Imidazole analytics

Imidazoles are heterocyclic aromatic compounds with two neighbouring nitrogen oxide atoms in the ring structure. The most important imidazole compounds in foodstuffs are 5-HMF (5-hydro-xymethylfurfural), THI (2-acetyl-4-tetrahydroxybutylimidazole) and 4-MEI (4-methylimidazole). Histamines, which trigger an intolerance reaction in around 1% of the population, are also imidazoles.

Occurrence and formation

Imidazoles are a side-product that forms in the process of caramel colour production and during non-enzymatic browning processes (Maillard reaction). Caramel colour is produced by heating glucose syrup with sulphuric acid in the presence of ammonia (ammonia caramel E 150c), for example. Caramel colour is a food colouring additive approved for many foods (E150 and E150ad). It is added to beverages (e.g. caffeine-containing soft drinks or malt beer), confectionery and bakery products.

5-HMF is frequently used as an indicator of a product having been heated. The most well-known example is honey. Its HMF content not only increases with heating but also with the duration of storage, so low levels are an indicator of freshness and naturalness.

Legal basis

The directive specifies a general maximum amount of  40 mg/kg of 5-HMF in honey at the European level. This directive has been translated into national law in the form of the German Honey Regulation. Other regulations can be found in the guidelines for honey, including special requirements for superior-quality honey (e.g. “Auslese”). Furthermore, the German Beekeeper’s Association (Deutscher Imkerbund) has defined a maximum level of 15 mg of 5-HMF/kg for honey that carries the quality seal "Echter Deutscher Honig“ (engl.: real German honey).

For 4-MEI, Regulation (EU) No. 231/2012 specifies maximum limits of 200 mg/kg in ammonia caramel (E 150c) and 250 mg/kg in sulphite ammonia caramel (E 150d), respectively. Additionally, a THI maximum limit of 10 mg/kg is set for ammonia caramel (E 150c). These maxmimum limits for 4-MEI and THI are expressed on equivalent colour basis, i.e. are epxressed in terms of a product having a colour intensity of 0.1 absorbance units.


ifp Institut für Produktqualität offers analytical services for imidazoles at their state-of-the art premises in Berlin-Adlershof. Analysis is performed after sample extraction using high-performance liquid chromatography coupled with two mass spectrometers (LC-MS/MS).