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HPLC fingerprint method for vanilla analysis Natural or Synthetic Vanilla?

From Anita Piper, Stephan Altmaier, Matthias Nold*

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In addition to natural vanilla, artificial vanilla flavors are also used in the food industry. A HPLC method and reference materials help to distinguish them. Because: product counterfeiting is not exactly rare when it comes to the supposed natural (and expensive) vanilla flavor.

1 Vanilla aroma often makes 
customers believe it is of natural 
1 Vanilla aroma often makes 
customers believe it is of natural 
(Source: ©oxie99 -

Vanilla is one of the most popular flavors in food and beverage products. The demand far exceeds the global supply of naturally grown vanilla; therefore, in addition to natural vanilla, artificial vanilla flavors are used in the food industry. Natural vanilla is commonly substituted for synthetically produced vanillin or by other compounds with a similar flavor such as ethyl vanillin. And because of the large price difference between natural and synthetic vanilla, this is a very attractive target for food criminals and frauds.

Analysis of the chromatographic fingerprint of a vanilla flavor represents an efficient method to detect these types of adulteration and mislabeling [1]. Characteristic markers for natural vanilla are vanillic acid, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde and vanillin. For artificially produced vanilla, cheap chemicals such as guaiacol or eugenol are typically used as starting materials. The presence of traces of these compounds are indicators of synthetically produced vanilla. Ethyl vanillin or coumarin are also often added to enhance the flavor.

Gallery with 7 images

Merck recently launched a vanilla extract set of two reference materials. They can be utilized to analyze vanilla authenticity by determining the chromatographic fingerprint of natural and synthetic vanilla samples. These two reference extracts are also available individually:

  • Vanilla extract, natural: Vanillin, Vanillic acid, Absence of Ethyl vanillin (qualitative); Vanillin, Vanillic acid (quantitative)
  • Vanilla extract, synthetic: Vanillin, Vanillic acid, Ethyl vanillin (qualitative); Vanillin, Vanillic acid (quantitative)

The products are developed and manufactured by HWI pharma services in Rülzheim, Germany, and are qualified as secondary standards, traceable to HWI primary reference standards quantified by
qNMR. These products add to the range of Mercks plant extract reference materials designed for rapid identification and quantification of typical constituents of plants used as food additives or as herbal medicinal products.

HPLC Fingerprint Method

In the following section an HPLC method for the detection of natural and synthetic vanilla markers using a Chromolith Performance RP-18 endcapped 100 x 2 mm column (s. Table 1) is presented. Results for both the synthetic and the natural vanilla reference material extract are shown. In addition, samples of food and beverage products containing vanilla flavor, such as bourbon vanilla, ice cream and Rooi­bos tea, were also tested (see Table 1 for sample preparation details).

For all the standards, extensive studies were made to determine LOD, LOQ, linearity, repeatability and standard deviation. The complete dataset incl. the data for Rooibos tea and validation data for the method can be viewed online in the full version of this article at, Issue 7.

Results & Discussion

The prepared standard solution was used for method development and validation. The chromatogram and retention data is shown in Figure 2. Vanillin is the main component of the natural vanilla extract reference material, in addition traces of 4-hydroxybenzoic acid, vanillic acid and 4-hydroxybenzaldehyde could be detected. No ethyl vanillin, guaiacol, coumarin or eugenol were present (s. Figure 3a). For comparison, the chromatogram of a commercial Bourbon vanilla sample (s. Figure 3b) is very similar to the natural vanilla extract reference material.

In contrast to natural vanilla, the synthetic vanilla extract reference material shows, besides vanillin as the major peak, ethyl vanillin, coumarin and traces of eugenol (s. Figure 4a). In the ice cream sample, guaiacol is the major peak (s. Figure 4b). In addition, traces of ethyl vanillin, coumarin and eugenol were detected, indicating the synthetic nature of the material.


The examples shown demonstrate the applicability and value of matrix reference materials to help detect food fraud and food mislabeling.


[1] E. Cicchetti, A. Chaintreau, J. Sep. Sci. 32 (2009) 3043–3052.

* A. Piper, S. Altmaier, M. Nold: Merck KGaA, 64293 Darmstadt/Germany


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