Proof of sugar syrup in honey using mass spectrometry

Development of a routine-capable method using non-targeted analytics

Honey is valued worldwide as a high-quality sweetener. In the European Union (EU) and in Germany, strict regulations apply to the product (EU Honey Directives 2001 and 2024, and Honey Guidelines 2011). Depending on the quality, honey can be sold at a high price, which increases the motivation to falsify it, for example, by adding sugar syrup. A study published by the EU in 2023 showed irregularities concerning compliance with legal requirements in 46 % of the tested honey samples. It was also found that there is a lack of harmonized methods for official control authorities. To close this gap, NRZ-Authent develops methods for authenticity testing of honey. Mass spectrometric methods are used, which are particularly suitable for detecting sugar syrup in honey.

Step-by-step development of a routine-capable detection method

The proof of sugar syrup in honey should be carried out using non-targeted analytical methods. For this, no predefined analytes are examined, but a wide range of small molecules are investigated. After sample preparation, the substances are separated by high-performance liquid chromatography and detected using high-resolution mass spectrometry. Due to the high complexity, the method is being developed step by step.

Simple sample preparation

Based on a literature review, scientists at NRZ-Authent developed a first test system for early harvest honey, summer honey and linden honey, as well as 50:50 mixtures of honey and rice syrup. A simple sample preparation was developed, in which the honey only needs to be dissolved, filtered and diluted. In the test, two liquid chromatographic separation methods were compared with each other: hydrophilic interaction and reversed phase. It was found that the hydrophilic interaction can distinguish a higher number of analytes, while the reversed phase can be more easily transferred to other devices. Both methods are able to distinguish pure honey from mixtures with rice syrup. The reversed phase method should be optimized as the final procedure.

Selection of marker substances and build-up of a database

Regarding marker substances, a general distinction is made between direct markers (import by adding sugar syrup) and indirect markers (for example, a decrease in endogenous honey substances through stretching with sugar syrup). Direct markers provide a clear evidence of the respective addition, indirect markers can possibly be applied to all additions of sugar syrup. According to the current state, oligosaccharides with a polymerization degree of four to seven and smaller analytes such as, for example, proline or choline are suitable for detecting rice syrup. Further potential markers could arise in the course of method development. In parallel with the determination of marker substances, a database should be set up that allows the analysis of honey without the need for a direct comparison with reference material for each measurement.

Official method for the determination of sugar syrup in honey

As soon as the method for the first test system (three honeys, rice syrup) is optimized, the number of samples to be tested will be gradually increased to ten honeys and sugar syrups each. After evaluating these experiments, it will be decided which additional honeys (different varieties, different countries of origin) are necessary for the generalization of the method. The goal is to standardize the method as an official method (§64 LFGB), which can be made available for food control authorities throughout Europe via DIN (German Institute for Standardization) and CEN (European Committee for Standardization). Together with the specific data sets, a comprehensive method for the detection of sugar syrup in honey will then be possible.

Q&A about the work of NRZ-Authent in the field of authenticity control of honey