The well-known and widely accepted ¹³C EA/LC-IRMS method uses carbon stable isotope ratios (¹³C/¹²C) of honey protein and individual sugar fractions to detect foreign sugars from C4 plant (e.g. sugar cane, corn) and C3 plant (e.g. rice, beet, wheat) origin. It has been in use for approx. 15 years for the routine control of honey samples.

The ¹H NMR profiling allows a fast screening for important quality parameters of honey (e.g. fructose, glucose, sucrose, HMF, proline), statistical verification models for the verification of a declared botanical and/or geographical origin, and the detection of foreign sugars. NMR cannot identify unknown origins and varieties or blends. The number of verification models is limited to the most frequent origins and varieties.

NMR is less sensitive in the detection of C4 sugars compared to ¹³C EA/LC-IRMS, but improves C3 sugar detection capabilities where ¹³C EA/LC-IRMS has some limitations.

NMR profiling relies on extensive databases of authentic honeys in order to assess authenticity by comparison of spectral profiles. The databases need regular updates and review of judgement criteria to reflect all honey sources and possible natural alterations in honey composition due to changes of environmental, climate or beekeeping conditions over time.

Findings of adulterations should always be confirmed by complementary methods, e.g. LC-HRMS. NMR can also detect fraudulent processing applied to honey by untypical NMR profiles. For almost 10 years, ¹H NMR profiling has been an important part of honey authenticity assessments complementing ¹³C EA/LC-IRMS analysis.

Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is the newest authenticity method applicable to honey, first developed and introduced by the Eurofins network in 2018 as an ISO 17025 accredited method. It was particularly developed to detect those adulterations with foreign sugars, which remain undetected with 13C EA/LC-IRMS and 1H NMR (tailored syrups).

LC-HRMS allows screening for multiple generic and syrup-specific marker molecules of sugar syrups used as adulterants. The method can be used both in a targeted and non-targeted mode, so that also yet unknown adulterants can be identified and characterised, and samples previously tested can be evaluated retrospectively for new adulterants.

The particular power of the method is based on the use of typical multi-marker profiles for each syrup registered in our syrup database, instead of single marker molecules employed by conventional methods, allowing an unequivocal and sensitive detection of hard to detect adulterants.

Besides the SOTA bundle, pollen microscopy and sensory is important to identify the origin(s) and/or variety of monofloral honeys and honey blends. The honey sediment can give valuable information regarding authenticity (e.g. foreign particles typical for adulteration, or improper processing practices). The sensory evaluation is equally useful in terms of authenticity (e.g. by match or mismatch between the microscopic picture and sensory properties).

In addition to the described analytical methods 13C EA/LC-IRMS, 1H-NMR profiling, LC-HRMS, pollen analysis and sensory evaluation, our specialist laboratory for honey analysis, Eurofins Food Integrity Control Services GmbH, offers a new method for the detection of foreign enzymes. This method can detect enzymes that are difficult or even impossible to identify with standard enzyme tests. Artificial enzymes can be added to honey to increase enzyme activity to meet the international criteria (amylase/diastase activity) required for honey marketing. 

This bottom-up proteomics method is based on the separation of proteins in honey from the main components (mono-, di-, and oligosaccharides) followed by the digestion of these proteins to small protein fragments (peptides) with a chain length between 6 and 30 amino acids. These peptides are detected by LC-MS/MS.

This new method is so specific and sensitive that even very small amounts of foreign enzymes can be detected. In addition to those enzymes, proteins of the honeybee such as the abundant Major Royal Jelly Protein MRJP1 as well as α-glucosidase Apis mellifera and α-amylase Apis mellifera are measured. This test is performed to identify irregularities of these proteins caused by the addition of sugar syrup or rigorous processing of the honey.