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22nd Conference “New Cryogenic and Isotope Technologies for Energy and Environment” - EnergEn 2018 Băile Govora, Romania, October 24 – 26, 2018
Characterization And Classification Of Honeys With Different Botanical
Sources Based On Organic Acids And Water-Soluble Vitamins In
Conjunction With Chemometric Analysis
Corina Teodora Ciucure, Elisabeta-Irina Geană*
National R&D Institute for Cryogenics and Isotopic Technologies – ICIT Rm. Valcea, 4th Uzinei Street, PO
Raureni, Box 7, 240050 Rm. Valcea, Romania
Honey is a supersaturated solution of sugars with about 18% water content and other
minor components such organic acids, vitamins, flavonoids, phenolic acids, proteins, free
amino acids, enzymes and minerals (León-Ruiz et al., 2013) with therapeutic potential. The
qualitative and quantitative characterization of the minor constituents like organic acids and
water soluble vitamins in sugar based matrices (e.g. honey) are still scarcely explored
research areas. Thus, development of reliable analytical methods for the determination of
organic acids and water soluble vitamins in honey as powerful tools for honey botanical
origin authentication is an important (Ciulu et al., 2011; Mato et al., 2006).
The aim of this work was to develop two chromatographic methods for separation and
quantification of the main organic acids and water soluble vitamins in different honeys types
in order to create databases for honey botanical origin discrimination.
A ultra-high-performance liquid chromatography (UHPLC) separation with diode
array (DAD) detection methods were developed and used for the characterization of organic
acids and water-soluble vitamins of 82 honey with different botanical origin (acacia,
sunflower, linden, rape, polyfloral and honeydew). Chromatographic separation of organic
acids (oxalic, formic, malic, lactic, ascorbic, maleic, succinic, citric, propionic and fumaric
acids) was performed at 15 °C with a Hypersil Gold aQ reversed phase column (250x 4 mm,
5 µm) and isocratic elution of water with 0.02% H2SO4 at 0.3 mL/min flow rate, while water
soluble vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C) were separated using a Syncronis C18
reversed phase column (250x 4.6 mm, 5 µm) and gradient elution of two solvents (A: water
with 0.025% TFA and B: ACN) at 0.8 mL/min flow rate. The organic acids were isolated and
pre-concentrated from the honey matrix by solid-phase extraction using ion-exchange
cartridge Sep-Pak (500 mg) (Suárez-Luque et al., 2002), while water soluble vitamins were
analysed directly after their complete dissolution from the honey matrix. Sample extracts
were injected into analysing system after filtering through a 0.45 µm pour size membrane
filter. Identification of organic acids and water-soluble vitamins was done using reference
standards and the amounts in the extracts were calculated as mg/100 g honey using external
calibration curves, which were obtained for each compound.
The proposed methods have been successfully applied for the separation and
quantification of organic acids and vitamins in honeys with different botanical origins. The
honey samples had similar, but quantitatively different, organic acids and water soluble
vitamins profiles. Relatively high amounts of citric and propionic acids were identified in all
honey extracts. Citric acid was predominant in sunflower honeys while propionic acid in
acacia and rape honeys. Formic acid was identified only in honeydew honeys and tree rape
22nd Conference “New Cryogenic and Isotope Technologies for Energy and Environment” - EnergEn 2018 Băile Govora, Romania, October 24 – 26, 2018
honeys. Oxalic acid was identified in all honey samples, in some cases in high amounts,
indicating residency after the bee treatment. The overall amount of the investigated water
soluble vitamins in honey samples was quite low or undetectable, in many cases, confirming
that honey is not a vitamin-rich food. Vitamin C seems to be not abundant in honey, due to
his low stability, the highest concentration observed in our study being for honeydew honey.
Vitamin B5 seems to be less common than vitamin C and B3, but in considerable amounts.
-6
-4
-2
0
2
4
6
8
-10 -8 -6 -4 -2 0 2 4 6
F2 (2
6.7
5 %
)
F1 (62.32 %)
acacia honeydew linden polyfloral rape sunflower
Figure 1. Scatter plot of the first two discriminant functions showing separation between
honey types
Keywords: honey, botanical origin, organic acids, vitamins, UHPLC
Acknowledgements
This work was performed within the framework of the research projects PN-III-P2-2.1-PED-2016-1656 –
“Alternative analytical approaches for detecting adulteration of honey with emphasis on its biologically active
compounds”, SAFE-HONEY, 194PED/2017, supported by the Romanian National Authority for Scientific
Research and Innovation, CNCS – UEFISCDI.
References Ciulu, M., Solinas, S., Floris, I., Panzanelli, A., Pilo, M.I., Piu, P.C., Spano, N., Sanna, G., 2011. RP-HPLC
determination of water-soluble vitamins in honey. Talanta 83, 924–929. doi:10.1016/j.talanta.2010.10.059
León-Ruiz, V., Vera, S., González-Porto, A. V., San Andrés, M.P., 2013. Analysis of Water-Soluble Vitamins
in Honey by Isocratic RP-HPLC. Food Anal. Methods 6, 488–496. doi:10.1007/s12161-012-9477-4
Mato, I., Huidobro, J.F., Simal-Lozano, J., Sancho, M.T., 2006. Analytical Methods for the Determination of
Organic Acids in Honey. Crit. Rev. Anal. Chem. 36, 3–11. doi:10.1080/10408340500451957
Suárez-Luque, S., Mato, I., Huidobro, J.F., Simal-Lozano, J., Sancho, M.T., 2002. Rapid determination of
minority organic acids in honey by high-performance liquid chromatography. J. Chromatogr. A 955, 207–
14.
22nd Conference “New Cryogenic and Isotope Technologies for Energy and Environment” - EnergEn 2018 Băile Govora, Romania, October 24 – 26, 2018