Trace analysis of vanillin and ethyl vanillin in food using LC-ESI-MS/MS

Detection with a LOQ of 15 µg/kg in infant formula, shortbread and other matrices

Apr 2021. Infant formula and follow-on formula for infants is subject to particularly high-quality requirements all over the world. Moreover, consumers increasingly desire foods that have been kept as natural as possible. The addition of certain additives should be avoided if possible. Such substances can include (artificial) aromas such as vanillin and structurally similar compounds. In China, these substances are considered contaminants when used in infant food. For this reason, a sensitive analytical method based on liquid chromatographic separation in combination with mass spectrometry was developed for its determination in infant formula and other foods.

Occurrence, production and use of vanillin and related compounds

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important natural or nature-identical flavourings worldwide. Natural sources of vanillin are mainly the capsule fruits of the spice vanilla. In addition, vanillin can be obtained biotechnologically or from the wood component lignin. Out of more than one hundred identified vanilla flavour compounds, vanillin is the most important. In addition to vanillin, there is a synthetic, non-naturally occurring variant, ethyl vanillin. Ethyl vanillin is used today as an artificial flavour in e.g. ice cream and baked goods.

Other compounds, which have not yet been implemented in the method described, are isomers of vanillin such as isovanillin and ortho-vanillin as well as methyl vanillin. The first two compounds mentioned are naturally occurring isomers of vanillin, while methyl vanillin is also produced synthetically and does not occur in nature. Isovanillin is an almost odourless compound that is formed in cassava roots, for example. Ortho-vanillin, on the other hand, is an important flavour substance in whiskey and red wine production. Here, the ortho-vanillin from the oak wood in which it occurs naturally is transferred to the alcoholic beverages mentioned during barrel storage.

Legislation in Europe and China

At national level (German food law), different terms are used in the list of ingredients depending on the source of the vanillin. According to Regulation (EC) No. 1334/2008 (Flavour Regulation), the designation natural vanilla flavour may only be used for extracts made from vanilla pods. If biotechnological processes were used to produce the aroma, its designation may only be declared as a natural aroma. If, on the other hand, synthetic compounds (methyl vanillin, ethyl vanillin) are used, the designation may not be used in the declaration. Accordingly, such compounds are referred to as vanilla flavouring or aroma.

In Germany, vanilla extract and biotechnologically produced (nature-identical) vanillin are not considered as additives but food ingredients and may therefore be used in food without quantity restrictions.

However, according to Chinese Regulation GB2760-2015 (Chinese Standards for Food Additives), the addition of flavours, including vanillin and ethyl vanillin, is completely prohibited in infant formula food for infants aged 0–6 months.

Analysis of vanillin and ethyl vanillin at trace level

Our experts from the Competence Center for Organic Contaminants have many years of experience with the LC-MS/MS analysis of organic compounds at trace levels. For the determination of vanillin and ethyl vanillin in food with a limit of quantification (LOQ) of 15 µg/kg, the latest chromatographic separation techniques ((U)HPLC) are available in combination with the latest and currently most sensitive mass spectrometers. The use of isotope-labelled internal standards for vanillin and ethyl vanillin represent the status quo in terms of precision and accuracy of the analytical results obtained. The daily measurement of calibration standards and food reference materials for the critical control of the analysis are additional quality standards that increase the reliability of the analysis.

Analysis of vanilla and flavours

In addition to the trace analysis of vanillin and ethyl vanillin, Eurofins also offers the analysis of higher contents of the aroma compounds as well as various analyses for the control and proof of the authenticity of the expensive raw materials. Further analyses for the confirmation of the quality and safety of vanilla and other aromas complement Eurofins’ comprehensive analytical portfolio.

Any questions about the analysis of vanillin and ethyl vanillin?

Please contact our expert for organic contaminants, Sindy Boehme, our expert for the analysis of flavours and aromas, Dr Thomas Gronewold or kindly refer to your personal account manager.