Accepted Manuscript

Application of Analytical Methods in Authentication and Adulteration of Honey

Amna Jabbar Siddiqui, Syed Ghulam Musharraf, M. Iqbal Choudhary, Atta-ur-

Rahman

PII: S0308-8146(16)31384-X
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.09.001
Reference: FOCH 19781

To appear in: Food Chemistry

Received Date: 11 May 2016

Revised Date: 31 August 2016
Accepted Date: 1 September 2016

Please cite this article as: Siddiqui, A.J., Musharraf, S.G., Iqbal Choudhary, M., Rahman, A-u., Application of
Analytical Methods in Authentication and Adulteration of Honey, Food Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.foodchem.2016.09.001

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Application of Analytical Methods in Authentication and
Adulteration of Honey

Amna Jabbar Siddiqui, *Syed Ghulam Musharraf, M. Iqbal Choudhary and Atta-ur-Rahman

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi-75270, Pakistan.

*
Corresponding author
Email address: musharraf1977@yahoo.com
Tel: + 92 21 34824924-5; 34819010; fax: + 92 21 34819018-9

1
Contents:
1. Introduction to honey
1.1 Chemical composition
1.2 Reported biological activities
2. Honey authenticity and related issues
3. Methods for confirmation of botanical origin
3.1 Estimation of physicochemical parameters
3.2 Various instrumental methods
3.2.1 Front phase fluorimetric spectroscopy
3.2.2 Infrared spectroscopy
3.2.3 Nuclear magnetic resonance
4. Geographical authentication study
4.1 Estimation of physicochemical parameters
4.2 Various instrumental methods for geographical authentication studies
4.2.1 Inductively coupled plasma-MS
4.2.2 Nuclear magnetic resonance
5. Controlling honey adulteration
5.1 Corn syrup adulteration
5.2 Beet syrup adulteration
5.3 Rice syrup adulteration
5.4 Inulin syrup adulteration
6. Conclusion
7. References

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Abstract
Honey is synthesized from flower nectar and it is famous for its tremendous therapeutic
potential since ancient times. Many factors influence the basic properties of honey including
the nectar-providing plant species, bee species, geographic area, and harvesting conditions.
Quality and composition of honey is also affected by many other factors, such as overfeeding
of bees with sucrose, harvesting prior to maturity, and adulteration with sugar syrups. Due to
the complex nature of honey, it is often challenging to authenticate the purity and quality by
using common methods such as physicochemical parameters and more specialized
procedures need to be developed. This article reviews the literature (between 2000-2016) on
the use of analytical techniques, mainly NMR spectroscopy, for authentication of honey, its
botanical and geographical origin, and adulteration by sugar syrups. NMR is a powerful
technique and can be used as a fingerprinting technique to compare various samples.

Keywords
Honey authenticity; adulteration; botanical origin; geographical origin; nuclear magnetic
resonance

3
1. Introduction to honey
Honey is a natural product produced by honey bees (Apis mellifera L.) from various
secretions of plants. There are two major types of honey, categorized on the basis of
secretions of plants used for their synthesis: (i) blossom honey made from the nectar of
flowers, and (ii) honeydew honey made from secretions of all living parts of plants other than
flower or excretions of sucker insects. The composition and properties of honey depend on
the botanical origin of the source of nectars or secretions (Bertelli et al., 2010), where as the
carbohydrates are the major constituents of honey.

1.1 Chemical composition

Two sugars, dextrose and levulose, are the chief ingredients of honey, with smaller
quantities of 22 other more complex sugars, including disaccharides maltose, sucrose,
maltulose, turanose, isomaltose, laminaribiose, nigerose, kojibiose, gentiobiose and β-
trehalose. The trisaccharides found include maltotriose, erlose, melezitose, 1-kestose,
isopanose, isomaltotriose, panose, and theanderose. Different plants contain minor quantities
of all these sugars (Bogdanov et al., 2004).
Many organic acids are also present in honey. These are lactic, formic, butyric,
tartaric, pyruvic, acetic, citric, oxalic, succinic, malic, maleic, α-ketoglutaric, glucose-6-
phosphate, pyroglutamic and glycolic acid. Among these acids, gluconic acid is the common
one present. It is produced from dextrose by the action of an enzyme, glucose oxidase . This
unique feature of honey, that is the presence of enzymes, makes it different from other
sweetening agents. These enzymes are derived from the yeasts, nectar, pollen, bee, and
micro-organisms. Some of the most important honey enzymes are glucose oxidase, catalase,
acid phosphatase, invertase, and diastase. Heating of honey can de-stabilize or destroy these
enzymes (Hebbar et al., 2003). Apparently the organic acids and enzymes play an important
role in defining the physical and biological properties of various honeys.
The mineral content or ash in floral honey varies from 0.02 to 0.1 %. Due to its richer
mineral content, honey is less suitable for storage at low temperatures . A list of average
composition of floral and honeydew honey is shown in supplementary table 1 (Olga et al.,
2012). Other constituents such as moisture content, reducing sugars, electrical conductivity,
free acids, sucrose content and hydroxymethylfurfural (HMF) may also influence the
nutritional quality, granulation, flavor and texture of honey. The medicinal value of honey is
also due to these constituents. Therefore, the International Honey Commission (IHC) has

4
proposed certain composition as quality criteria for honey. The physico-chemical parameters
of product have great importance in the honey industry.

1.2 Reported biological activities

Honey is a popular carbohydrate resource for athletes, generally taken before and
after resistance exercise . The lower glycemic index of honey also allows its modest use in
types I and II diabetes. It elevates hemoglobin concentration, stimulates insulin secretion,
decreases blood glucose levels, and improves lipid profile . Several studies have reported the
antioxidant activity and phenolic content in honey (Piljac-Žegarac et al., 2009; Jamróz et al.,
2014), and established a correlation between phenolic content, antioxidant activity, and color
intensity. Due to the presence of phenolic constituents, honey exhibits anti-carcinogenic, anti-
inflammatory, anti-atherogenic, anti-thrombotic, immune modulating, and analgesic
activities. Studies have also shown that natural honey decreases cardiovascular risk factors
without any increase in body weights (Yaghoobi et al., 2008). The inhibitory effects of honey
against 60 gram-positive and gram-negative bacteria, including aerobes and anaerobes was
studied, and it therefore helps in cleaning up wounds (Molan, 2006). Apart from its use in
cooking, the anti-microbial, anti-inflammatory and antioxidant effects of honey make it a
useful substance. In the medical papyri of Egypt, back to 1553-1550 B.C., there are signs that
honey heals wounds, causes urination, and serves as weight reducing agent. Similarly, Galen,
a great Roman physician, regarded honey as a cure for all kinds of poisoning and intestinal
diseases. The greatest medical authority of mediaeval times, Ibn-Sina (Ave-Sina) wrote that,
“honey helps you when you have a runny nose, cheers you up, makes you feel fit, facilitates
the digestion of food, gets rid of wind, and improves the appetite. It is almost a provision for
retaining youth, making the memory better, sharpening the wits, and loosening the tongue.”
Modern research indicates that honey can serve as a remedy against various diseases.
Due to the complex mixture of sugars, acids and proteins, honey improves the resistance
against pathogenic organisms . Honey is also a useful remedy against helminth diseases, in
particular intestinal nematodes, including ascariasis, hookworm infection, etc (Sajid and
Azim, 2012). In another study, honey glycoproteins and peptides were found to exhibit
immunomodulatory properties by interfering with molecules of the innate immune system in
humans (Mesaik et al., 2014).
Due to the high dietary value and the exclusive flavor character of natural bee honey,
it is much expensive than other sweetener, and it is ultimately a target of adulteration for

5
economic profit. In contrast to other synthetic sweeteners, adulteration in honey is relatively
easy, but difficult to detect. Thus authenticity of honey is a global important concern for
consumers as well as for commercial producers.

2. Honey authenticity and its related issues

Authorities such as Codex Alimentarius standard, the EU Honey Directive and other
legislations, regulate food authenticity at national levels. However, growing global trade of
honey and its medicinal uses make it important to have international standards for honey
authenticity. Honey authenticity has two major aspects: (i) origin of the honey, and (ii) the
mode of production of the honey. The origin of the honey includes geographical origin and
botanical origin, while production is related to the harvesting of honey hives and processing.
International Honey Commission recently reviewed the Codex Alimentarius (CA) standards
and European Community standards. Supplementary table 2 shows these compositional
standards for honey.

3. Methods for confirmation of botanical origin

The botanical origin may be determined if the honey originates totally or largely from
a single specific source (unifloral honey), and thus mimic the physico-chemical, organoleptic
and microscopic characteristics of the source. However as bees feed on various plants, pure
unifloral honeys are generally very rare. Various unifloral honeys exhibit usual sensory,
physico-chemical and melissopalynological properties. However, the analyses of these
properties are protracted, and involve dedicated experts. Based on organoleptic, physico-
chemical and pollen analysis, the International Honey Commission has developed standards
for the main European unifloral honeys (Persano Oddo et al., 2004).

3.1 Estimation of physicochemical parameters

The color analysis is a valuable categorization for unifloral honeys. For example,
alfafa yields a white honey, heather produces reddish-brown, and acacia and citrus produces a
straw colored honey. The honey color is also associated with its flavor. Light colored honey
is placid, whereas darker types have sharper tastes. Dark colored honeys are claimed to have
more phenolic acid derivatives but fewer flavonoids than the light colored ones (Bogdanov et
al., 2004). The electrical conductivity factor has also been integrated in recent times in the
new international standards for honey for discrimination between honeydew and blossom

6
honey, 0.5-0.8 mS/cm is the range of electrical conductivity for mixed blossom honeys, while
less than 0.5 mS/cm is that for pure blossom honeys, with numerous exceptions (Saxena et
al., 2010). Electrical conductivity and Fourier Transform Infrared (FT-IR) spectrometry have
been used in combination to differentiate between honeydew and blossom honey, mixed with
honeydew (Etzold and Lichtenberg-Kraag, 2008). Normally, the pH of honey is between 3.5
and 5.5 due to the presence of organic acids that give flavor to the honey, and protection
against microbial damage. In addition to this, the pH values also help in the identification of
the botanical origins of honey (Sanz et al., 2005). The botanical source of honey is also
evaluated by microscopic pollen analysis (Ohe et al., 2004). In a quantification study of
Co ea honey, excellent correlation was found between quantified caffeine, theobromine,
and trigonelline markers and the relative amounts of Coffea pollen measured in samples
(Schievano et al., 2015). However, due to the significant difference of the pollen content, it is
now considered as a minor method.
Another approach involves the chemometrical estimation of physicochemical
parameters (sugars, optical rotation, nitrogen content, electrical conductivity, etc). These
factors allowed a good segregation of a few unifloral honeys (Terrab et al., 2003). However,
they may not distinguish between unifloral and polyfloral honeys. Both quantitation and
chemometric analysis of the volatile components can also be used (Bogdanov, 2004).
Evaluation of the botanical origin of honey, employing an analysis of the volatile compounds,
is a useful approach. Current methods that use solvent extraction or analyze headspace (static
and dynamic) have shown good approximation. However, these methods are complex and
require expensive instrumentation. Among them, solid phase micro extraction (SPME) is a
flexible, simple and a relatively economical extraction technique. The results depend on both
the fiber characteristics, and extraction conditions used for the analysis. However, further
studies are required before it is accepted as an authentic technique for honey aroma
extraction, and floral determination (Cuevas-Glory et al., 2007).

3.2 Various instrumental methods

3.2.1 Front Phase Fluorimetric Spectroscopy

Recently, different spectroscopic techniques, combined with chemometric analysis,
have been successfully used for the identification of botanical origin of honey. Ruoff K. et al.
in 2006 used Front Phase Fluorimetric Spectroscopy on 11 unifloral and polyfloral honey

7
types for the authentication of honey (Ruoff et al., 2006b). By using statistical techniques,
principal component analysis and linear discriminant analysis, it was shown that the
fluorescence characteristics of honey depend on their botanical origin.

3.2.2 Infrared spectroscopy

Different ranges of infrared absorptions can be used for the estimation of botanical
origin. Near-infrared spectroscopy has been used for the authentication of eight unifloral and
polyfloral honey types (Ruoff et al., 2006a). Similarly, a quantitative analysis of twenty
different physical and chemical parameters in honey by mid-infrared spectrometry was
carried out in 2006 (Ruoff et al., 2005). Chemometrics and FTIR spectroscopy were
successfully employed in the botanical origin studies (Kelly et al., 2004; Sivakesava and
Irudayaraj, 2001). However, still no biomarker could be identified from infrared
spectroscopy.

3.2.3 Nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) has been used by several researchers for the
estimation of the floral origin of honeys. NMR is a powerful technique for structure
determination. Hence it can also provide better understanding of the complex structures in
complicates systems, such as food (Cazor et al., 2006). There are several advantages of the
NMR technique as compared to other methods, including its non-invasive nature, the relative
ease, and rapidity of data execution (Caligiani et al., 2007) and the ability to present
information about a broad range of metabolites in a single run. Due to the complex nature of
obtained data, hence multivariate analysis can be useful to extract the useful information.
Several papers have published, indicating the high competence of these methods to categorize
honey samples according to their botanical origin. For instance in 2008, 71 Italian honey
samples of 5 different botanical origin was successfully distinguished using 2D high
resolution NMR approach (Lolli et al., 2008). While in a recent study of 2014, researchers
utilized low field nuclear magnetic resonance (LF 1H NMR) for differentiating eighty
samples of honey from 8 different botanical sources of Brazil (Ribeiro et al., 2014a).
Differentiation of honey from Eucalyptus, citrus and wildflower, produced in São
Paulo, Brazil, was achieved using 1 H-NMR spectroscopy along with chemometric methods
1
(Boffo et al., 2012). H-NMR data was subjected to principal component analysis.
Hierarchical clustering analysis and pattern recognition models revealed that it can categorize

8
the commercial honey samples based on their botanical origin. Prediction of the commercial
honey samples by SIMCA, KNN and PLS-DA models suitably classified 22.2, 66.7 and
72.2% of the samples, respectively. Supplementary figure 1 shows the predicted data for the
commercial samples and their classification as (A) wildflower, (B) Eucalyptus, and (C) citrus
class.
In another study, chloroform extracts of nonvolatile organic honey components were
subjected to a new pulse sequence for NMR profiling (Schievano et al., 2010). The modified
double pulsed field gradient spin echoes (DPFGSE) pulse sequence helped to reduce the scan
time to half with the same signal-to-noise (S/N) ratio. The spectrum, generated from the older
sequence, was dominated by signals in the 0-2 ppm region. Thus these signals mask the
actual resonances responsible for potential markers of botanical origin and, moreover, restrict
the practical receiver gain so that the very weak resonances are imprecisely sampled and
result in poor integration (Supplementary figure 2).
In a study by Beretta et al., solid-phase extraction (SPE), combined with 1H-NMR,
successfully identified sources and contamination of honey from different botanical origins
(Beretta et al., 2008). 44 commercial Italian honey samples from 20 selected botanical
sources were processed. Three varieties of honey showed specific and highly resolved signals
representing chemical markers specific to origin. Kynurenic acid was identified as marker for
chestnut origin. Similarly the mono-terpene derivative cyclohexa-1,3-diene-1-carboxylic acid
(CDCA) and 1-O-β-gentiobiosyl ester (CDCA-GBE) were identified as linden honey
markers. Aliphatic signals in the spectra were identified as honeydew markers.
Supplementary table 3 presents the semi-quantitative data relative to the proposed marker
compounds in the honeys analyzed.
Another NMR-based profiling for quick discrimination of oak honeydew honey from
other floral and honeydew honeys was used by Simova and co-workers (Simova et al., 2012).
This study was based on the identification of the protons and the carbon of the methylene
group of quercitol in the 1 H- and 13
C-NMR spectra, and on TOCSY spectroscopy of honey
(Supplementary figure 3). Quercitol was found to be present in all samples of oak honeydew
honey, while it was absent in other honeys.
In another study, nineteen saccharides were identified in the aqueous extracts of
honey of 5 different botanical species. This study concluded that the saccharide content could
be employed to characterize honey samples and to construct an identity card of saccharides
for each floral source (Consonni et al., 2012). Quantification of selected components of

9
honey was also carried out along with qualitative analysis. However, extensive overlap of
signals in the spectra causes difficulty in direct quantification with integration. Lachenmeier
et al. have successfully quantified thirteen metabolites in honey samples with R2>0.99
(Ohmenhaeuser et al., 2013). Due to the overlapping of signals more advanced techniques
were essential for quantification for instance multivariate regression or curve deconvolution.
Furthermore, the two major carbohydrates, glucose and fructose, showed relatively higher
peak intensities in comparison to other compounds, and consequently, ambiguous the
remaining spectrum. A total of 328 samples from variety of botanical origins were randomly
selected. It was suggested from the PCA score plot that this model not only differentiates
between the two major groups of honey (uni- and polyfloral), but clusters from several
unifloral honeys were also clearly separated from each other (Figure 1). It should be noted
that the 13C-NMR spectra are less useful as compared 1H-NMR spectra because of the lower
sensitivity. However, extensive quantification of saccharides in authentic Greek honey
13
samples was achieved by C NMR. The method was rigorously validated (accuracy,
linearity, range, limit of detection, etc.) using either single sugar molecules, or artificial
mixtures of isoglucose (glucopyranose and fructose) and global mixtures of fourteen model
compounds (Kazalaki et al., 2015). Apart from carbohydrates; carboxylic acids, amino acids,
ethanol and hydroxymethylfurfural in honey was also quantified by 1 H-NMR in various
monofloral honey (del Campo et al., 2016).
In early 2009, two new pyrrolidinyl quinoline alkaloids from chestnut honey was
identified for the first time named as 3-pyrrolidinyl-kynurenic acid (3-PKA) and γ-lactamic
derivative (γ-LACT-3-PKA) (Beretta et al., 2009b). Quantification of these quinoline
alkaloids and their biosynthetic precursor i.e. kynurenic acid (KA) in honey of different
botanical sources was performed using HPLC-DAD-ESI-MS, and multidimensional
diffusion-ordered (DOSY) NMR spectroscopy afterwards (Beretta et al., 2009a). In principal
component analysis of botanical sources of honey, it was clearly found that the developed
method separated chestnut honey from others on the basis of concentration level of these
quinoline alkaloids. In another study kynurenic acid was identified as marker of chestnut
honey of Corsica using one-dimensional 1H NMR spectroscopy (Donarski et al., 2010a). In
2015, two new quinolinone alkaloids including a kynurenic acid derivative and 13 known
compounds were also isolated from chestnut honey (Cho et al., 2015). Similarly in another
study, kynurenic acid and 4-quinolone-2-carboxylic acid were identified as chestnut honey
marker by 1H NMR and 13C NMR (Truchado et al., 2009). In continuation for marker

10
identification of unifloral honey, lumichrome (7,8-dimethylalloxazine) in thistle (Galactites
tomentosa Moench) unifloral honey was extracted by SPE using C18, SiOH, and NH2 phases
(Tuberoso et al., 2011). Its structure was elucidated on the basis of extensive 1D and 2D
NMR experiments as well as HPLC-MS/MS and Q-TOF analysis. Lumichrome is known to
be the main product of degradation obtained in acid medium from riboflavin (vitamin B2),
and this was the first report of the presence of lumichrome in honeys. Similarly, strawberry
honey markers were also identified using combined methodologies of HPLC-MS/MS 1D and
2D NMR (Tuberoso et al., 2010). Applying the same techniques on asphodel monofloral
honey revealed methyl syringate as a chemical marker and identified methylglyoxal as
marker in manuka honey (Tuberoso et al., 2009; Donarski et al., 2010b).
In many recent studies, NMR based metabolomics strategy with PCA and OPLS-DA
classification models was applied on monofloral and polyfloral honey discrimination. The
predictive components of the statistical models reveal not only the principal but also the
secondary floral origins present in a sample of honey, a novel feature with respect to the
methods present in the literature that are able to confirm the authenticity of monofloral
honeys but not to characterize a mixture of honey types (Schievano et al., 2016; Zielinski et
al., 2014; Schievano et al., 2013; Gresley et al., 2012; Schievano et al., 2012; Zheng et al.,
2016). A data fusion approach was applied to a 56 honey data set analysed by 1H-NMR and
LC-MS (with an Orbitrap-MS and a time of flight (TOF)-MS). It was suggested that the
discriminating potential is increased through the data fusion, allowing for a better separation
of eucalyptus, orange blossom and lavender (Spiteri et al., 2016). For the chemical
composition studies, black honeybees’ honey including sulla honey (Hedysarum coronarium
L.) and dill honey (Anethum graveolens L.) were studied with a multi methodological
approach, which consists of HPLC-PDA-ESI-MSn and NMR spectroscopy (Mannina et al.,
2015).
For the determination of the botanical origin of honey, a criterion for the
determination of unifloral as well as polyfloral origin needs to be developed. However, it is
very complex to identify consistent chemical markers for the differentiation of honey of
different floral sources due to their chemical composition which is influenced by a number of
other factors, including geographic origin, bee species, collection season, mode of storage.
Hence, different markers have been reported for honey of the same floral origin.
Additionally, sample preparation and analytical techniques also contribute to results for
chemical analyses of honey constituents. A more reliable categorization of honey floral class,

11
therefore, involves a panel of compounds, preferably combine with modern tools of
interpretation of the data, for instance, principal component analysis or cluster analysis.

4. Geographical authentication study

4.1 Estimation of physicochemical parameters

In the past decade, pollen analysis has been employed for the discrimination of honey
of various closer geographical zones using special statistical software (Arvanitoyannis et al.,
2005; Jamil Noor et al., 2015). Unfortunately, in most of the pollen-based analysis, the
geographical origin of the honey samples cannot be established. This means that
discrimination among honeys of various geographical origins was not possible on samples of
the same botanical origin using pollen analyses. Moreover, the number of samples studied
was usually small, or acquired from a small geographical zone. Any differences found were
as a result of variations of the local vegetation type rather than due to the geographical
regions. If the geographical zones are closer, variations are more complicated to identify. In
this situation more sophisticated methods should be employed.
Among phytochemicals, volatile organic compounds (VOCs) have been proposed as
the key markers for differentiating honeys from different geographical origins (Stanimirova
et al., 2010). In a recent study, VOCs combined with melissopalynology was used to identify
floral origins of honeys from four different countries (Panseri et al., 2013).
The aroma and taste of honey are associated to the nature of the volatile compounds.
They can vary as a result of the seasonal conditions and geographical origin. Hence in a study
by Silvano and coworkers, the physicochemical parameters (moisture, color, free acidity,
electrical conductivity, glucose, fructose, sucrose and hydroxymethyl furfural) and the
sensory properties were analyzed in honeys from different geographical areas of the south
east region of Buenos Aires province (Argentina) (Silvano et al., 2014). Cluster and linear
discriminant analyses were carried out. Samples were grouped together based on sampling
regions also illustrated by the principal component analysis. This study suggested that
geographic categorization of honeys on the basis of physicochemical parameters showed
potential. Though, the sensory properties could not be predicted properly.

4.2 Various instrumental methods for geographical authentication studies

12
4.2.1 Inductively coupled plasma mass spectrometry
Trace element determination in food samples by atomic absorption spectrometric
techniques have been used as a key analytical method. The use of inductively coupled plasma
mass spectrometry (ICP-MS) is becoming more popular in food analysis. This technique have
advantage of multi-element measurement at low detection limits when compared to graphite
furnace atomic absorption spectrometry (GF AAS) or inductively coupled plasma atomic
emission spectrometry (ICP-AES). Furthermore, due to a wider linear dynamic range, this
makes easy to determine major as well as trace elements in the same sample injection.
Moreover, it provides simpler spectral interpretation, and isotopic information as compared to
ICP-AES, ICP-MS. The technique has been effectively useful to vegetables (Ariyama et al.,
2006), nuts (Gomez-Ariza et al., 2006), tea (Moreda-Piñeiro et al., 2003) and wines (Coetzee
et al., 2005). ICP-MS has been used in combination with data mining approaches for the
estimation of the geographical origin of Brazilian honeys (Batista et al., 2012). Forty-two
chemical elements were identified and three machine learning tools were applied for the class
discrimination. It proved that it is probable to utilize data mining tools to identify the area
from where the honey is originated.

4.2.2 Nuclear magnetic resonance spectroscopy

NMR spectroscopy is a powerful technique for the analysis of complex mixtures. 23
poly-floral and 18 acacia honey samples from different countries (geographical origins) were
subjected to 1H-NMR spectroscopy, coupled with multivariate data analysis (Consonni and
Cagliani, 2008). 13C-NMR spectroscopy was also used with the aim to identify possible sugar
isoforms. PCA and Hierarchical PLS-DA model was generated to classify geographical
differentiation of polyfloral and Acacia honeys from different countries (European
Community (EC) and non-EC) as shown in figure 2.
1
H-NMR spectroscopy can play an important role in saccharide analysis.
Geographical origin determination using saccharide analysis was carried out by Consonni and
co-workers (Consonni et al., 2013). A total of 57 samples from Italy, Hungary, South
America and China were successfully differentiated by multivariate methods. Using 1D 1H
NMR and 2D HSQC (heteronuclear single quantum correlation) spectroscopy with spiking
experiments of standards, a total of 19 saccharides were identified. The OPLS-DA model,
built on 22 samples, resulted in satisfactory separation of various saccharide contents as
shown in figure 3.

13
 Determination of geographical origin in case of Corsican (France) and Non-Corsican
1
honey (Australia, Germany, Ireland and Italy) was carried out by using H-NMR
spectroscopy and mathematical modeling methods (Donarski et al., 2008). A total of 182
samples from 10 different regions of five countries were analyzed at 500 MHz NMR
spectrometer using a cryoprobe. The three supervised statistical approaches, projection to
latent structures-linear discriminant analysis (PLS-LDA), two-stage genetic programming
(two stage GP), and an innovative combination of PLS and GP (PLS-GP) were used to build
up a model for the discrimination of geographical origin of honeys. The results from the PLS-
GP model were afterwards used to conclude the existence of peaks that specify the
geographical origin of Corsican honey. 96% of samples were successfully cross validated by
using this model. The variables used in this model were transformed back to their analogous
1D 1H-NMR chemical shifts. Both 1D and 2D NMR techniques were employed for the final
identification of compounds. Certain resonances were due to trigonelline (Supplementary
figure 4). A spiking experiment confirmed the presence of trigonelline as a biomarker. It is a
plant hormone, usually present in salt-stressed plants.
In 2015, a worldwide study on honey authenticity (botanical and geographical origin)
and sugar adulteration was performed using proton-NMR profiling (Spiteri et al., 2015). In
addition to this, quantification procedures and statistical models were also defined to check
the authenticity of both mono- and multi-floral honey. The reference data set used was a
worldwide collection of more than 800 honeys, and from more than 35 countries. Typical
plant nectar markers can be used to check monofloral honey labeling. Spectral patterns and
natural variability were established for multifloral honeys, and marker signals for sugar
syrups were identified by statistical comparison with a commercial dataset of ca. 200 honeys.
Although the results are qualitative, spiking experiments have confirmed the ability of the
method to detect sugar addition down to 10% levels in favorable cases. Within the same
NMR experiments, quantification of glucose, fructose, sucrose and 5-HMF (regulated
parameters) was performed. Finally markers showing the onset of fermentation were also
described.
The chemical analysis of honey can improve the reliance of customers on certified
regional products. The geographical origin is a criterion of quality with certified description
of origin. Therefore, the geographical categorization of honeys can raise its commercial
value, and contribute to the micro economy of the region.

14
5. Controlling honey adulteration
Food industry is a rapidly growing world-wide due to increase in population and in
increase in the consumption of high quality products. In USA, almost three fourth of the daily
income is used for the purchase of food and for its preparation. However, consumption of low
quality and junk foods may cause risk to the human health. The risk increases multifold, if the
nature of the food is changed by adulteration with foreign bodies . The act of deliberately
degrading the quality of food presented for sale, either by the addition or exchange of low-
grade materials or by the elimination of various important constituent is termed as “food
adulteration”. Food is declared adulterated if cheaper substances which injuriously affect the
health are added to real product.
Fraudulent practices in food sector have been frequent since ancient times. Recently, food
fraud has become more complicated due to the use of unusual or artificial adulterants, which
has rung the alarm regarding related health risks (Krska et al., 2012).
The imbalance between increasing consumption/demand and the limited availability
of high quality honey supply has resulted in increase in price, as well as made it more prone
to adulteration. Therefore, techniques for efficient quality control and detection of
adulteration are of great importance to ensure quality and safety.
The most frequent method of honey adulteration is the addition of sucrose, which
should not be surplus 1% of its dried mass. Over feeding of bees with sucrose is also a
method of adulteration (Guler et al., 2007; Cotte et al., 2003). Other methods of adulteration
include the addition of fructose or glucose which alters the fructose/glucose ratio (1- 1.2 in
pure honey). It can be suggested that honey has been adulterated while this fructose to
glucose ratio diverge .
Bees collect the nectar mostly from the flowers of C3 plants, and to a lesser extent
from the flowers of C4 plants. Maize/corn and sugar cane belong to C4 plants, while beet,
rice, wheat and cichory belong to C3 plants. Along with the addition of low-cost sweeteners
such as corn syrup (CS), invert sugar syrup (IS), and high fructose corn syrup (HFCS), honey
adulteration with rice syrup has also been appeared in the market recently. The rice syrup
adulteration is not easy to identify by the current analytical methods.

5.1 Corn syrup adulteration

Sugar syrups derived by the C4 metabolic pathway exhibit a 13C/12C ratio (expressed
as δ13C) different from sugars derived from the C3 pathway. This ratio is near to -10% for C4

15
syrups, while themean value for honey is -25.4%. However, these values are not credible
criterion for the detection of C4 plant sugar in honey. A pure honey sample is likely to be
adulterated if the bees use C4 plants along with other plants, and the δ13C value exceeds the
limit. In a similar way, even if sugar is added to the honey, δ13C value may still be more
negative than -23.5% depending on the addition ratio and the honey sample may be declared
as unadulterated. Hence, it is necessary to consider the carbon isotope ratios of both raw
honey and protein fractions (δ13Chon and δ13Cpro) in order to detect the addition of sugar. The
minimum difference between these two values (δ13Chon-δ13Cpro) in an unadulterated honey is
expected to be -1.0%, and of C4 sugar is 7%. Recently, isotope ratio mass spectrometry
(IRMS) has been employed to measure this ratio in pine honey of Turkey (Çinar et al.,
2014). 100 different pine honey samples harvested in 3 different years (2006, 2007 and
2008) from 9 different localities in Mugla province were examined in this study. A
controlled adulteration of high fructose corn syrup (HFCS) in pure pine honey (PPH) was
carried out to estimate HFCS content in the mixture by means of 13C analyses and C4 sugar
value. For this purpose, the samples containing different amounts of HFCS + PPH were
analyzed (supplementary table 4) and C4 sugar % was calculated, based on the following
equation (Padovan et al., 2007):

C4 Sugar (%) = [(δ13Cpro - δ13Chon) / (δ13Cpro - δ13Csug)] x 100

In the above equation, δ13Cpro represents 13

C/12C ratio corrected as per standard of protein
fraction, while δ13Chon and δ13Csug represent 13
C/12C ratio of raw honey, and 13
C/12C ratio of
C4 sugar (-9.7 for corn sugar), respectively. It is clear from the results that as HFCS
concentration increased in the sample, the C4 sugar value also increased. In other words, C4
sugar value, calculated from δ13Cpro - δ13Chon, is a reliable indication of the HFCS addition in
pine honey. In another study, D/H ratio would be used to differentiate certain single-flower
varieties and application of the proposed method on adding a C4 syrup to authentic samples
showed limitation that the detection of a syrup spike was starting only at 20%, which is far
from satisfying (Cotte et al., 2007).
In another study, Fourier transform infrared (FTIR) spectroscopy was used to quantify
corn syrup (CS), high fructose corn syrup (HFCS), and inverted sugar (IS) in honeys of four
different locations of Mexico (Gallardo-Velázquez et al., 2009). The optimal calibrations for
the three adulterants were developed with partial least squares (PLS). The developed models
were validated using separate sets of data and found the standard error of prediction (SEP)

16
between 1.5–2.1; 2.1–3.0 and 1.4–2.5 for CS, HFCS, and IS respectively. It shows that these
adulterants can easily be detected and quantified in honey using this method. External
validation of the method using Mexican honeys of four various states gave 100% selectivity
with no false positive results.
Carbohydrate analysis is also a common approach to detect honey adulteration. High
performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD)
technique was used in an study on oligosaccharides, fractionated by activated charcoal, for
detecting honey adulteration by corn and high fructose corn syrups (Morales et al., 2008).
This method was able to detect honey adulterations with corn syrup down to 5%.
Adulteration with HFCS of various degrees of isomerisation (20% and 40%) was also
identified.
In another study of HFCS adulteration in honey, low field nuclear magnetic resonance
spectroscopy (LF 1H NMR) was used (Ribeiro et al., 2014b). In this study, proton relaxation
was used involving the relaxation time constants T1 (longitudinal) and T2 (transverse). T2
relaxation decay in foods is multi-exponential, indicating the presence of different water
concentrations in the foods matrices. Different water concentrations can be studied because
protons in different environments show different T2 relaxation properties. Honey is a very
complex multi-component system, and its LF 1 H NMR relaxation profile can be modeled as a
linear combination of characteristic relaxation times from the measurable hydrogens present
in their structures. The transverse relaxation time (T2) was calculated by a spin echo T2 edited
experiment known as Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence. Analysis of the
relaxation curve fitness results in multi-exponential curve and specifies that two exponentials
were adequate to explain the whole system in all samples. This bi-exponential fitting hence
suggested two water concentrations, by means of equivalent relaxation times T21 and T22. The
ranges of the honey samples, adulterated with HFCS, and of unadulterated pure blossom
honey were analyzed by LF-NMR data (supplementary table 5). Comparison of the
continuous distributed curves showed noticeable diversity in the distribution of water
mobility among different percentages of adulterated honeys. The 100% adulterated samples
tend to show a slightly broader T2 distribution than other adulterations. This behavior can be
explained on the basis of high water activity in fructose syrup. By using biexponential fitting
of the transverse relaxation data (Supplementary figure 5), it was possible to discriminate
between the two water populations in all samples, one with a faster relaxation time T21 in the
range of 1.26–1.60 ms, and another with a slower relaxation time T22 in the range of 3.33–

17
7.38 ms, depending on the percentage of adulteration with high fructose syrup. T21 was
slower in pure blossom honey (i.e. 1.60 ms), indicating that water mobility was lower in
unadulterated honey as compared to the adulterated honey. The results indicate that
adulterated honey with HFCS can be correctly discriminated from pure blossom honey by
using LF 1 H-NMR as the relaxation times were significantly decreased by increasing the
concentration of the adulterant in pure honey.

5.2 Beet syrup adulteration

The carbon isotope ratio method for honey adulteration detection does not properly
work when honey is adulterated with C3 plant syrups because nectars bearing flowering
plants are more or less entirely of C3 plant origin. Beet is also a C-3 plant and therefore is not
easy to identify by the carbon isotope ratio method. However, deuterium to hydrogen ratio of
sugars varies in C3 plants. Therefore, hydrogen isotope ratio can be used for detection of
adulteration of honey from a sugar of C3 plant.
An FT-Raman approach was used by Paradkar and Irudayaraj to anticipate the degree
and the type of adulteration in honey (Paradkar and Irudayaraj, 2001). Various quantities of
invert beet and cane syrups was added to the pure honey samples of clover, orange and
buckwheat. FT Raman spectrum of the adulterated samples in the region between 200 and
1600 cm─1 was used for the generation of model. PLS and PCA were applied for
quantification, whereas PLS-DA and canonical variate analyses (CVA) were applied for
discriminant analysis (Supplementary figure 6). FT-Raman spectroscopy was found to be an
efficient technique in predicting the adulteration with beet and cane invert (R2>0.91) in
clover, orange and buckwheat honeys. CVA model yielded satisfactory classification rate
with 96% accuracy for honey adulteration.

5.3 Rice syrup adulteration

Another C3 syrup adulterant which is becoming very common nowadays is rice
syrup. Rice syrup is very complex to identify by current analytical methods. It is not probable
to identify rice syrup by common stable carbon isotopic ratio analysis (SCIRA), as it also
belongs to C3 plants. Moreover, in the process of rice syrup production of rice syrup,
different polysaccharides and oligosaccharides were hydrolyzed. This makes it tricky to
identify the presence of rice syrup via thin-layer chromatography (TLC) and high-
performance anion exchange chromatography with pulsed amperometric detection

18
(HPAEC−PAD). Currently honey adulteration with rice syrup has become a serious trouble
both in terms of quality assurance, and safety of the product.
In 2013, a biomarker from rice syrup adulteration was identified by Xue and co-
workers (Xue et al., 2013). Thirty two rice syrup samples and 160 natural honey samples
were analyzed by high performance liquid chromatography with diode array detection
(HPLC−DAD); the comparative chromatogram is shown in Figure 4. The characteristic
compound in figure was identified as 2-acetylfuran-3-glucopyranoside (AFGP) with the help
of NMR and MS analyses. 186 honey samples from various origins were analyzed for the
determination of AFGP using an easy and rapid HPLC−DAD method. The developed method
showed limit of detection of 10% rice syrup adulteration in honey.
Three-dimensional fluorescence spectroscopy (3DFS) was also found to be a powerful
analytical tool that can provide fluorescence information of mixed materials. The 3DFS
technique could be used to detect rice syrup adulterant concentration in honey. In recent
years, Chen et al. systematically studied different multivariate calibration methods for the
analysis of 3DFS (Chen et al., 2014). In this study, two different multivariate calibrations,
back propagation artificial neural network (BP-ANN) and partial least squares (PLS), were
used as discrimination models. Finally BP-ANN was selected as the optimal model for
detecting honey adulteration by rice syrup (Supplementary figure 7).

5.4 Inulin syrup adulteration

Inulins are naturally occurring polysaccharides, belong to a class of dietary fiber
known as fructans. Basically fructans are polymers of fructose with a terminal glucose. The
linkage position of the fructose residues determines the type of the fructan. In inulin, the
fructosyl residues are linked through β-2,1-linkages. It is commonly present in wheat, onion,
bananas, garlic, asparagus, sunchoke and chicory; however, syrups of various levels of
polymerization are also formed commercially by incomplete enzymatic hydrolysis of inulin.
Recently a gas chromatographic mass spectrometric (GC-MS) method is developed for the
detection of honey adulteration with low level of high fructose inulin syrups (HFIS) (Ruiz-
Matute et al., 2010). It is clearly shown from the profile of the adulterated sample in
supplementary figure 8 that the major variations were appeared in the trisaccharide zone.
Inulotriose was not detected in any honey sample and appeared in high concentrations in all
HFIS, therefore it could work as the finest marker for detecting inulin syrup adulterations.

19
The detection limit (LOD) was 0.03 mg g-1 honey, which would represent that an addition of
less than 1% syrup can be detected by this method.
In another study, a method was developed to detect adulteration in honey falsified by
intentional addition of three different concentrations of seven commercial sugar syrups, using
one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) coupled
with multivariate statistical analysis (Bertelli et al., 2010). Sixty-three authentic and 63
adulterated honey samples were analyzed. The best discriminant model was obtained by 1D
spectra, and leave one-out cross-validation showed a predictive capacity of 95.2%. 2D NMR
also furnished acceptable results (cross-validation correct classification 90.5%), although the
1
H NMR sequence is preferable because it is the simplest and fastest NMR technique.
All the techniques presented above have their advantages and disadvantages with
reference to the technique itself or with the honey authentication and adulteration. Table 1
shows a summary of key merits and demerits of the various techniques. While table 2
summarizes the sample preparation strategies, pulse sequence and other NMR parameters
used in different studies. Generally, honey sample was simply diluted in solvent or pH was
adjusted using phosphate buffer. Majority of the studies involve standard pulse sequence for
1
H NMR spectroscopy. However, for a modified pulse sequence liquid-liquid extraction via
chloroform was used for sample preparation. Overall, NMR spectroscopy provides simple
and fast methods of sample preparation to analysis for food analysis.

6. Conclusion:

In the current global manufacturing and marketing, food authentication is an important

challenge for ensuring better quality food. Among modern techniques, 1H-NMR spectroscopy
is an important tool for mixture analysis. In combination with chemometric techniques, the
application of NMR has largely improved the capability for sample classification. Moreover,
the NMR technique has a major advantage as it allows the distinction and assignment of
inconsistent signals to specific molecular markers. Besides this, the NMR allows
quantification of various sugars simultaneously in order to estimate the correct level of
adulteration with high precision and accuracy. The easy sample preparation and the high
quality outcomes in NMR spectroscopy represent a suitable substitute to other complex and
protracted analytical techniques. Unfortunately, due to the high cost of instrumentation and
the complex nature of the technique, use of NMR is relatively less common and only

20
available used in large industries and R&D institutions. It is expected that in the coming
years, the availability of bench-top NMR instruments, and falling price of routine NMR
instruments (300 MHz) will help to boost up this technique in honey authentication. It is
therefore likely that the use of NMR techniques will be more common in the future as a
routine technique for the analysis of honey.

Conflict-of-interest disclosure: All the authors declare no competing interests relevant to

this study.

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25
Figure legends

Figure 1: Scatter plot of the PCA scores of different types of unifloral honeys, obtained from
1
H-NMR (9–0.25 ppm; Pareto scaling (a)) and 13C-NMR (200–0.25 ppm; no scaling (b)).
Figure 2: Hierarchical PLS-DA performed using 13 polyfloral honeys of different origins
(training set) with reprojection of polyfloral test set sample scores (10 samples). Filled
symbols represent training set honey samples from Hungary (H, diamond), Italy (I, circle),
and Argentina (RA, triangle), while open symbols represent test set honey samples from
Hungary (H, diamond), Italy (I, circle), and Argentina (RA, triangle) from different EC
countries (star), and from different EC and non-EC countries (inverted triangle).
Figure 3: Score plot of OPLS-DA performed by using 22 multifloral honey samples: filled
boxes, dots, triangles, and diamonds represents Chinese, Hungarian, Italian and South
American multifloral honey samples, respectively. R2X ¼ 91.5%, R2Y ¼ 85.5% e Q2 ¼
73.2%.
Figure 4: Chromatograms of rice syrup and natural honey. Arrow indicates 2-acetylfuran-3-
glucopyranoside (AFGP) (marker) of rice syrup.

26
Table 1: Key merits and demerits of the discussed techniques.
Technique Merits
Melissopalynological Simple or no sample preparation;
analysis and other best for uni-floral honeys of same
physicochemical geographical origin
parameters detection
Protein characterization To identify the honey bee origin
Chromatographic analysis Complex, volatile and non-
volatile, and wide variety of
analytes are readily analysed
High-performance anion- did not require derivatization;
exchange shorter total analysis time
chromatography with
pulsed amperometric
detection (HPAEC-PAD)
Front phase fluorimetric Botanical origin of polyfloral
spectroscopy honeys can be identified easily;
highly sensitive in comparison to
other spectroscopic technique
Fourier Transform Botanical origin of polyfloral
Infrared spectroscopy honeys can be identified easily;
short analysis time
Fourier transform raman No water interference and
spectroscopy minimal fluorescence
interference; detect adulteration
from the same plant source
Isotope ratio mass wide applicability and versatility
spectrometry (IRMS) to be coupled with several
different interfaces
Inductively coupled In comparison to other atomic
plasma-mass spectroscopic techniques, it
spectrometry provides multi-element analysis
with low detection limits
Nuclear magnetic Fingerprint technique so easy to
resonance identify a specific biomarker for
a class of sample; minimal
sample processing; non-
destructive nature;
27
Demerits
Wide range of thresholds;
could not work for honey
from close geographical
zones
Artificial marker protein
standard of honey has to be
prepared.
Honey origin is difficult to
be identified
Need specialized equipment
to handle to high-pH mobile
phases; no method flexibility
to resolve an interfering
peak
Geographical origin
estimation could not be done
accurately
Geographical origin
estimation could not be done
Aqueous, dark colored
samples at high temperatures
increase interferences
Lack of availability of IRMS
standards and standardized
methods; not suitable for
routine analysis
Exogenous addition of
sugars could not be
identified
Extensive chemometric
analysis is required which
makes it complicated for
routine analysis
Table 2: Summary of sample preparation and NMR parameters of honey sample analysis using NMR
spectroscopy
S. Sample NMR parameters
Aim of the study Reference
No. Preparation Pulse sequence NMR parameters
1 Solid phase DOSY using 16k data points,32 To investigate the (Beretta
extraction using bipolar gradient spectra in the second quantitative profile of et al.,
silica-bonded C- pulses for dimension, scans 24; Kynurenic acid in honeys 2009a)
18 phase diffusion relaxation delay 1 s; from different botanical
measurements eddy current delay (Te) sources
5 ms
2 Honey dilution in (1) 1H NMR Data points 16K, To detect adulteration in (Bertelli
solvent (DMSO) spectra acquisition time1.71s, honey falsified by et al.,
using the delaytime 10s, scans, 8, intentional addition of 2010)
presaturation spectral width 2003.205 different concentrations
technique for Hz, 10 s of commercial sugar
water signal HMBC delay time syrups
suppression
(2) HMBC
3 Honey dilution in low power water spectral width 4664 Hz, To discriminate the (Boffo et
solvent (D2O) signal 65,536 data points, pulse monofloral honeys al., 2012)
suppression width 8.5 µs, relaxation produced in the state of
delay 1.5 s, acquisition São Paulo.
time 7.0 s, 64 scans
1
4 Honey dilution in H NMR spectra 7500 Hz spectral width geographical (Consonn
solvent (D2O) using the and 32 k of data points differentiation of i and
presaturation polyfloral and acacia Cagliani,
technique for honeys from different 2008)
water signal countries
suppression
1
5 Honey dilution in H NMR spectra 7500 Hz spectral width Determination of the (Consonn
solvent (D2O) using the over 32 K data points saccharide contents of i et al.,
presaturation Italian honeys of 2012)
technique for different botanical
water signal origins
suppression

6 Honey dilution in HSQC 7500 Hz and 31,400 Hz To distinguish Italian (Consonn

solvent (D2O) of spectral width for 1H multifloral honey from i et al.,
and 13C nuclei those of other countries 2013)
respectively, with 512
experiments over 2K
points
7 Fermentation of SNIF-NMR for acquisition time 6.8 s To create databases on (Cotte et
honey sugars and the determination with spectral bandwidth the isotopic al., 2007)
extraction of of (D/H) ratio 1205 Hz characteristics of the
ethanol honey of certain floral
varieties
8 Water dilution and 1D NOESY with 128 scans of 64 K data Simultaneous (del
pH adjustment water signal points spectral width of quantification of thirteen Campo et
suppression 8012 Hz, recycle delay analytes in monofloral al., 2016)
of 2.0 s honey
1
9 Honey dilution in H NMR on 14 s delay time, 32K data Geographical (Donarsk
solvent (D2O) and cryoprobe using points with spectral discrimination of i et al.,
pH adjustment by the presaturation width of 14 ppm Corsican and non- 2008)
phosphate buffer technique Corsican honey
1
10 Honey dilution in H NMR on 14 s delay time, 32K data Identification of (Donarsk
solvent (D2O) and cryoprobe using points with spectral botanical markers of i et al.,
pH adjustment by the presaturation width of 14 ppm Corsican honey 2010a)

28
 phosphate buffer technique
11 Honey dilution in DOSY NMR Delay time 3 s, Application of DOSY (Gresley
solvent (D2O) WATERGATE WATERGATE pulse NMR as a technique for et al.,
sequence duration was 1000 µs the separation of manuka 2012)
(STEBPGP1S19) with 64 linear gradient honey components
. steps
12 Freeze drying and (1) 1H NMR (1) data points was 16K, Classification of Italian (Lolli et
reconstitution into spectra with the delay time 10 s, Honeys by HR-NMR al., 2008)
D2O for 1 H and presaturation spectral width 2003.205
DMSO for 13C (2) 1H-13C Hz
HMBC (2) 4K datapoints for 1H
and 256 13C; delay time,
1.0 s; HMBC delay time,
65 ms.
1
13 Honey dilution in H NMR spectra 64 transients with a To study the chemical (Mannina
solvent (D2O) using the recycle delay of 3 s. composition of different et al.,
presaturation botanical origin honeys 2015)
technique for produced by Sicilian
water signal Black honeybees
suppression
14 Honey dilution in NOESY- 64 scans, 65 k points, Botanical origin (Ohmenh
solvent (D2O) and presaturation spectral width authenticity and aeuser et
pH adjustment by pulse sequence 19.9914 ppm, receiver quantification of 13 al., 2013)
phosphate buffer gain 22.6, acquisition analytes
time 4.096 s.
15 No sample Carr–Purcell– 10 scans, 256 points, To investigate changes in (Ribeiro
preparation Meiboom–Gill 100 ms between scans, the distribution of water et al.,
(CPMG) pulse and 100 µs between using LF 1H NMR in 2014a)
sequence pulses of 90° and 180° comparison with
physicochemical
measurements of
different floral origins
honey
16 No sample Carr–Purcell– 10 scans, 256 points, To investigate (Ribeiro
preparation Meiboom–Gill 100 ms between scans, adulteration by HFCS et al.,
(CPMG) pulse and 100 µs between using LF 1H NMR and 2014b)
sequence pulses of 90° and 180°. physicochemical
analytical methods.
17 Liquid-liquid modified double- 64K points, spectral monofloral and polyfloral (Schieva
extraction via pulsed field width 14ppm, recovery honeys authentication no et al.,
chloroform gradient spin delay 2 s 2016)
echo sequence
18 Liquid-liquid modified double- 64K points, spectral To isolate and identify (Schieva
extraction via pulsed field width 14 ppm, relaxation compounds of honeys no et al.,
chloroform gradient spin time 2 s responsible for statistical 2013)
echo sequence discrimination of
different botanical
origins
19 Liquid-liquid modified double recycle time 2 s, spectral monofloral and polyfloral (Schieva
extraction via pulsed field window 6000 Hz; scans honeys marker no et al.,
chloroform gradient spin 256, data points 32K, identification and 2010)
echoes receiver gain 8K authentication
(DPFGSE)
sequence
20 Liquid-liquid modified double 64K points, spectral To create an NMR (Schieva
extraction via pulsed field width 14 ppm, relaxation spectra library of no et al.,
chloroform gradient spin time 2 s authentic monofloral and 2012)
echoes polyfloral Italian honeys
(DPFGSE)
29
 sequence
1
21 Honey dilution in H NMR spectral width 10 ppm, To differentiate oak (Simova
solvent (D2O) 32 scans, 32 K data honeydew honey from et al.,
points, relaxation delay other honey 2012)
10 s,
22 Honey dilution in NOESY- 64 scans, 65 k points, data fusion from NMR (Spiteri
solvent (D2O) and presaturation spectral width 20 ppm, and HRMS (TOF-MS et al.,
pH adjustment by pulse sequence receiver gain 32, and Orbitrap-MS) for the 2016)
phosphate buffer acquisition time 4.096 s botanical classification of
honey
23 Honey dilution in NOESY- 64 scans, 65 k points, botanical and (Spiteri
solvent (D2O) and presaturation spectral width 20 ppm, geographical origin et al.,
pH adjustment by pulse sequence receiver gain 32, authenticity 2015)
phosphate buffer acquisition time 4.096 s,
recycling delay 8 s,
mixing time 0.01 s
24 Honey dilution in water suppressed 32 K data points, Qualitative and (Zheng et
solvent (D2O) and NOESYPR1D 6000-Hz spectral width, quantitative analysis of al., 2016)
pH adjustment by pulse sequence acquisition time 2.5 s, Chinese honeys from
phosphate buffer recycle delay time 3.0 s, different botanical and
32 scans geographical origin
25 Honey dilution in Standard 1D 1H relaxation delay 3.5 s, To determine the (Zielinski
solvent (D2O) and NMR spectra 256 scans, 32K data botanical origin of polish et al.,
pH adjustment by with water points, 20.55 ppm monofloral and polyfloral 2014)
HCl suppression spectral width honeys

30
Figure 1
Figure 2
Figure 3
Figure 4
Highlights:

• Honey is prone to adulteration foodstuff due to its various medical uses.

• Various analytical methods are introducing in honey authentication and adulteration.

• Specially NMR combined with chemometric techniques improved the sample classification.

• NMR allows the confirmation of marker compounds specific to honey and adulterants.

31