Talanta Open 3 (2021) 100022

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Talanta Open
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Potential of FTIR- ATR diamond in discriminating geographical and

botanical origins of honeys from France and Romania
François Guyon a,b,∗, Enora Logodin a, Dana Alina Magdas c,d,∗∗, Laetitia Gaillard a
a
Service Commun des Laboratoires, 3 avenue du Dr. Albert Schweitzer, 33600 Pessac France
b
Service Commun des Laboratoires, 146 Traverse Charles Susini, 13388 Marseille, France
c
National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat Str., 400293 Cluj-Napoca, Romania
d
Cluster Agro-Food-Ind Napoca, Cluj-Napoca, Romania

a r t i c l e i n f o a b s t r a c t

Keywords: Soft independent modeling class analogy (SIMCA) was applied to infrared spectra, obtained by attenuated total
FTIR-ATR reflection – Fourier transform infrared (ATR - FTIR) spectroscopy, to develop discrimination models for the flo-
Honey ral and geographical origin of honeys. Mono-floral honey samples, originated from France and Romania, were
Mono-floral
analyzed for this purpose. The meaningful spectral areas which were selected for the development of discrim-
Geographical origin
inating models were: 500 - 1610 and 1700 - 3000 cm−1 . A second derivative and an “autoscale” processing
Multidimensional analysis
was performed to experimental spectra before the statistical classifications. The honeys varieties were accurately
classified, independently of the geographical origin of honeys, suggesting the high potential of this technique
for botanical recognition. The obtained interclass distances among honey varieties were significant and the main
identified discrimination markers, from FTIR spectra, are especially related to the floral origin through the pollen
gathered by bees. A global geographical classification of honeys provided a correct classification with a weak in-
terclass distance showing the model limitations for the authenticity control purposes. Nonetheless, geographical
classifications inside a single class of honey variety lead to a correct classification with a significant interclass
distance.

1. Introduction
Honey is a natural sweet commodity produced by bees – Apis Millifera
– from the plants (nectar or plant secretion). Honey contains about 75%
of carbohydrate, mainly glucose and fructose and very small fraction of
oligosides. The remaining 20% are formed by water (15–17%), proteins
(0.1 - 3.3%), organic acids (0.6%) and a high number of compounds at
trace level (i.e. mineral, volatile compounds) [1]. Honey contains more
than 200 substances [2].
Honey is appreciated by consumers for its sweetness and taste. Those
features are mainly linked to the botanical origin of the plants involved,
as raw materials, in honey elaboration. Because of the increasing envi-
ronmental and sanitary concerns, some consumers are looking for prod-
ucts bought through short producing chains, paying an intense attention
to the regional food commodities. Therefore, a rigorous control of foods
botanical and geographical origin become mandatory. From the com-
mercial point of view, the honey pot price is related to country origin
and, within a same country origin, to specific regions but also to the
honey floral origin; the floral origin brings added value to the product
through the specific aroma and taste as well as for specific medicinal
virtues [3]. Another important aspect is related to the honey chemical
composition which requirements are listed in the Codex Alimentarius
and in a EU regulation which specify authorized limits for some com-
pounds as well as the mandatory information to be provided on the label
[4, 5].
For many reasons, honey is a potential candidate for falsification.
Among these reasons, it can be mentioned here, the decrease of the
production level, the high cost of production in some countries, the
specificities of some honeys [6] and consumers’ interest for particular
botanical and geographical origins. Sixty percent of honey consumed
in France is represented by imported honey. In 2019, Romania has the
largest honey production within the European Union (EU) and represent
3% of French honey importation [7].
Because of the highest market demand and the limited available
quantities of honey, different types of falsification can be found. From
these, the most commune are related to: partially or totally product sub-
stitution, mixture of products or wrong floral and geographical declara-

∗
Corresponding author at: Service Commun des Laboratoires, 3 avenue du Dr. Albert Schweitzer, 33600 Pessac France.
∗∗
Corresponding author at: National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat Str., 400293 Cluj-Napoca,
Romania.
E-mail addresses: labo33@scl.finances.gouv.fr, guyonfra@gmail.com (F. Guyon), alina.magdas@itim-cj.ro (D.A. Magdas).

https://doi.org/10.1016/j.talo.2020.100022
Received 28 September 2020; Received in revised form 13 November 2020; Accepted 14 November 2020
2666-8319/© 2021 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
F. Guyon, E. Logodin, D.A. Magdas et al.
tion. In order to detect unfair practices, various markers have been in-
volved to check honey authenticity like: stable isotopes [8-10], mineral
concentration [11] and more recently 1 H NMR [12–14]. Other studies
used combination of various sources of data, but in all cases the use
of multidimensional analysis for an advanced data treatment became
essential [15].
Because of the fast commercial exchanges, nowadays control author-
ities require fast, portable and environmentally friendly techniques to
allow the control of imported honey on site i.e. at the country border
and on the domestic market. Spectrophotometric techniques present all
the previous required characteristics: the spectral information can be
discriminant, some systems have been miniaturized and the direct anal-
ysis, without sample preparation, makes them environmentally friendly
techniques. Some previous studies applied these techniques to honey
matrices with different purposes, starting from the detection and quan-
tification of some specific compounds to the classification of honeys.
Honey composition was quantified using infrared spectra and partial
least square model [16]. Mid Infrared (MIR) has been used for qual-
ity control measurements [17] and adulterations detection [18]. Honey
botanical origin was assessed by a targeted approach of the IR spectra
[19, 20]. For most of the spectroscopic studies, honey samples were geo-
graphically localized at the country level like Mexico [21], Switzerland
[22], Turkey [23, 24], Uruguay [25].
In this study, the impact of longer distance was envisaged by the
analysis of 103 mono-floral honeys from France and Romania spread
over 7 main floral origins. Part of this work was devoted to the model
elaboration by selecting the discriminant parts of the spectra obtained
by attenuated total reflection – Fourier transform infrared (ATR - FTIR)
and the optimization of the data treatment. Then, two models were con-
structed to evaluate the discriminating potential of FTIR spectra for the
differentiation of: i) 7 honey varieties independently of their geographi-
cal origin and ii) French from Romanian honeys, independently of their
floral origin.
2. Material and method
2.1. Samples
For this study, 103 authentic honey samples, collected during 2018
harvest and directly from producers located in France (53) and in Roma-
nia (50), were used. French and Romanian honey samples were collected
all around the countries. According to their floral origin and country,
honey distribution was for France (acacia −18; linden - 3; colza - 6; sun-
flower - 6; lavender - 7; chestnut - 5; coriander - 2; other mono-floral
honeys - 6) and for Romania (acacia −17; linden - 10; colza - 6; sunflower
- 6; coriander - 1; other mono-floral honeys - 10). The mono-floral origin
was confirmed by melissopalynology analysis which is, up to now, the
recognized method for pollen analysis.
2.2. FTIR-ATR spectroscopy
Fourier transform MIR spectra were recorded on a Nicolet iS5
(Thermo Scientific, Waltham, Massachusetts, USA) equipped with a
thermic detector DTGS (Deuterated triglycerine sulfate) and an ATR di-
amond accessory. The system resolution was 0.8 cm−1 . At room temper-
ature, samples were placed on the cell measurement and scanned from
4000 to 500 cm−1 . For each sample, 32 scans were run and the mea-
surements were performed in duplicate to check the honey homogene-
ity. Data were extracted from the Omnic software (Thermo Scientific,
Waltham, Massachusetts, USA) and then transformed in a Galactic file,
readable by the data treatment software, using MestReNova software
(Mestrelab Research, Santiago de Compostela, Spain).
Honey homogeneity was checked on 10 pots (diameter: 8 cm; height:
10 cm), containing solid honeys, performing thus a depth profile of the
pot. FTIR-ATR measurements were performed on the first honey layer,
then the equivalent of 4 cm of honey were taken off and a new analysis
2
Talanta Open 3 (2021) 100022
was performed at this level, this measurement was repeated after a new
removal of 4 cm. These 10 samples were also melted at 43 °C for two
hours, being then manually homogenized before analyses.
2.3. Multidimensional analysis
Pirouette software (Infometrix, USA) was used for multidimensional
data treatment. The matrix is divided in three parts: the first one cor-
responds to the sample identification, the second one to the variables
(spectral intensities obtained for each honey), and the third one to the
class variable (Romania and France for the geographical classification
and each mono-floral type for the botanical classification). The spec-
tral range included in the model corresponds to the full recorded range
500–4000 cm−1 from which, two areas were excluded: 1610–1700 and
3000–4000 cm−1 . A second derivative was firstly applied to the selected
data set, followed by an “autoscale” preprocessing which corresponds
to a mean-centering and a subsequent variance scaling of the signal. A
soft independent modeling classification analogy (SIMCA) was applied
to this set of data using a factor number of 2 and a local scope. SIMCA
is a supervised classification method indicating that the mathematical
algorithm will check for similarity among classes to find discriminat-
ing parameters. A leave one-out cross validation (LOOCV) was used for
the evaluation of the model accuracy [26]. In LOOCV, a sample is ex-
cluded from the matrix, and an intermediate model is created. The ex-
cluded sample is tested with this intermediate model to observe its class
prediction. This step was automatically repeated for all samples of the
matrix providing a misclassification table. The table diagonal is given
by the number of correctly classified samples revealing the potential of
the model to distinguish among different classes. The second provided
information was the interclass distance, which is directly related to the
power of the discrimination model. When this distance is small, reliable
classifications could be difficult to obtain. A distance lower than “1″
shows a low capability of the model to distinguish among the classes,
while a distance of “3″ or higher reveals the model potential for food
control applications [27].
3. Result and discussion
3.1. Experimental data preprocessing and optimization
The application of a model for laboratory routine analysis as well as
for field quality control is linked to the matrix homogeneity, particu-
larly when it is a solid matrix as honey. In the preparation process, hon-
eys are melted and homogenized before commercialization. Nonethe-
less, the “spectroscopic” homogeneity was check on 10 honey pots con-
taining solid honeys. FTIR-ATR measurements, performed at different
depth profile of the pot and after a mild heating homogenization, did
not show any significant differences, all spectra were fully reproducible,
confirming the expected homogeneity of honeys.
For the development of reliable recognition models, in addition to a
representative sample set and accurate measurements, two main issues
need to be focus on: data preprocessing and identification of meaningful
spectral range. In order to address the first issue, various data process-
ing (none, autoscale, variance and range scale, Pareto), available under
Pirouette software, were applied to the entire spectral range from 500 to
4000 cm−1 after a second derivative treatment of the transmittance val-
ues (Fig. 1). Based on the interclass distance, discriminating and mod-
eling power, the best preprocessing treatment was proved to be the “au-
toscale” preprocessing which consists in a mean-centering followed by
a variance scaling. Data selection is a critical step in multidimensional
analysis, particularly in spectroscopy, where large data sets are gener-
ated and also, in most of the cases, the recorded differences among the
spectra are very subtle. In this case, the exclusion of some spectral areas
with high transmittance intensity but low discrimination potential can
provide a better classification. These particular areas are linked to the
water absorption bands. The main water bands are centered at 3920,
F. Guyon, E. Logodin, D.A. Magdas et al.
Fig. 1. FTIR spectra of the 103 honeys where the excluded areas of the model
are in gray (A). Graphs B and C represent the discriminating wavelengths power
for the mono-floral (B) and geographical (C) models.
3490 and 3280 cm−1 for O–H stretching and 1645 for H–O-H bending
vibrations, respectively [28]. Moreover, some spectral areas have no
meaningful discriminant potential due to the absence of the signal, as is
the case of the spectra tail after 3700 cm−1 . For this reason, a spectral
range selection was performed with the aim of classification improve-
ment. As illustrated on Fig 1, the data treatments were performed on the
spectra after excluded two wavelength ranges: 1610 – 1700 cm−1 and
3000 – 4000 cm−1 (ranges marked in gray correspond to the excluded
spectral areas). Based on above mentioned considerate, all honey spec-
tra (103 samples) were preprocessed by applying a second derivative
and an “autoscale” processing, followed by a soft independent model-
ing of class analogy (SIMCA) which is a supervised classification method
(Fig. 2).
3.2. Botanical origin model
For this classification, only honeys elaborated from the gathering of
flowers pollens and able to create a first draft of a mono-floral group
were considered to estimate the potential of FTIR-ATR diamond spec-
troscopy for this purpose. A class variable was arbitrarily attributed to
each honey, according to its floral origin and independently of its ge-
ographical origin. The LOOCV results are listed in Table 1. A correct
classification was obtained for all mono-floral honeys. Near infra-red
spectra are linked to the chemical composition of the honey, which is
further related to the floral origin. The algorithm of the multidimen-
sional software is able to detect and discriminate each kind of mono-
floral honey which was used in this study. The information provided
by the interclass distance (Table 1) is useful to evaluate the separation
degree among the classes, the higher the better. According to the ob-
tained separation for coriander honey, it was observed that this variety
is very specific as compared to other honey sorts. This fact is not really
surprising taking into account its carrot-like color, probably a result of
the high carotene content of the plant itself [29, 30], which makes it
3
Talanta Open 3 (2021) 100022
Fig. 2. Three dimensional representation of botanical classification according
to honeys’ geographical origin. Solid and light symbols: Romanian and French
honeys respectively, acacia (A) and sunflowers (B) honeys.
visually significantly different from other honeys. Nonetheless, the set
of coriander honeys needs to be significantly enlarged in order to have
a clear repartition of this mono-floral type of honey. The model clearly
separates chestnut variety from colza and lavender honeys. The color
difference among honeys cannot explain the classification as, for exam-
ple, the interclass distance between chestnut and acacia or sunflower
is significantly lower. The classification among sunflower, acacia and
colza is not really efficient for control purposed as the interclass dis-
tance is lower than 1. The number of samples should be increased to
confirm whether or not MIR spectrum contains enough discriminant in-
formation to allow the classification among these three different floral
honeys.
Fig. 1B is a representation of the discriminating power of each wave-
length of the FTIR-ATR spectra of honeys. Wavelength attribution to a
specific molecule is not straight forward by IR, particularly in a complex
mixture like honey. This tentative assignment was established based
on the available honey literature. The most powerful predictors (dis-
crimination power >40), which allowed the botanical origin classifi-
cation were located in the spectral range situated between 880 and
1600 cm−1 . These results are in a good agreement with other previous
works [23] which obtained the best classification of Anatolian honeys
in the spectral area from 750 to 1800 cm−1 . It was previously reported
[16] that the most sensitive absorption region of the major constituents
from honey like sugars and organic acids is located between 750 and
1500 cm−1 while, the region situated between 900 and 1500 cm−1 con-
tains absorption bands specific to sucrose, glucose ad fructose.
F. Guyon, E. Logodin, D.A. Magdas et al. Talanta Open 3 (2021) 100022

Table 1
Result of the model LOOCV classification (C) of mono-floral honeys independently of the
geographical origin; in parenthesis, the interclass distance.

CAca CChest CCol CLav CLin CSun CCor No Match %

Acacia 35 0 0 0 0 0 0 0 100
Chestnut (2.0) 5 0 0 0 0 0 0 100
Colza (1.6) (3.1) 12 0 0 0 0 0 100
Lavender (2.0) (4.0) (1.2) 7 0 0 0 0 100
Linden (1.3) (1.7) (1.2) (1.5) 12 0 0 0 100
Sunflower (0.7) (2.3) (0.9) (1.2) (1.0) 12 0 0 100
Coriander (5.3) (11.1) (5.4) (4.9) (6.0) (4.7) 3 0 100

Abbreviations: Aca: Acacia; Chest: Chestnut; Col: Colza; Lav: Lavender, Lin: Linden; Sun:
Sunflower; Cor: Coriander.

Table 2 Table 3
Result of the LOOCV model classification according to the 4 main Result of the model LOOCV classification (C)
botanical origins after the exclusion of a test set, in parenthesis, the according to honey geographical origin inde-
interclass distance (A) and classification prediction obtained by the pendently of the floral origin; in parenthesis,
model on the test set formed by the randomly excluded samples (B). the interclass distance.

(A) CAca CCol CLin CSun No Match % CRo CFr No match %

Acacia 28 0 0 0 0 100 Romania 53 0 0 100

Colza (1.8) 9 0 0 0 100 France (0.4) 50 0 100
Linden (1.3) (1.5) 9 0 0 100
Sunflower (0.8) (1.2) (1.4) 9 0 100 Abbreviations: Fr: France; Ro: Romania.
(B) CAca CCol CLin CSun No Match %
Acacia 5 0 0 0 0 100 3.3. Geographical origin model
Colza 0 3 0 0 0 100
Linden 0 0 3 0 0 100
Sunflower 0 1 0 2 0 67 The model was constructed considering the entire set of honey and
two compared groups (class variable): France and Romania. The LOOCV
Abbreviations: Aca: Acacia; Col: Colza; Lin: Linden; Sun: Sunflower. results are listed in Table 3. A high classification percentage was ob-
tained as all the honeys were attributed to the correct country. This
result suggests that there are discriminant parameters providing this ac-
In the present work, the best discrimination potential proved to have, curate classification. Nonetheless, the interclass distance (Table 3 ) was
in this order, the spectral ranges: 1580–1600; 1075–1195 and 1450– found to be 0.4 which is below the value of 1 indicating that beside
1458 cm−1 . The 1580–1600 cm−1 region corresponds to C=O vibration, the LOOCV result, there is not enough discrimination power for control
mainly of carbohydrates [21]. In the spectral area situated around 1075 purposes between the two classes. This is verified by the discriminating
−1195 cm−1 , characteristic signals of C–O stretching vibration of phe- power of the FTIR spectra wavelengths presented on Fig. 1C. The dis-
nolic compounds [31] as well as to C–C and C–O stretching vibrations of criminating wavelengths are similar as in the previous classification, all
lipids from pollen [20] were identified. The transmittance band around in the same magnitude range but with a discriminating power that is 10
1450–1458 cm−1 was previously attributed to the combination of O–H times lower as compared with the one obtained for botanical differen-
bending of the C–OH group and C–H bending of the alkenes [21]. Other tiation. This result confirms previously reported results which claimed
two areas, with a discrimination power higher than 40, were found to that MIR potential is limited for geographical classification of honeys
be 1305–1310 and 880–900 cm−1 . The discriminant region, situated [22].
around 1300 −1310 cm−1 , can be attributed to the amide III (the in- Tentatively, alternative classifications were performed considering
phase combination of N–H deformation and C–N stretching vibration) one floral origin for both geographical origins. The classification re-
[20]. According to some previous works [32], the spectral range sit- sults of 3 mono-floral honeys (acacia, colza and sunflower) are listed
uated between 750 and 900 cm−1 is specific for the anomeric region in Table 4. A correct classification is obtained for the 3 mono-floral
characteristic of the saccharide configuration. honeys with an interclass distance of 1.1 for acacia, 2.6 for colza and
As a result, it appears that the floral discrimination is based on carbo- 2.1 for sunflower. These interclass distances are much higher than the
hydrate content, lipids from pollen, phenolic compounds and amino acid one obtained for the global geographical model (0.4) which considers all
composition. This result is not surprising because it has been demon- the floral origins. The differentiation of acacia honey originated from the
strated that phenolic compounds are linked to the flower type [33, 34]. two countries was mainly based on the signals from 876, 1056 and 1182
Pollen is another characteristic of botanical origin, being previously re- cm−1 . The signal from 876 cm−1 could be attributed to saccharide con-
ported that its chemical composition distinctly differs from one plant figuration [32] while, the bands at 1056 and 1182 cm−1 were attributed
species to another [35, 36]. to C–OH group as well as the stretching vibration C–C and C–O in the
In order to estimate the potential of FTIR-ATR diamond to carbohydrate structure and C–O in the phenol [31]. For colza honey, in
discriminate among floral origins, a model was built considering the addition to the signals responsible for the geographical discrimination
4 main mono-floral honeys only, i.e. acacia, colza, linden and sunflower. of acacia honeys, bands around 1313 cm−1 attributed to the amide III
The model was built after the exclusion of a test set, excluded samples [20] and 1715 cm−1 related to phenolic molecules [37] were discrim-
were randomly selected. The results listed in Table 2 showed the cor- inant. The geographical discrimination of sunflower honeys was based
rect LOOCV classification of the model as well as the classification of on some predictors which were also discriminating either for acacia or
honeys used in test set: acacia, colza and linden honeys were correctly for colza honeys. These results showed that mono-floral FTIR-ATR pat-
classified and among the 3 sunflower honeys, 1 was classified as colza tern prevails on the geographical origin but once a single floral origin
honey. Even if the model and the test set contains a limited number of is considered in the model and the varietal effect is taken out, the FTIR-
samples, these results show the potential of this method for the floral ATR spectra contains also discriminant information related to honey
origin discrimination. geographical origin.

4
F. Guyon, E. Logodin, D.A. Magdas et al.
Table 4
Result of the model LOOCV classification (C) according to the geographical origin and considering
three floral origins; in parenthesis, the interclass distance.
CAcaFr CAcaRo
Acacia Fr 18 0 (1.1)
Ro 1 17
Colza Fr
Ro
Sunflower Fr
Ro
Abbreviations: Fr: France; Ro: Romania ; Aca: Acacia; Col, Colza; Sun: Sunflower.
4. Conclusion
In this work, a preprocessing data treatment, consisting in the ap-
plication of second derivative, followed by autoscale was applied on a
meaningful selection of spectral ranges in order to improve the discrim-
ination power of the developed models. It was proved that the main
discrimination spectral range for botanical and geographical fingerprint
was the area 880–1600 cm−1 . Despite that the meaningful discrimina-
tors for both classifications are situated in the same spectral region, their
discriminating power is 10 times lower for geographical discrimination
as compared with the floral origin differentiation. Also, the modest in-
terclass distance, which was obtained for geographical differentiation of
honeys, reflected that this approach is mainly suitable for botanic dis-
crimination. The main differentiation markers for honey botanical ori-
gin were mainly based on the characteristic signals of the lipids from:
pollen, phenolic compounds and amino acids. This fact proved the re-
liability of this approach for honey floral discrimination being known
that these compounds are acknowledged botanical markers.
Nonetheless, it appears that the geographical discrimination seems to
be possible among a similar floral origin with other markers not linked
to floral characteristics. This findings show the relevance of data treat-
ment using a limited number of parameters, i.e. geographical among
one floral origin preferable than geographical classification with multi-
floral honeys. Then to develop an arborescence of “small” model tests.
This work need to be confirm by the use of a larger set of mono-floral
honeys.
Declaration of Competing Interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
Honey cooperatives(in alphabetical order): ADA Bretagne (GIE Ele-
vage), ADA Poitou Charentes, cooperative France miel, Syndicat AOP
miel Corse, Provence miel and Syndicat des miels Provence et Alpes
du Sud (SYMPAS) are warmly thanked for providing honey samples for
this study. D.A.M. acknowledge the financial support offered through
Program NUCLEU, PN 19 35 02 02 and by AgroTransilvania Cluster,
through the project “AgroTransilvania Cluster - Cluster specialized in
bioeconomy”, SMIS Code 104449. Philippe Paulin is warmly thanked
for helpful advices on MIR measurements.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.talo.2020.100022.
Talanta Open 3 (2021) 100022
CColFr CColRo CSunFr CSunRo No class %
0 100
0 94
6 0 (2.6) 0 100
0 6 0 100
6 0 (2.1) 0 100
0 6 0 100
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