EU Coordinated action to deter certain 
fraudulent practices in the honey sector  
Analytical testing results of imported honey  
 
Ždiniaková, T., Lörchner, C., De Rudder, O., 
Dimitrova, T., Kaklamanos, G., Breidbach, A., 
Respaldiza, A., Vaz Silva, I.M., Paiano, V.,  
Ulberth, F., Maquet, A. 
2023 
EUR 31461 EN
ISSN 1831-9424

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Contact information  
Name: Lourdes ALVARELLOS 
Address: Retieseweg 111, 2440 Geel, Belgium 
Email: lourdes.alvarellos@ec.europa.eu 
Tel.: +32(0)14-571316 
 
EU Science Hub 
https://joint-research-centre.ec.europa.eu 
 
 
JRC130227 
 
EUR 31461 EN 
 
 
PDF 
ISBN 978-92-68-01292-5 
ISSN 1831-9424 
doi:10.2760/184511 
KJ-NA-31-461-EN-N 
 
 
 
 
 
      
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How to cite this report: Ždiniaková, T., Lörchner, C., De Rudder, O. Dimitrova, T., Kaklamanos, G., Breidbach, A., Respaldiza, A., Vaz Silva, I.M., 
Paiano, V., Ulberth, F. Maquet, A., EU Coordinated action to deter certain fraudulent practices in the honey sector - Results of analytical 
testing of imported honey, Publications Office of the European Union, Luxembourg, 2023, doi:10.2760/184511, JRC130227.

i 
Contents 
Acknowledgements .......................................................................................................................................................................................................................................... 1 
Abstract ....................................................................................................................................................................................................................................................................... 2 
1 Context ................................................................................................................................................................................................................................................................ 3 
2 Scope of the EU coordinated action ........................................................................................................................................................................................ 3 
3 Participation ................................................................................................................................................................................................................................................... 4 
4 Origin of honey consignments and trade flow ............................................................................................................................................................. 5 
5 Laboratory testing .................................................................................................................................................................................................................................... 6 
5.1 Test methods ................................................................................................................................................................................................................................... 6 
5.2 Compliance assessment ....................................................................................................................................................................................................... 6 
6 Analytical testing results ................................................................................................................................................................................................................... 6 
7 Conclusions ..................................................................................................................................................................................................................................................... 9 
List of abbreviations and definitions ........................................................................................................................................................................................... 11 
List of figures ..................................................................................................................................................................................................................................................... 12 
List of tables ........................................................................................................................................................................................................................................................ 13 
Annex 1. Analytical methods and decision mechanism applied to identify suspicious samples of honey in the 
frame of the Coordinated Control Plan for Honey .......................................................................................................................................................... 14

1 
Acknowledgements 
The cooperation of the competent authorities of the Member States of the European Economic Area is kindly 
acknowledged. 
Authors 
Ždiniaková, Tereza 
Lörchner, Carolin 
De Rudder, Olivier 
Dimitrova, Tatyana 
Kaklamanos, Georgios 
Breidbach, Andreas 
Respaldiza, Arantza 
Vaz Silva, Ines M. 
Paiano, Viviana 
Ulberth, Franz 
Maquet, Alain

2 
Abstract 
In the frame of the EU coordinated action 15 EU Member States (BE, BG, CZ, DE, DK, EL, ES, FR, HU, IE, IT, LT, 
PL, RO, SE) plus CH and NO randomly sampled 320 honey consignments originating from 20 exporting 
countries, which were sent to JRC for analysis to detect the presence of exogenous sugar syrup in honey. 
Several methods (EA/LC-IRMS, HPAEC-PAD, 1H-NMR) were used by the JRC to detect markers indicative for the 
presence of exogenous sugar syrups. 
Of the 320 samples received from the competent authorities of the participating countries, 147 (46 %) were 
suspicious of being non-compliant with the provisions of the EU Honey Directive 2001/110/EC. 
The suspicion rate was considerably high in comparison to an earlier EU-wide coordinated control plan 
conducted in 2015-17, where 14 % of the analysed samples did not comply with established benchmark 
criteria to assess honey authenticity. However, a different set of methods with improved detection capability 
was used in the present study, which may explain this difference. 
Stable carbon isotope ratio analysis by EA-IRMS (AOAC method 991.41), a method that has frequently been 
used in the past to detect sugar syrups made of maize starch or sugarcane, was not effective in detecting 
honey suspicious of being adulterated. This is a clear indication that such sugar syrups are no longer used to 
extend honey and have been replaced by syrups made mostly from rice, wheat or sugar beet. 
The highest absolute number of suspicious consignments originated from China (66 out of 89, 74 %), 
although honey originating from Turkey (14 out of 15, 93 %) had the highest relative proportion of suspicious 
samples. Honey imported from the United Kingdom had an even higher suspicion rate (10 out of 10, 100 %). 
However, the available traceability information suggests that this could be the result of honey produced in 
other countries and further processed in the United Kingdom before its re-export to the EU. 
Although a substantially high number of honey consignments imported into the internal market was tested, 
the obtained results represent the situation during the sampling period (10/2021 to 02/2022) and shall not be 
generalised or extrapolated to other situations.

3 
1 Context 
Honey is a natural product which has been valued for its sweetening properties since ancient times. The 
provisions of the EU Honey Directive1 aim at preserving the purity of honey as an unprocessed raw 
agricultural product, excluding modifications to its chemical composition. In the EU, market demand for honey 
is higher than domestic production and a substantial amount of honey is imported. Unfortunately, not all 
honey placed on the market is genuine. The results of a Coordinated Control Plan organised in 2015-17 at the 
EU level plus Norway and Switzerland, showed that at least 14 % of the checked samples did not conform to 
purity benchmarks2. As a follow-up the JRC organised a Round Table Discussion with stakeholders of the 
honey production chain and representatives of the competent authorities in the Member States as well as 
from European Commission services to identify gaps in knowledge related to authenticity testing of honey 
and possibilities to address them3. Extension of honey with sugar syrups was seen as the most widely 
observed malpractice. 
Producer organisations4 as well as consumer protection associations5 have repeatedly raised concern over the 
presence on the market of honey not complying with regulatory requirements. These concerns are supported 
by the number of notifications regarding adulterated honey in the Administrative Assistance and Cooperation 
– Food Fraud system in 20206.  
The EU average unit value for imported honey was 2.32 €/kg (excluding honey from New Zealand) in 20217, 
whereas sugar syrups made from rice are available at around 0.40 – 0.60 €/kg, depending on the purity of 
the syrup. The price difference between authentic honey and sugar syrups and the difficulty of detecting 
extension of honey with syrups provides attractive fraud opportunities for dishonest business operators. 
Various sources indicated that particularly imported honey does not comply with provisions of the EU Honey 
Directive 2001/110/EC, specifically with the requirement that honey shall not have added to it any food 
ingredient, including food additives, nor shall any other additions be made other than honey (Annex II, 
Composition criteria for honey). 
Therefore, an action at EU level was organised to gather intelligence on the incidence of non-conforming 
honey imported into the Union through sampling and analysis that served to target further investigations 
within the EU. The action was initiated and coordinated by the Directorate-General for Health and Food Safety 
(DG SANTE) of the European Commission and implemented by the members of the EU Food Fraud Network 
who received the technical assistance of the Joint Research Centre and the support of the European Anti-
Fraud Office (OLAF). 
  
2 Scope of the EU coordinated action 
The EU Food Fraud Network agreed to focus in the first phase of the coordinated action on imported honey 
through sampling at border control posts (BCPs) and analysis of the samples by the JRC. In case that a 
consignment presented to BCP control consisted of more than one production lot, only one lot was subjected 
to sampling. Several aliquots from a lot had to be taken to form an aggregate sample which had to be sent to 
JRC for analysis. Each aggregated sample had to be identified with the corresponding Common Health Entry 
Document (CHED)8 reference number of the sampled consignment. This allowed retrieving all necessary 
                                           
1  
Council 
Directive 
2001/110/EC 
of 
20 
December 
2001 
relating 
to 
honey. 
https://eur-lex.europa.eu/legal-
content/EN/TXT/PDF/?uri=CELEX:32001L0110&from=EN  
2  
https://ec.europa.eu/food/safety/agri-food-fraud/eu-coordinated-actions/coordinated-control-plans/honey-2015-17_en 
(accessed 
24/06/2022) 
3  
Technical Round Table on Honey Authentication, JRC-Geel, Belgium, 25 January 2018 Ares(2018)1677606 - 27/03/2018 
4  
Copa-Cogeca Position Paper on the European Honey Market. Action Plan to Rectify the Alarming Situation. Brussels, February 2020 
https://www.copa-cogeca.eu/downloadThread.aspx?threadID=2153212 (accessed 24/06/2022) 
5  
Un miel correct pour moins de 6€ Test Achats - avr. 2021 - N° 662 https://www.test-achats.be/sante/alimentation-et-
nutrition/aliments-et-complements-alimentaires/news/miel-authenticite (accessed 24/06/2022) 
6  
2020 Annual Report – The EU Agri-Food Fraud Network and the Administrative Assistance and Cooperation System. 
https://ec.europa.eu/food/system/files/2021-09/ff_ffn_annual-report_2020_1.pdf  
7  
https://ec.europa.eu/info/sites/default/files/food-farming-fisheries/animals_and_animal_products/documents/market-presentation-
honey_en.pdf   
8  
The Common Health Entry Document (CHED) is a mandatory document that must be presented at border control post in order to 
carry out security checks when these goods enter the EU market: animals, products of animal origin, products of plant origin, feed 
and food products.

4 
control and traceability data, including the place of first destination of the consignment within the EU, from 
the TRACES9 system. 
Detection of extension of honey with sugar syrup(s) and related labelling issues was the primary aim of the 
coordinated action. Other means of adulterating or misdescribing the true nature of honey, e.g. related to 
geographic or botanical origin, were outside the scope of the action. 
The JRC had to analyse the honey samples sent by the participating countries in the coordinated action using 
stable carbon isotope ratio isotope analysis, which would allow detecting adulteration with sugar syrups 
produced from maize or sugarcane at levels more than 1 %, and at levels of more than 10 % for syrups 
produced from rice, wheat or sugar beet. In addition, for the detection of addition of sugar syrups obtained 
from rice or wheat a profiling method targeting the presence of exogenous oligo- and polysaccharides and 
other adulteration markers in honey had to be employed. 
 
3 Participation 
Fifteen EU Member States (BE, BG, CZ, DE, DK, EL, ES, FR, HU, IE, IT, LT, PL, RO, SE) plus CH and NO 
participated in the coordinated action. They sampled in total 320 consignments of honey (Figure 1). Based on 
the information reported by the competent authorities the majority of samples was taken at BCPs (84 %); the 
rest either at the premises of honey packers/distributors or unknown. JRC received the samples between 
28/10/2021 and 11/02/2022 for laboratory analysis.  
Figure 1. Participation in the EU coordinated action to control imported honey. 
 
 
                                           
9  
TRACES is the European Commission's online platform for sanitary and phytosanitary certification required for the importation of 
animals, animal products, food and feed of non-animal origin and plants into the European Union, and the intra-EU trade and EU 
exports of animals and certain animal products.

5 
4 Origin of honey consignments and trade flow 
The majority of sampled honey consignments originated from China (89), Ukraine (74), Argentina (34), Mexico 
(22), Brazil (18) and Turkey (15) (Figure 2) and most of them were declared as ‘polyfloral’ (77 %) or 
‘monofloral’ honey (11 %); the rest was of unknown botanical origin. The consignments were randomly 
sampled and offered a good representation of the trade flows during the sampling campaign. Figure 3 
informs about the complex trade flows of honey from exporting countries to the place of first destination of 
the sampled consignments in the EU. Flow patterns vary to a large degree; whereas honey imported by Poland 
stayed in the country, a larger part of honey imported by Germany was re-exported to other EU Member 
States.  
Figure 2. Geographical origin of sampled honey consignments. 
 
Figure 3. Flow of sampled honey consignments from exporting countries to importing EU Member States and place of 
first destination in EU Member States. Origin, importer and destination information according to the CHED documentation.

6 
5 Laboratory testing   
5.1 Test methods 
The JRC used several methods to detect honey suspicious of containing added sugar syrup(s) (Table 1). 
Method details are given in Annex I. 
 
Table 1. Detection methods for sugar syrup(s) added to honey. 
Method principle 
Benchmark values / Markers of adulteration 
Elemental Analyser/Liquid Chromatography – Isotope 
Ratio Mass Spectrometry (EA/LC-IRMS) 
Benchmark values for differences between 13C/12C 
stable carbon isotope ratios of protein and sugar 
compounds as proposed by Elflein and Raezke10 
High-Performance Anion Exchange Chromatography -  
Pulsed Amperometric Detector (HPAEC-PAD) 
Polysaccharides with a degree of polymerisation (DP) 
equal or larger than ten (DP≥10)11 
Liquid Chromatography -  High Resolution Mass 
Spectrometry (LC-HRMS)* 
Oligosaccharides 
with 
DP≥6 
and 
<1012 
2-Acetylfuran-3-glucopyranoside 
(AFGP)12 
Difructose anhydride (DFA)12 
Proton 
Nuclear 
Magnetic 
Resonance 
(1H-NMR) 
Spectroscopy 
Mannose13 
Honey-Profiling™14 
 * Insufficient sensitivity of the method to detect polysaccharides with DP≥10. 
 
5.2 Compliance assessment 
Honey offered on the internal market needs to comply with Council Directive 2001/110/EC, particularly with 
the composition criteria given in Annex II. If extended with appropriate sugar syrups imitating the sugar 
composition of genuine honey, the adulterated products will in most cases comply with the provisions. 
Therefore, the presence of an adulteration marker in honey or a 13C/12C isotope ratio not respecting the 
benchmark values of Table 1 led to the conclusion that the honey was suspicious of not being in compliance 
with the requirement that honey shall not have added to it any food ingredient, including food additives, nor 
shall any other additions be made other than honey. 
 
6 Analytical testing results 
Of the 320 samples received from the competent authorities of the EU Member States, 147 (46 %) were 
suspicious of being non-compliant with the provisions of the EU Honey Directive 2001/110/EC because at 
least one marker of extraneous sugar sources was detected (Figure 4). However, the used techniques 
provided qualitative information (presence/absence of markers) and, therefore, it was not possible to estimate 
the level of exogenous syrups present in honey.  
                                           
10  
Lutz Elflein, Kurt-Peter Raezke: Improved detection of honey adulteration by measuring differences between 13C/12C stable carbon 
isotope ratios of protein and sugar compounds with a combination of elemental analyzer - isotope ratio mass spectrometry and 
liquid chromatography - isotope ratio massspectrometry (δ13C-EA/LC-IRMS). Apidologie 39 (2008) 574–587 
11  
V. Morales, N. Corzo, M.L. Sanz: HPAEC-PAD oligosaccharide analysis to detect adulterations of honey with sugar syrups. Food 
Chemistry 107 (2008) 922–928  
12  
Bing Du, Liming Wu, Xiaofeng Xue, Lanzhen Chen, Yi Li, Jing Zhao, Wei Cao: Rapid Screening of Multiclass Syrup Adulterants in Honey 
by Ultrahigh-Performance Liquid Chromatography/Quadrupole Time of Flight Mass Spectrometry. Journal of Agricultural and Food 
Chemistry 63 (2015) 6614−6623 
13  
J. Missler, T. Wiezorek, G. Beckh: Mannose: a marker for adulteration with syrup or resin treatment of blossom honey. Proceedings of 
the XIII International Conference on the Applications of Magnetic Resonance in Food Science, 2016 
14  
https://www.bruker.com/en/products-and-solutions/mr/nmr-food-solutions/honey-profiling.html (accessed 05/07/2022)

7 
 
Figure 4. Geographical origin of suspicious honey consignments 
 
 
Oligo/polysaccharides and mannose were the markers which most frequently flagged a suspicious honey, 
either in combination or as single marker. In 44 % of suspicious cases, even two to five marker substances 
were present.  
Stable carbon isotope ratio analysis by EA-IRMS (AOAC method 991.41), a method that has frequently been 
used in the past to detect sugar syrups made of maize starch or sugarcane , was not effective in detecting 
honey suspicious of being adulterated. This is a clear indication that sugar syrups made of maize starch or 
sugarcane are no longer used to extend honey. 
The highest absolute number of suspicious consignments originated from China (66 out of 89, 74 %), 
although honey originating from Turkey (14 out of 15, 93 %) had the highest relative proportion of suspicious 
samples (Figure 5).  
Honey imported from the United Kingdom had an even higher suspicion rate (10 out of 10, 100 %). However, 
the available traceability information suggests that this could be the result of honey produced in other 
countries but further processed and re-exported by the United Kingdom.  
Honey consignments sampled in Romania had the lowest level of suspicion of non-compliance (3 out of 15, 
20 %), while the highest rate was observed for France (17 out of 21, 81 %). Trade flows differed also to a 
certain degree reflecting the importance of major sea ports for trade routes; honey from The Americas 
entered the EU through ports in Belgium, Germany, France and Spain, while Ukrainian honey was mostly 
exported to neighbouring Poland and Moldavian honey to Romania (Figure 6). However, Chinese honey was 
sampled by nearly all EU Member States participating in the coordinated action.

8 
Figure 5. Proportion (%) of suspicious honey consignments of major honey exporting countries (for statistical 
reasons, countries with less than 10 samples were not considered). 
 
 
 
 
Figure 6. Geographical origin of honey consignments entering the EU (for statistical reasons, Member States 
with less than 10 samples were not considered; bars in orange indicate number of suspicious samples, bars in 
green number of non-suspicious samples).

9 
 
 
 
 
 
 
7 Conclusions 
The EU coordinated action launched by the European Commission confirmed the assumption that a significant 
part of honey imported into the internal market is suspicious of not complying with the provisions of the EU 
Honey Directive 2001/110/EC. Of the 320 samples received from the competent authorities of the EU 
Member States, Norway and Switzerland, 147 (46 %) were suspicious of being non-compliant. The suspicion 
rate was considerably high in comparison to an earlier EU wide coordinated control plan conducted in 2015-
17, where 14 % of the analysed samples did not comply with established benchmark criteria to assess honey 
authenticity. However, a different set of methods with improved detection capability was used in the present 
study, which may explain this difference. 
Stable carbon isotope ratio analysis by EA-IRMS (AOAC method 991.41), a method that has frequently been 
used in the past to detect sugar syrups made of maize starch or sugarcane, was not effective in detecting 
honey suspicious of being adulterated. This is a clear indication that sugar syrups made of maize starch or

10 
sugarcane are no longer used to extend honey and that they have been replaced by syrups made mostly from 
rice, wheat or sugar beet. Consequently, improved, harmonised and generally accepted analytical methods are 
still needed to increase the capability of official control laboratories to detect honey adulterated with tailor-
made sugar syrups that imitate to a large extent the characteristic sugar profile of genuine honey. Once 
available, such improved detection methods will constitute an effective deterrent to reduce fraud 
opportunities in international honey trade.

11 
List of abbreviations and definitions 
DP 
Degree of polymerisation 
EA/LC-IRMS 
Elemental Analyser/Liquid Chromatography – Isotope Ratio Mass Spectrometry 
1H-NMR 
Proton Nuclear Magnetic Resonance Spectroscopy 
HPAEC-PAD 
High-Performance Anion Exchange Chromatography -  Pulsed Amperometric Detector 
LC-HRMS 
Liquid Chromatography -  High Resolution Mass Spectrometry

12 
List of figures 
 
Figure 1. Participation in the EU coordinated action to control imported honey. . ...... ...................................................... 4 
Figure 2. Geographical origin of sampled honey consignments.................................................................................................... 5 
Figure 3. Flow of sampled honey consignments from exporting countries to importing EU Member States and 
place of first destination in EU Member States. Origin, importer and destination information according to the 
CHED documentation................................................................................................................................................................................................ 5 
Figure 4. Geographical origin of suspicious honey consignments. .............................................................................................. 7 
Figure 5. Proportion (%) of suspicious honey consignments of major honey exporting countries (for statistical 
reasons, countries with less than 10 samples were not considered). ......................................................................................... 8 
Figure 6. Geographical origin of honey consignments entering the EU (for statistical reasons, Member States 
with less than 10 samples were not considered; bars in orange indicate number of suspicious samples, bars in 
green number of non-suspicious samples). ................................................................................................................................................ 8

13 
List of tables 
 
Table 1. Detection methods for sugar syrup(s) added to honey. .................................................................................................. 6

14 
Annex 1. Analytical methods and decision mechanism applied to identify suspicious samples of 
honey in the frame of the Coordinated Control Plan for Honey 
 
JRC’s quality system is ISO 9001 certified and certain testing activities of JRC Geel are ISO 17025:2017 
accredited. However, the methods of analysis used for generating the reported data are outside the scope of 
accreditation. This fact has to be considered in case a competent authority wishes to initiate regulatory action 
and might bring a requirement for retesting for regulatory or enforcement action at Member State level. 
 
A1. Elemental Analyser/Liquid Chromatography – Isotope Ratio Mass Spectrometry (EA/LC-IRMS) 
The combination of elemental analyser with an isotope ratio mass spectrometer (EA-IRMS) to determine the 
δ13C values of protein isolated from honey together with liquid chromatography coupled to an isotope ratio 
mass spectrometer (LC-IRMS) to determine the δ13C values of fructose, glucose, disaccharides and 
trisaccharides was used to detect addition of sugar syrups made from C4 plants, notably from maize, and 
from C3 plants, notably from rice, wheat or potato. 
The AOAC 998.12 method was used to determine δ13C values of protein isolated from honey using a NCS 
2500 Elemental Analyser (Thermoquest Italia S.p.A., Milan, Italy) coupled to a Thermo Fisher Scientific Delta 
Plus XP IRMS operated using ISODAT NT 2.0 software..  
LC-IRMS analyses were performed using a Dionex Ultimate 3000 HPLC system equipped with a pump, an 
auto-sampler and a column compartment for temperature control. The HPLC was linked to a Thermo Fisher 
Scientific LC-ISOLINK interface and analyses of CO2 isotopic ratios were carried out using a Thermo Fisher 
Scientific Delta V Advantage IRMS operated using ISODAT 3.0 software. 
Honey samples were prepared by diluting honey with Ultrapure water (Fluka Analytical) to a concentration of 
4 mg/mL. The HPLC column was a 300 x 7.8 mm Phenomenex Rezex RCM Monosaccharide (Phenomenex, 
Utrecht, NL). A flow rate of 0.3 mL/min and a temperature of 70 °C were used during analysis. Ultrapure 
water from VWR Chemicals was used as an eluent. The IRMS was calibrated using two-point linear 
normalisation with two certified reference standards IAEA-601 benzoic acid and IAEA-CH6 sucrose. 
The benchmark values proposed by Elflein and Raezke15 were used to assess honey purity based on the 
EA/LC-IRMS results. The differences in the δ13C values (Δδ13Cmax) of the individual sugars of authentic honey 
are distributed around a value close to zero. Based on the analysis of 451 authentic honey samples Elflein 
and Raezke calculated purity criteria (confidence level 99.7%) that which flag a honey sample as non-
compliant if one of the Δδ13C falls outside the cut-off limit. Measurement uncertainty of the LC-IRMS method 
was estimated using the intra-laboratory reproducibility standard deviation to estimate the expanded 
uncertainty (U) of the Δδ13C values used for compliance testing (Table A1) was calculated according to:  
𝑈𝑈= 2 ∗ ඥ𝑆𝑆𝑆𝑆2(𝑎𝑎) + 𝑆𝑆𝑆𝑆2(𝑏𝑏) 
𝑆𝑆𝑆𝑆2(a) and 𝑆𝑆𝑆𝑆2(b) being the intra-laboratory reproducibility variances of the sugars used for calculating the 
differences. Only if the obtained Δδ13C value exceeded the decision threshold, a sample was flagged as being 
suspicious. 
 
 
 
                                           
15 Elflein, L. and K.-P. Raezke, Improved detection of honey adulteration by measuring differences between 13C/12C stable carbon isotope 
ratios of protein and sugar compounds with a combination of elemental analyzer - isotope ratio mass spectrometry and liquid 
chromatography - isotope ratio mass spectrometry (δ13C-EA/LC-IRMS). Apidologie, 2008. 39(5): p. 574-587.

15 
 
Table A1. Benchmark criteria and decision thresholds for assessing honey purity on the basis of EA/LC-IRMS 
measurements. 
Parameter 
Benchmark 
Decision threshold taking 
account of measurement 
uncertainty 
Δδ13C (fructose – glucose) 
±1.0 
± 1.33 
Δδ13C (fructose – disaccharides) 
±2.1 
± 2.52 
Δδ13C (fructose – trisaccharides) 
±2.1 
± 2.61 
Δδ13C (fructose – protein) 
±2.1 
± 2.49 
Δδ13C (glucose – disaccharides) 
±2.1 
± 2.53 
Δδ13C (glucose – trisaccharides) 
±2.1 
± 2.62 
Δδ13C (glucose – protein) 
±2.1 
± 2.50 
Δδ13C (disaccharides – trisaccharides) 
±2.1 
± 2.68 
Δδ13C (disaccharides – protein) 
±2.1 
± 2.58 
Δδ13C (trisaccharides – protein) 
±2.1 
± 2.66 
 
 
 
 
A2. High Performance Anion Exchange Chromatography – Pulsed Amperometric Detector (HPAEC-
PAD) 
HPAEC-PAD was used to detect polysaccharides with a degree of polymerisation (DP) of 10 and higher in 
honey as an indicator for adulteration with sugar syrups. Such polysaccharides are the result of incomplete 
enzymatic breakdown of starch and/or incomplete removal of them during syrup purification. 
A BioLC system ICS-2500 (Dionex, Sunnyvale, USA) including an ED50A electrochemical detector was used. 
Saccharides were separated with a CarboPac PA200, 3 × 250 mm column (Thermo Fisher Scientific, 
Merelbeke, Belgium). Separation of the oligo-/polysaccharides was achieved with a gradient program of 100 
mM NaOH (eluent A) and 100 mM NaOH/1000 mM NaOAc (eluent B) at a flow of 0.45 ml/min. The 
temperature for the column was set to 30 °C. 
A3. Liquid Chromatography – High Resolution Mass Spectrometry (LC-HRMS) 
LC-HRMS was used to flag suspicious samples via the presence of marker compounds (mannose16, difructose 
anhydride (DFA) and 2-acetylfuran-3-glucopyranoside (AFGP)17) indicating the presence of sugar syrups. It 
was also used to confirm the presence of oligosaccharides (DP 6 and higher). 
Separation was afforded by a Waters BEH Amide 100 x 2.1 mm, 1.7 µm particle size, column in HILIC mode. 
Gradient conditions were optimized to obtain baseline separation of mannose. The high resolution mass 
spectrometric detection was done with an Orbitrap Elite (Thermo Scientific) in positive mode electro spray 
ionisation with single MS full scan range from m/z 50 to m/z 2000. Compounds were identified by comparison 
to reference materials of known purity. 
 
                                           
16 J. Missler, T. Wiezorek and G. Beckh: Mannose: a marker for adulteration with syrup or resin treatment of blossom honey.  Magnetic 
Resonance in Food Science 2016 Proceedings. doi: 10.1255/mrfs.4 
17 Bing Du, Liming Wu, Xiaofeng Xue, Lanzhen Chen, Yi Li, Jing Zhao, and Wei Cao: Rapid Screening of Multiclass Syrup Adulterants in Honey 
by Ultrahigh-Performance Liquid Chromatography/Quadrupole Time of Flight Mass Spectrometry. J. Agric. Food Chem. 2015, 63, 
6614−6623

16 
 
A4. Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR) 
1H-NMR was applied as a confirmatory analysis in case the marker compound mannose (indication for 
presence of sugar syrups) could be detected in honey samples by LC-HRMS.  
The measurements were performed with a 400 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, 
Germany). The specific parameters for the 1H-NMR single experiment are listed in Table A2. 
Table A2: Specific parameters for single experiment of analysis of honey.  
Pulse programme 
AQ [s] 
Temperature 
NS 
DS [s] 
TD 
RG 
noesygppr1d 
3.985 
300.00 K ± 0.1 K 
32 
4 
65536 
16 
AQ [s] – acquisition time in s; NS – number of scans; DS – dummy sans; TD – time domain size; RG – receiver 
gain. 
After the calculation of a free induction decay (FID) for each sample, the FID’s were automatically pre-
processed in TopSpin (Bruker BioSpin GmbH, Rheinstetten, Germany), which included a multiplication with an 
exponential line broadening (LB 0.3 Hz) function, Fourier transformation, phase and baseline correction. The 
chemical shift axis of the generated spectra was referred to the signal of 3-(trimethylsilyl)-propanoic-2,2,3,3-
d4 acid sodium salt (TSP) at δ 0.00 ppm. 
On a case-by-case basis, NMR-based profiling in combination with a commercially available databank and 
software tool (Bruker Honey Profiling™) was used as supportive evidence before taking a final decision. 
 
Decision rules for flagging suspicious samples 
A sample was considered as being suspicious of adulteration with a sugar syrup if: 
The results of the EA/LC-IRMS analysis fell outside the decision threshold(s) reported in Table A1, or; 
Oligosaccharides with a degree of polymerisation (DP) between 6 and 9 were detected by LC-HRMS, or; 
Polysaccharides with a DP between 10 and 19 were detect by HPAEC-PAD, or; 
Mannose was detected by LC-HRMS and its presence confirmed by NMR, or; 
Difructose anhydride (DFA) was detected by LC-HRMS, or;    
2-Acetylfuran-3-glucopyranoside (AFGP) was detected by LC-HRMS.

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