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

*irina.geana@icsi.ro

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