Handbook of Vanilla Science and Technology

vanillin to the extract. These include high pressure liquid chromatography (HPLC) and methods based on the isotopic composition of the vanillin found in the vanilla product.

10.4.1 Liquid chromatographic methods

Ever since the proposal that the vanilla HPLC constituent ratios could be used to verify the origin of vanilla in the early 1980s, a lot of effort has been expended on the application of this concept. Initially, the vanillin/p-hydroxybenzaldehyde ratio was proposed but later the use of the ratios between all of the four major vanilla phenolics, including vanillin/vanillic acid and vanillin/p-hydroxybenzoic acid as well as other constituent ratios were incorporated. Researchers from around the world investigated a variety of HPLC eluents and columns as well as a variety of HPLC conditions (Figure 10.2). Most used reverse phase columns and methanol, acetonitrile and water, in a variety of combinations, as the eluant and a variety of acids as modifiers. However, it appeared that if the analysis was effective in the separation and quantification of these constituents, the various ratios were reasonably consistent, no matter the geographic origin of the vanilla. It was proposed in the 1980s and 1990s that these ratios should be used as a means to authenticate vanilla and eventually these ratios began to be incorporated into the national regulations of various countries, primarily in Europe. The ratios were eventually promulgated into EU regulations for vanilla. While regulations make these absolutes, it soon became clear that there were natural vanilla exceptions that fell outside the established ranges. Because this is a natural product, natural constituent variability is inevitable, so the use of rigid ratio limits as a regulatory standard may be asking too much of Nature. However, the ratio of vanillin/p-hydroxybenzaldehyde has shown the most consistency with fewer natural outliers. The usefulness of this ratio is primarily due to the greater concentrations of these two vanilla constituents, which results in lower statistical error. A better use of these HPLC ratios, especially the vanillin/p-hydroxyben-zaldehyde ratio, would be their use as an initial check. When the vanilla product falls beyond the established ratio limit, it should be analyzed by another more definitive and more expensive method, such as SIRA, NMR, etc.

Fig.10.2 Typical HPLC profile of a vanilla extract from the author’s laboratory (courtesy of Alan Harmon).

HPLC methods are based primarily on separating four key vanilloid type constituents. These constituents are often summed and compared to the common UV result for total “vanillin”, expressed as g/dL and/or to establish ratios in order to authenticate beans or extracts. The four major non-volatile constituents separated and quantified are vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and vanillic acid (Figure 10.3). Other constituents and non-vanilla adulterants are also often separated and identified, for example, coumarin, ethyl vanillin, piperonal, etc. These constituents and others may provide further information as to the quality and source of the vanilla and can be helpful to the analyst for assessing the overall age, purity, quality, or sensory properties of vanilla products (Kahan et al. 1997; Taylor 1993; Hoffman et al. 2005). The ratios used for authentication are fairly consistent. The most significant ratio is the vanillin to p-hydroxybenzaldehyde ratio, which falls within the 10 to 20 range for authentic vanilla products. These four constituents in vanilla products are often measured to resolve many requirements. In pursuit of this information, many HPLC method variations have been developed but most involve aqueous methanol as the eluant, several include acetonitrile and most include an acid modifier and use a variety of reverse phase columns. Many investigators have contributed to the number of HPLC methods (Sinha et al. 2008).

Fig. 10.3 Structures of the four vanilloid compounds used for calculation of component ratios, typically from HPLC analysis.

Among the many published methods, some might be adopted for use by quality control chemists or researchers, if applicable to their needs for either routine or investigative studies. Clear methods and simple procedures are preferred in the quality lab; commercial labs may use a number of methods to fulfill their purpose for customer studies and the researcher may apply methods which maximize the chemical information or provide accurate analysis of a key analyte.