Vanilla – Natural or Out of the Reaction Flask

Anita Piper , Stephan Altmaier , Matthias Nold

HPLC fngerprint method and reference materials help to distinguish natural from synthetic or adulterated vanilla

Vanilla Ice Cream

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 (Figure 1).

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 vanilin or coumarin are also often added to enhance the flavor (Figure 2).

4-Hydroxybenzoic acid

4-Hydroxybenzoic acid (Cat.No. 92596)


4-Hydroxybenzaldehyde (Cat.No. 91554)

Vanillic acid

Vanillic acid (Cat.No. 68654)


Vanillin (Cat.No. 30304)

Figure 1. Compounds found in natural vanilla.


Guaiacol (Cat.No. PHR1136)

Ethyl vanillin

Ethyl vanillin (Cat.No. 75042)


Coumarin (Cat.No. 72609)


Eugenol (Cat.No. 79891)

Figure 2. Markers for artificial vanilla.

We recently launched a set of two reference materials for natural and synthetic vanilla extracts for the testing of vanilla authenticity by chromatographical fingerprint. These two reference extracts are also available individually (Table 1).

Table 1. Vanilla Extract Reference Materials (Natural and Synthetic)

The products are developed and manufactured by HWI pharma services GmbH in Rülzheim, Germany, and are qualified as secondary standards, traceable to HWI primary reference standards quantified by qNMR. These products add to a range of 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 we present an HPLC method to detect natural and synthetic vanilla markers using a Chromolith® Performance RP-18 endcapped 100x2 mm column (Table 2). Results for both the synthetic and the natural vanilla extract are shown. In addition, samples of food and beverage products containing vanilla flavor, such as ice cream, rooibos tea and Bourbon vanilla, were tested.

For all the standards, extensive studies were made to determine LOD, LOQ, linearity, repeatability and standard deviation (see below under Method Validation Data).

Table 2.Experimental conditions & sample preparation

Blank run:

Blank Run

Results & discussion

The prepared standard solution was used for method development and validation. The chromatogram and retention data is shown in Figure 3. Vanillin is the main component of the natural vanilla extract reference material (cat. no. 06261501), in addition traces of 4‑hydroxybenzoic acid, vanillic acid and 4‑hydroxybenzaldehyde could be detected. No ethyl vanillin, guaiacol, coumarin or eugenol were present (Figure 4a). For verification purposes also a spiked extract was injected (Figure 4b). For comparison, the chromatogram of the commercial Bourbon vanilla sample (Figure 5) is very similar to the natural vanilla extract reference material.

In contrast to natural vanilla, the synthetic vanilla extract reference material ( 06271501) shows, besides vanillin as the major peak, ethyl vanillin and coumarin as well as traces of eugenol (Figure 6a). For verification purposes also a spiked extract was injected (Figure 6b). In the ice cream sample, guaiacol instead of vanillin is the major peak (Figure 7). In addition, traces of ethyl vanillin, coumarin and eugenol were detected, indicating the synthetic nature of the used material. the Rooibos tea sample the coumarin peak is pronounced (Figure 8)

Chromatographic data

Standard solution

Figure 3. Standard solution

Matrix standard solution natural

Figure 4a. Matrix standard solution natural (vanilla extract, natural, cat. no. 06261501).

Matrix standard solution natural

Figure 4b. Matrix standard solution natural (vanilla extract, natural, cat. no. 06261501) spiked with 100 µL standard solution.

Commercial bourbon vanilla sample

Figure 5. Commercial bourbon vanilla sample.

Matrix standard solution synthetic

Figure 6a. Matrix standard solution synthetic (vanilla extract, synthetic, cat. no. 06271501).

Matrix standard solution synthetic

Figure 6b. Matrix standard solution synthetic (vanilla extract, synthetic, cat. no. 06271501) spiked with 100 µL standard solution.

Ice cream sample

Figure 7. Ice cream sample

Rooibos tea sample

Figure 8. Rooibos tea sample

Method validation data

1. Specificity: inject standard solution and determine the retention time and content of desired analyte @280 nm

2. Standard repeatability sample solution of Rooibos tea in mAU*min


4. Linearity (area mAU*min)



The examples shown demonstrate the applicability and value of matrix reference materials to help detect food adulterations and mislabeling. The developed and validated method in conjunction with the presented reference materials allow an efficient and reliable analysis of vanilla flavored samples.



Cicchetti E, Chaintreau A. 2009. Quantitation of the main constituents of vanilla by reverse phase HPLC and ultra-high-pressure-liquid-chromatography with UV detection: Method validation and performance comparison. J. Sep. Sci.. 32(17):3043-3052.