla beans (Fu et al. 2002a,b). In the first study they used SCO₂ at two different pressures, 35 and 30 MPa. In the second study they compared the SCO₂ isolates to those from a 95% ethanol extract. WithNIST107LTB andNIST21LIB databases, 28 and 32 constituents were identified in the first study, and 28 and 31 in the second, respectively.

Da Costa and Pantini (2006) investigated Tahitian vanilla with International Flavor & Fragrances’ Generessence® program. Imported beans were analyzed by GC/MS and dynamic headspace directly, and after methylene chloride extraction, and with sorptive stir-bar extraction (Twister™). These analyses identified 276 compounds. Sixteen were targeted by this program as being of interest, including several not previously reported in vanilla. Table 10.1 contains the newer components discovered and Figure 10.1 the structures of the novel anisyl esters. Joulian et al. (2007) used a variety of techniques, including stir-bar solvent extraction (SBSE), GC-MS, and LC/MS/MS, in order to confirm that thep-methoxy aromatic compounds, including anisaldehyde, are the major aroma contributors to characteristic Tahitian vanilla. Most interestingly, after careful analysis, heliotropin (piperonal) was shown not to occur naturally in this product. This confirmed what Ehlers et al. (1994) had reported.

Table 10.1 Components of interest in Tahitian vanilla. (Reproduced from Da Costa and Pantini 2006, with permission from Elsevier.)

Component | Concentration ppt[3] | Component | Concentration ppt

Anisyl alcohol | 225.8 | Caffeine | 0.1

Anisic acid | 87.4 | Theobromine | 0.1

Anisaldehyde | 25.0 | a-Ionone | 0.4

Dianisyl ether | 3.1 | b-Ionone | 0.4

Anisyl ethyl ether | 15.0 | Dihydroactinidiolide | 0.2

Anisyl methyl ether | 0.8 | Vitispirane | 0.3

Anisyl anisate | 6.6 | Anisyl 4-hydroxybenzoate | 7.4

Anisyl trans-cinnamate | 0.5 | Anisyl cis-cinnamate | 0.2

Fig. 10.1 Novel compounds identified in Tahitian Vanilla. (Reproduced from Da Costa and Pantini, 2006, with permission from Elsevier.)

An Indian research group (Sharma et al. 2007) developed a reverse-phase HPLC (RP-HPLC) method, which separated 10 phenolic compounds efficiently and could be used for analytical research as well as for quality assurance. The compounds were 4-hydroxybenzyl alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and piperonal.

FDA researchers (de Jager et al. 2007) developed a liquid chromatography mass spectrometer (LC-MS) method to determine coumarin, vanillin, and ethyl vanillin in vanilla extract products. Both UV and MS signals were used to quantify the data. Electrospray ionization (ESI) for MS detection was utilized with selected ion monitoring (SIM). Limits of detection (LOD), accuracy, and precision were determined for each compound. Twenty-four commercial vanilla extracts were analyzed, 9 were domestic and 15 were imported. It was determined that the adulteration of Mexican vanilla was not widespread, as was reported in 1988. Further investigation of these vanilla products with HS-SPME and GC-MS methods at CFSAN (FDA) was also undertaken. A survey of these 24 vanilla products revealed that not only could the method be used for analysis of coumarin, vanillin, and ethyl vanillin, but also could screen for 18 other vanilla flavor constituents. This data, when compared with LC-ESI-MS, demonstrated that these two conceptually different methods could be confirmatory of each other.

Sharma et al. (2007) developed an RP-HPTLC method for the quantitative determination of vanillin and related phenols. The method also involves an accelerated solvent extraction (ASE). Silica gel coated aluminum plates were used with a methanol/water/isopropanol/ acetic acid mobile phase. This was a simple, fast, sensitive method useful for analytical research and for on-line analyses of vanilla extracts for QC. A Chinese group (Zhou et al. 2007) investigated the use of different solvents during the ultrasonic extraction of vanilla beans. They identified acetone as the best solvent. The extract was separated with SPE and the analysis by GC-MS showed a detection limit of vanillin of 0.04 mg/L and that of p-hydroxybenzaldehyde of 0.06 mg/L.