
==== Front
J AOAC Int
J AOAC Int
jaoac
Journal of AOAC International
1060-3271
1944-7922
Oxford University Press

38490244
10.1093/jaoacint/qsae021
qsae021
Short Communication
Food Authenticity/Food Fraud, Food Allergens
AcademicSubjects/SCI00030
AcademicSubjects/SCI00980
AcademicSubjects/SCI01060
AcademicSubjects/SCI01140
AcademicSubjects/SCI01180
Purity Assessment of Honey Based on Compound Specific Stable Carbon Isotope Ratios Obtained by LC-IRMS
https://orcid.org/0009-0004-0401-3578
Ulberth Franz European Commission, Joint Research Centre (JRC), 2440 Geel, Belgium

Aries Eric European Commission, Joint Research Centre (JRC), 2440 Geel, Belgium

De Rudder Oliver European Commission, Joint Research Centre (JRC), 2440 Geel, Belgium

Kaklamanos Georgios European Commission, Joint Research Centre (JRC), 2440 Geel, Belgium

https://orcid.org/0000-0002-2109-9594
Maquet Alain European Commission, Joint Research Centre (JRC), 2440 Geel, Belgium

Corresponding author’s e-mail: franz.ulberth@ext.ec.europa.eu.
Sep-Oct 2024
15 3 2024
15 3 2024
107 5 884887
7 12 2023
29 2 2024
2 3 2024
28 8 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The use of stable carbon isotope ratios (δ13C) of sugar fractions of honey is a powerful tool to detect adulteration with sugar syrups. This is accomplished by calculating differences of the δ13C values between individual honey saccharides and comparing them to published purity criteria. A liquid chromatography–isotope ratio mass spectrometry (LC-IRMS) method for the determination of δ13C values of sugars in honey was previously validated by an interlaboratory comparison, but no further guidance was given how to include the obtained precision figures of the compound-specific δ13C values in the purity assessment of honey.

Objective

To use existing data to estimate the standard deviation of the repeatability (sr) and reproducibility (sR) of differences (Δ δ13C) between the δ13C values of individual honey saccharides.

Methods

Previously published δ13C values were used to calculate differences (Δ δ13C values) between δ13C fructose—δ13C glucose, δ13C glucose—δ13C disaccharides, etc. in a honey sample; sr and sR of Δ δ13C values were calculated according to ISO 5725–2:2019.

Results

The Δ δ13C sr and sR values were essentially of the same magnitude as the sr and sR values of δ13C values of the sugar fractions. The precision of the Δ δ13C values was used to estimate the critical difference for comparing a test result with a reference value according to ISO 5725–6:1994. This varied between 0.26 and 1.10‰.

Conclusion

The estimated critical differences can be used to determine whether a honey test result complies with published Δ δ13C purity criteria.

Highlight

The proposed procedure will increase confidence in decisions based on compound-specific δ13C values regarding the conformity of honey with published purity criteria.
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pmcThe economically motivated adulteration of honey compromises its quality and integrity and is of increasing prevalence. Among the most prevalent malpractices is the addition of sugar syrups or feeding bees with syrups during the nectar flow to increase volume and lower production costs for financial gain. Such deceptive practices erode the trust and reputation of the honey value chain, leading to consumers mistrusting the manufacturers’ claims of product authenticity, purity, and quality (1). Recognizing the need to address this critical issue, the scientific community has developed advanced analytical methods to detect honey adulteration (reviewed in 2–4). Among them, liquid chromatography–isotope ratio mass spectrometry (LC-IRMS) has emerged as a powerful tool for the detection of honey adulteration with sugars derived from C3 plants (rice, wheat, sugar beet), and increased the sensitivity for detecting sugars from C4 plants (corn, sugar cane) (5–7). The addition of 1–2% sugar syrups derived from C4 plants, a level where the AOAC Official MethodSM  998.12 fails (8), can be reliably detected using the LC-IRMS method. However, the detection capability for syrups from C3 plants is poorer and varies between 10 and 20%, depending on the type of sugar syrup.

Recently, the LC-IRMS method was validated through interlaboratory comparison (ILC) (9), and the precision of the stable carbon isotope ratios (δ13C) of mono-, di-, and trisaccharides formed the basis for the standardization of the method by the European Committee for Standardization (CEN) (10). The standard provides instructions for the determination of δ13C values but does not give guidance how the obtained data can be used for the purity assessment of honey. International marketing standards for honey such as the FAO/WHO Codex Alimentarius Standard 12-1981 (11), the EU Honey Directive (12), or the USP Honey Standard (13) do not describe reference values of compound-specific δ13C values that can be employed for purity assessment. The most common approach builds on the strong correlations between the δ13C values of individual saccharides, which are altered when exogenous sugars are added. For purity assessment, the differences between the δ13C values of sugar pairs (Δ δ13C) are calculated, i.e., Δ δ13C [fructose—glucose], Δ δ13C [fructose—disaccharides], etc., and compared to cutoff values estimated by analysis of authentic honey samples. Elflein and Raezke (6) analyzed 451 authentic honeys and determined cutoff values, termed purity criteria, that are ±1‰ for Δ δ13C [fructose—glucose], and ±2.1‰ for all other Δ δ13C values of LC separated sugar fractions (Δ δ13C max). These purity criteria are widely used by private and official control laboratories; yet no agreement exists how to take into account measurement uncertainty in the purity assessment.

The Joint Research Centre (JRC) of the European Commission carried out two larger studies to detect sugar syrups in honey by using LC-IRMS (14, 15), making use of the published purity criteria (6). To take account of the uncertainty of the measured Δ δ13C values, the variances of the δ13C values of the sugar fractions obtained from a honey quality control material (=intra-laboratory reproducibility) were combined to give [1] uncertainty u=variancesugarA+variancesugarB

If the test results minus the expanded uncertainty (k = 2) exceeded the respective purity criteria, the honey was deemed suspicious of adulteration. Obviously, this approach does not take account of between-laboratory sources of uncertainty. Therefore, this article explores the opportunity to use the data from the multi-laboratory validation of the LC-IRMS method to estimate repeatability and reproducibility of Δ δ13C values, which can be used for comparing an obtained measurement result with a reference value as described in Clause 4.2.3 of ISO 5725–6:1994 (16).

Experimental

The setup of the ILC to validate the LC-IRMS method for the determination of δ13C values of honey saccharides is described in (9). Most of the 14 participating laboratories employed columns filled with polymeric styrene-divinylbenzene resins in the Ca2+ form and followed the protocol described in (6) for separation; one participant used an anion-exchange (CarboPac) column. Honey samples were selected from the JRC collection of honeys (Supplementary Table S1).

Precision (repeatability and reproducibility) of Δ δ13C values was estimated according to ISO 5725–2:2019 procedures (17); calculations were done with the AOAC Interlaboratory Study Workbook—Blind Replicates (18).

Results and Discussion

The precision data of the LC-IRMS method to determine compound-specific δ13C values of honey saccharides have already been reported (9). However, the results of such an analysis are usually used to assess honey purity by calculating all possible differences (Δ δ13C values) between fructose, glucose, di-, and trisaccharides and comparing them with the purity criteria proposed in (6). In a conformity assessment, the uncertainty of the test results, in this case the Δ δ13C values, has to be taken into account. One way of doing this is to combine mathematically an estimate of the uncertainty of δ13C values of the two sugars used to calculate the difference (Equation 1). An alternative approach is to use the uncertainty of the calculated Δ δ13C values directly, which aligns more closely with the primary objective of the LC-IRMS analysis using the Elflein and Raezke (6) purity criteria. To this end, the collaborative study data were used to calculate the Δ δ13C values per laboratory and honey sample. Standard deviations for repeatability (sr) and reproducibility (sR) were then calculated according to ISO 5725–2:2019 (17). Once sr and sR are known, they can be used to calculate the critical difference (CD) as given in Clause 4.2.3 of ISO 5725–6:1994, which is [2] CD=122.8sR2-2.8Sr2 (n−1n)

If the absolute difference between the mean of the Δ δ13C values of n LC-IRMS measurements and the reference value (purity criterion) exceeds the CD, this indicates a suspect sample at the 95% probability level. A prerequisite for this approach is that the testing laboratory’s sr values are consistent with the precision estimates from the collaborative study. ISO 21748:2017 provides guidance how to verify this requirement (19).

Table 1 summarizes the sr and sR of Δ δ13C values calculated from the data obtained in the ILC; Supplementary Tables S1–S7 contain more detailed information on the outcome of the data evaluation. The precision estimates of the Δ δ13C values are essentially of the same magnitude as the δ13C values of the sugar fractions estimated in the ILC (9) (Table 2).

Table 1. Precision data for the differences between δ13C values of honey saccharides (Δ δ13C, absolute values) and critical differences for comparing the mean of duplicate test results (n = 2) with a reference value: A (lemon), B (polyfloral), C (honeydew), D (honeydew), E (acacia), F (lavender)

	Honey sample		
	A	B	C	D	E	F	Mean	
Δ δ13C (‰) [fructose—glucose]	
sr	0.07	0.10	0.10	0.15	0.07	0.06	0.09	
sR	0.25	0.43	0.31	0.25	0.21	0.14	0.27	
CD	0.49	0.69	0.60	0.45	0.40	0.26	0.48	
Δ δ13C (‰) [fructose—disaccharides]	
sr	0.15	0.14	0.22	0.18	0.13	0.14	0.16	
sR	0.31	0.34	0.31	0.31	0.29	0.34	0.32	
CD	0.57	0.65	0.53	0.56	0.54	0.64	0.58	
Δ δ13C (‰) [fructose—trisaccharides]	
sr	0.15	0.21	0.16	0.27	0.18	0.39	0.23	
sR	0.26	0.47	0.28	0.31	0.50	0.55	0.40	
CD	0.47	0.88	0.51	0.48	0.96	0.94	0.71	
Δ δ13C (‰) [glucose—disaccharides]	
sr	0.14	0.12	0.26	0.12	0.16	0.14	0.16	
sR	0.24	0.47	0.30	0.37	0.21	0.31	0.32	
CD	0.43	0.92	0.47	0.71	0.35	0.58	0.58	
Δ δ13C (‰) [glucose—trisaccharides]	
sr	0.33	0.08	0.27	0.25	0.18	0.22	0.22	
sR	0.35	0.56	0.40	0.50	0.50	0.44	0.46	
CD	0.52	1.10	0.70	0.93	0.96	0.81	0.84	
Δ δ13C (‰) [disaccharides—trisaccharides]	
sr	0.50	0.15	0.11	0.14	0.24	0.40	0.26	
sR	0.57	0.40	0.25	0.27	0.52	0.54	0.43	
CD	0.89	0.76	0.47	0.50	0.97	0.91	0.75	

Table 2. Precision data for δ13C values of fructose, glucose, disaccharides, and trisaccharides determined by LC-IRMS in honey (9): A (lemon), B (polyfloral), C (honeydew), D (honeydew), E (acacia), F (lavender)

	Honey sample	
	A	B	C	D	E	F	
δ13C (‰) fructose							
sr	0.13	0.09	0.09	0.08	0.08	0.12	
sR	0.38	0.28	0.19	0.27	0.38	0.37	
δ13C (‰) glucose							
sr	0.10	0.10	0.12	0.13	0.12	0.12	
sR	0.31	0.43	0.34	0.33	0.29	0.42	
δ13C (‰) disaccharides							
sr	0.18	0.08	0.24	0.15	0.14	0.12	
sR	0.35	0.34	0.36	0.24	0.36	0.31	
δ13C (‰) trisaccharides							
sr	0.68	0.19	0.23	0.25	0.15	0.38	
sR	0.68	0.36	0.35	0.50	0.45	0.57	

The standard deviation of repeatability (sr) of Δ δ13C values was, with few exceptions, independent of the magnitude of the Δ δ13C values (correlation coefficient r = 0.134, P > 0.1). On the other hand, the standard deviation of reproducibility (sR) increased with increasing Δ δ13C values (correlation coefficient r = 0.700, P < 0.05). As expected, the Δ δ13C values of [fructose—trisaccharides], [glucose—disaccharides], [glucose—trisaccharides], and [disaccharides—trisaccharides] were higher than the Δ δ13C values of [fructose—glucose] and [fructose—disaccharides] (Figure 1). The CD values for duplicate analyses (n = 2) calculated according to Equation 2 varied between 0.26 and 1.10‰, with means ranging from 0.48‰ for Δ δ13C [fructose—disaccharides] to 0.84‰ for [glucose—disaccharides] (Table 1). Using the mean CD values to compare a test result to the reference value may raise the risk of false-positive assessments. It may, therefore, be more prudent to use the highest CD values for comparison: 0.7‰ for Δ δ13C [fructose—glucose] and 1.1‰ for all other differences.

Figure 1. Relationship between the differences (Δ) of the δ13C values between the LC separated sugars and the repeatability and reproducibility standard deviation obtained by an interlaboratory comparison (9). Symbols: open circles, Δ δ13C [fructose—glucose]; filled circles, Δ δ13C [fructose—disaccharides], rectangles, Δ δ13C [fructose—trisaccharides]; diamonds, Δ δ13C [glucose—disaccharides]; triangles, Δ δ13C [glucose—trisaccharides]; stars, Δ δ13C [disaccharides—trisaccharides].

The CD values can also be incorporated into the approach proposed by Elflein and Raezke (6) for assessing the purity of honey. In other words, if the mean of duplicate LC-IRMS analysis of a honey sample results in an absolute Δ δ13C [fructose—glucose] value that is greater than 1.7‰ and/or an absolute Δ δ13C max value greater than 3.2‰, the sample should be considered noncompliant with the reference values at a confidence level of 95%.

Conclusions

We propose to include the CD in the evaluation of honey purity based on published Δ δ13C purity criteria (6), which takes the uncertainty of δ13C values of honey saccharides into account. The CD for Δ δ13C [fructose—glucose] was estimated as 0.7‰ and for Δ δ13C max as 1.1‰ provided the mean of duplicate LC-IRMS analyses is used for calculating CD. For practical reasons, laboratories may decide to carry out a single determination first, and if δ13C [fructose—glucose] and/or Δ δ13C max is above the purity criteria, to repeat the analysis for confirming the suspicion.

Supplementary Material

qsae021_Supplementary_Data

Acknowledgments

The contributions of the participants in the validation study (Czech Agriculture and Food Inspection Authority (CAFIA), Inspectorate Brno, Brno, Czech Republic; Department of Management Science and Engineering, Akita Prefectural University, Akita, Japan; Elementar Analysensysteme GmbH, Langenselbold, Germany; Eurofins Food Integrity Control Services GmbH, Ritterhude, Germany; FERA Science Ltd, York, United Kingdom; Floramo Corporation Srl, Rocca de’Baldi (CN), Italy; Imprint Analytics GmbH, Neutal, Austria; Institut des Sciences Analytiques (ISA), Villeurbanne, France; Intertek Food Services GmbH, Bremen, Germany; LAVES Lebensmittel- und Veterinärinstitut Oldenburg, Oldenburg, Germany; MAPAMA—Laboratorio Arbitral Agroalimentario, Madrid, Spain; QSI—Quality Services International GmbH, Bremen, Germany; Service commun des laboratoires (SCL) de Bordeaux, Pessac, France) are kindly acknowledged.

Supplemental Information

Supplemental information is available on the J. AOAC Int. website.

Conflict of Interest

All authors declare no conflict of interest.
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References

1 Gustafson C.R. , ChampetierA., TuyizereO., GitungwaH. (2024) Food Control  155 , 110070. doi: 10.1016/j.foodcont.2023.110070
2 Zhang G. , AbdullaW. (2022) Food Control  138 , 108992. doi: 10.1016/j.foodcont.2022.108992
3 Se K.W. , WahabR.A., Syed YaacobS.N., GhoshalS.K. (2019) J. Food Compos. Anal. 80 , 16–32. doi: 10.1016/j.jfca.2019.04.001
4 Ulberth F. (2016) Advances in Food Authenticity Testing, Woodhead Publishing, Cambridge, UK, pp 729–753. doi: 10.1016/B978-0-08-100220-9.00026-6
5 Cabañero A.I. , RecioJ.L., RupérezM. (2006) J. Agric. Food Chem. 54 , 9719–9727. doi:10.1021/jf062067x 17177492
6 Elflein L. , RaezkeK.-P. (2008) Apidologie  39 , 574–587. doi: 10.1051/apido:2008042
7 Biswas A. , NareshK.S., JaygadkarS.S., ChaudhariS.R. (2023) Food Chem. 416 , 135825.doi: 10.1016/j.foodchem.2023.135825 36924528
8 Official Methods of Analysis ( 2005) AOAC Official Method 998.12: C-4 Plant Sugars in Honey, Internal Standard Stable Carbon Isotope Ratio Method, AOAC INTERNATIONAL, Gaithersburg, MD, Chapter 44 , 27–30
9 Aries E. , De RudderO., KaklamanosG., MaquetA., UlberthF. (2021) J. AOAC Int. 104 , 1698–1702. doi: 10.1093/jaoacint/qsab091 34550371
10 CEN ( 2023) Food authenticity—Determination of the δ13C value of mono- (fructose and glucose), di-, and trisaccharides in honey by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) (prEN 17958:2024) European Committee for Standardization, Brussels, Belgium
11 FAO/WHO Codex Alimentarius ( 1981) Revised Codex Standard for Honey (CODEX STAN 12–1981, Rev.2 (2001))
12 Council Directive  2001/110/EC of 20 December 2001 relating to honey. OJ L 10, 12 January 2002, 47–52.
13 USP Food Chemicals Codex ( 2023), Thirteenth Edition
14 Aries E. , BurtonJ., CarrascoL., De RudderO., MaquetA. (2016) Scientific support to the implementation of a Coordinated Control Plan with a view to establishing the prevalence of fraudulent practices in the marketing of honey (2016) JRC Technical Report, JRC104749, https://food.ec.europa.eu/safety/eu-agri-food-fraud-network/eu-coordinated-actions/honey-2015-17_en (accessed October 26, 2023)
15 Ždiniaková T. , LörchnerC., De RudderO., DimitrovaT., KaklamanosG., BreidbachA., Respaldiza HidalgoM.A., Vaz SilvaI. M., PaianoV., UlberthF., MaquetA. (2023) EU coordinated action to deter certain fraudulent practices in the honey sector, EUR 31461 EN, Publications Office of the European Union, Luxembourg. doi: 10.2760/184511, JRC130227
16 ISO ( 1994) Accuracy (trueness and precision) of measurement methods and results—Part 6: Use in practice of accuracy values (ISO 5725-6:1994)
17 ISO ( 2019) Accuracy (trueness and precision) of measurement methods and results—Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method (ISO 5725-2:2019)
18 AOAC Interlaboratory Study Workbook—Blind Replicates (2023) https://www.aoac.org/resources/?topic=Methods+%26+Standards&type=&key= (accessed October 24, 2023)
19 ISO (2017) Guidance for the use of repeatability, reproducibility and trueness estimates in measurement uncertainty evaluation (ISO 21748:2017)
