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MINERAL CONTENT ANALYSIS OF GREEK HONEY
BY INDUCTIVELY COUPLED PLASMA ATOMIC EMISSION
SPECTROSCOPY (ICP-AES):
A TOOL FOR BOTANICAL AND GEOGRAPHICAL DISCRIMINATIONS
Chalhoub Cherifa, Lydakis - Simantiris Nikosb, Naxakis Georgea, Arvanitoyannis Ioannisc & Gotsiou Panagiotaa*
a

Mediterranean Agronomic Institute of Chania (M.A.I.Ch.), Laboratory of the Chemistry of Natural Products and of Analytical
Chemistry, Chania, Greece
b

Technological Education Institution of Crete, Department of Natural Resources and the Environment, Chania, Greece

c

University of Thessaly, School of Agricultural Sciences, Department of Agriculture and Animal Production and Aquatic
Environment, Volos, Greece
*[email protected]

bstract
The variation in the concentrations of Ca, Mg, Cu, Mn, Zn & P has been studied in honey samples from
beekeepers in Greece, in relation to their botanical and geographical origin. The honey samples were analysed
by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), after their digestion in a laboratory
microwave oven. Data will be presented on the quantitative differences which they have been observed among
samples of different botanical origin (nectar, pine honeydew, fir honeydew) as well as among samples of the
same botanical but different geographical origin. The use of Atomic Spectroscopy on the botanical and
geographical discrimination of honey will be discussed.
Introduction
Food authenticity has become of great economic importance for the sectors involved in food production and marketing and
for the consumers, who are looking for guaranteeing the quality of their food. Honey could not be the exception in this case.
The proof of authenticity of honey according to its botanical and geographical origin has become an important quality
parameter in the market and a hard task for food scientists!
Honey is produced by honeybees from nectar of flowering plants as well as from honeydew (for e.g. pine or fir honeydew).
Some of its components are due to the maturation process, some are added by the bees and some are derived from the
plants (which in turn, absorb several components from their environment such as soil, air, water). The routine method for
the determination of the botanical and geographical origins of honey has been, traditionally, since long time ago, the
microscopical examination of it, i.e. the melissopalynological analysis, which however is considered to be defective or
subjective in several cases and requires particular skills and knowledge. However, during the last 20-30 years, research on
the various chemical components of honey has proved that other methods can be also used, complementary to the
melissopalynological analysis, such as the analyses of volatile components, amino acids, enzymes, flavonoids, sugars,
minerals (and trace elements) of honey, or combinations of the above, as most of these components are derived from the
plants from which honey originates.
The current work represents the first study with ICP-AES on the mineral profile of Greek honeys from various botanical
origins, regarding initially 6 basic elements. This has been an effort for an initial screening and this is why not all botanical
groups of honey are well represented. However, this technique can be applied to all possible matrices and analytes,
provided that suitable sample pre-treatments are resorted to. It is also multielemental in nature and possesses high
sensitivity and appropriate detection power.

Table 2

Table 3

Results & Discussion
The variation in the concentration of the studied elements according to the botanical origin of the honey samples is shown
in Tables 4-6 and Fig.1-6. The observed differences in the mean values proved to be statistically significant (a=0.05) for
all elements except for zinc (Zn), which is actually considered as a pollutant from the beekeeping equipment and storage
containers.
It is observed that honeydew honeys (either from pine or fir) were clearly distinct from nectar honeys for their high
concentrations of phosphorus (P), magnesium (Mg) and manganese (Mn) and relatively lower concentration of calcium
(Ca), with the exception of manganese and magnesium been also important in cotton and heather honeys, that are known
as exceptions among the nectar group for their particularly high values of electrical conductivity. The three first elements
have been proved to have also significantly (a=0.05) different concentrations among pine and fir honeydew honeys; a
significant observation reported for the first time in literature of honey research.
Nectar honey groups were in total characterized by relatively higher concentrations of calcium (Ca) compared to the
honeydew honeys, and only minor differences have been observed among the different classes of nectar honeys (with the
exception of cotton and heather honeys as mentioned earlier).
Finally, the blends of nectar and honeydew honeys have shown intermediate concentrations for most elements, as it was
expected.
A significant (a=0.05) positive correlation between the concentration of P and Mg has been observed as shown in Fig.7.
The combination of these two elements can be used for the clear distinction of honeydew honeys from
nectar honeys (even from cotton and heather honeys that possess similar electrical conductivity with
honeydew honeys) and for the unambiguous separation of fir honeydew honeys.
Concerning the geographical discrimination of honey samples from the same botanical origin, comparison has been
possible only for the botanical classes that had representative number of honey samples, i.e. blends and pine honeydew
honeys. Statistical analysis has shown that the relatively lower concentration of P could discriminate pine honeydew
honeys of Crete from pine honeydew honeys of other places from Greece (fig.8, tables 4-6). In the case of blends, no
statistically significant differences have been observed. One can see only a trend for the blends from Crete to possess
higher Ca concentration, lower Mg, Cu, Mn & P again as for pine honeys (fig.9-13).
Figures 9-13

Figures 1-6

blend N+HDW

300
180

160

200

140
Crete

N.Greece

120

100

100

Ca(ppm)

Ca (ppm)

80

0

60
N=

N=

1

2

7

1-orange blossom

9

2

3-multifloral

2-cotton

6

5-heather

4-thyme

11

3

3

3

mainland Greece

Crete

7-pine HDW

6-blend N+HDW

8-fir HDW

geographical origin

botanical category

blend N+HDW
80

160
70

140

60

120
100

50
C. & S.Greece
N.Greece

80

40

C. & S.Greece

60

Mg (ppm)

40

Crete
Crete

20
N=

20

3

3

mainland Greece

Crete

0
N=

1

2

7

1-orange blossom

9

2

3-multifloral

2-cotton

6

5-heather

4-thyme

11

geographical origin

3

7-pine HDW

6-blend N+HDW

blend N+HDW

8-fir HDW
2,5

botanical category
2,0

3,5

1,5

3,0

C. & S.Greece

2,5

1,0
Crete

2,0

,5
Crete

1,5

Cu(ppm)

0,0

1,0

N=

Cu (ppm)

0,0

3

3

mainland Greece

Crete

geographical origin

-,5
N=

1

2

7

1-orange blossom

9

2

3-multifloral

2-cotton

6

11

5-heather

4-thyme

blend N+HDW

3

7-pine HDW

6-blend N+HDW

4

8-fir HDW

botanical category

3

8

2

6

1

0

4
Mn(ppm)

C. & S.Greece

-1

2

N=

3

3

mainland Greece

Crete

Crete

geographical origin

Crete

0

blend N+HDW
120

-2
N=

1

2

7

1-orange blossom

9

2

3-multifloral

2-cotton

6

5-heather

4-thyme

11

3

7-pine HDW

6-blend N+HDW

110

8-fir HDW
100

botanical category
90

300

16
80

C. & S.Greece

14

70

12

200

P(ppm)

60

10

Crete

50
N=

8
6

3

3

mainland Greece

Crete

geographical origin
Crete

4

fir HDW

50

2

40

N.Greece
30

0

20

P (p p m )

N=

pine HDW

1

2

7

1-orange blossom

9

3-multifloral

2-cotton

2

6

5-heather

4-thyme

11

3

7-pine HDW

6-blend N+HDW

8-fir HDW

blend N+HDW
nectar
0

20

40

60

80

100

120

140

160

200
C. & S.Greece

Figure 7
pine HDW
100

200
P (ppm)

Crete

C. & S.Greece

Crete

0
N=

160

1

2

1-orange blossom

7

botanical category

P (ppm )

120

100
80
N=

7

4

mainland Greece

Crete

geographical origin

Fi
gure 8

9

3-multifloral

2-cotton

140

10

0
N =

3

21

adulter ated

authentic

300

Mg (ppm)

180

Conclusions
The present work, which is the first to be done for the mineral profile of Greek
honeys with different botanical origins, has given the first indications for the
possible use of quantitative differences in the concentrations of some elements
(Ca, Mn, Mg, P) for the discrimination of nectar and honeydew honeys and
moreover for the distinction of pine honeydew honeys from fir honeydew
honeys that cannot be done by melissopalynological analysis. The distinction of
the two latter groups have been based so far on the organoleptic examination
of these honeys which however is known to be a quite subjective method.
In addition, first data appear on the possibility of the geographical
discrimination among pine honeydew honeys from different areas of Greece,
which would give an added value for pine honeys labeled accordingly for their
geographical origin.
Finally, some unacceptable high values have been reported for Zinc (Zn) which
is considered a toxic element and attention has to be given for the equipment
used by honey producers and packagers.

Figure 14

botanical category

0

M g (p p m )

100

Zn (ppm)

nectar

major botanic group

Compared to the values reported in literature for nectar and honeydew
honeys from other countries (it is worthy mentioning that there are no reports
for pine honeydew honeys) (ref.1, 3-8, 10-21, 23) the concentrations of the
elements examined in the present work present similarities and differences but
it has to be taken into account the different methodologies used in each case
(for e.g. HR-ICP-MS, GFAAS, ETAAS, INAA, XRF, and other) and the different
botanical groups of honey compared, for which in most cases there were not
reports in literature (e.g. cotton, thyme, pine honeydew honeys).

-,5

,5

Mn (ppm)

Materials & Methods
In total, 47 different honey samples provided by beekeepers and one sample of honey from the market, that has proved to
be pure isoglucose syrup (called as syrup below), have been analyzed. The botanical origin and their authenticity have
been verified through melissopalynological examination and physico-chemical analyses. Among these samples, 24 were
nectar honey, 9 blends of nectar and conifers honeydew, 11 pine honeydew honeys and 3 fir honeydew honeys (see Table
1). According to the preparative analyses for their authenticity, 6 honeys have been characterized as adulterated (3 out of
the 10 multifloral honeys and 3 out of the 6 blends from Crete) and they have been examined separately.
Sample preparation & analysis
Honey samples have been preserved in the freezer (-18C) until their use.
In order to reduce sampling errors and to make the sampling more representative, aliquots of 10g of honey were diluted in
high purity deionised water at a 1:1 (w/w) ratio. From the previous solution, an aliquot of 0.6 g (equivalent to 0.3 g of non
diluted honey) was then transferred into high-pressure vessels HQ 50 (quartz glass vessels) and digested in a microwave
oven after the addition of 5 ml of nitric acid 65% and 0,2ml of hydrochloric acid 37%.
During the digestion and after 20 minutes approximately, the maximum temperature reached was 300oC, and the
maximum pressure reached was 71.5 bars. After digestion, samples were cooled at room temperature and made up to
11mL with 2% nitric acid in plastic tubes.
The analysis was performed with ICP-AES. Quantification has been done with the use of standard solutions in 2% nitric acid
of the examined elements (Ca, Mg, P, Cu, Mn, Zn) and the final concentrations are expressed as ppm per honey weight.
The conditions of the microwave oven and of the ICP-AES are given in the Tables 2 & 3 respectively.

Mg(ppm)

30

Regarding the comparative study between the 41 authentic honey
samples and the 6 adulterated ones, it was found that only the
concentration of Mg was significantly lower among the nectar honeys of the
adulterated group (fig.14). No significant differences have been detected
among the authentic and adulterated blends of nectar and honeydew honey
samples. But this may be also due to the limited number of cases we had.
Another important point of this study was the observation for the syrup
analyzed: it was clearly distinct from all samples for the combination of low
concentrations for 3 elements (Cu=0.0ppm, Mn=0.3ppm and P=15.3ppm, with
a minimum value of Mn and P for authentic honeys equal to 1.18ppm and
29.8mm respectively) (see tables 4-6).

4-thyme

2

6

5-heather

11

3

7-pine HDW

6-blend N+HDW

8-fir HDW

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