1 Int. J. Biomol. Biomed.

International Journal of Biomolecules and Biomedicine (IJBB)

ISSN: 2221-1063 (Print), 2222-503X (Online)
http://www.innspub.net
Vol. 12, No. 1, p. 1-6, 2021

RESEARCH PAPER OPEN ACCESS

Determination of hydroxy methyl furfural concentration in

honey using ultra violate-visible spectrometry in West Gojjam
Zone of Amhara Region, Ethiopia

Tezera Alehegn*, Kusse Gudishe Goroya

Department of Physics, College of Natural and Computational Sciences, Wolaita Sodo University,
Wolaita, Ethiopia

Key words: Concentration, HMF, Honey, Quality

Article Published: 30 January 2021

Abstract

This paper aimed to determine the concentration of hydroxyl methyl furfural (HMF) using UV-visible
spectroscopy to assess the quality of honey. The honey samples were collected from three honeys productive
temperature zones: temperate, sub-tropical and tropical. Following the procedure of white method, the
concentration of HMF of temperate, sub-tropical and tropical zone honey are found to be 11.18 ± 0.052mg/kg,
24.95± 0.119mg/kg, and 56.94±0.366mg/kg respectively. There is statistically significance differences between
the groups in HMF concentration at 95% confidence level (p<0.05). All the samples are found to have HMF value
less than the maximum concentration of HMF in honey set by standard controlling international organizations,
which shows good quality of the honey in the study areas.
*Corresponding Author: Tezera Alehegn  [email protected]

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2 Int. J. Biomol. Biomed.

Introduction for the indication of quality of honey at Dembecha

Honey is the natural sweet substance produced by Woreda in West Gojjam, Ethiopia.
honey bees from the nectar of plants which bees
collect and transform by combining with specific Materials and methods
secretions of their own and deposit, store and leave in Sample collection techniques
the honey comb to ripen and mature (Gebiru et al., The study area has three temperature zones namely
2015). Honey is considered as healthy and wholesome Temperate (Dega), Sub-tropical (Woyna Dega) and
food with curative properties as it contains plenty of Tropical (Kola). Sample representatives were
nutrients and plays effective antimicrobial effects collected from each temperature zone. Samples were
against many bacteria (Buba et al., 2013). collected directly from the bee keepers. Each honey
sample was assigned a code that was used throughout
Honey consists mainly of three sugars: Fructose, the experimental trail.
Glucose and Sucrose together with water with about 1%
made up of the pollen grains, amino acids and other Apparatus
minerals (Tadesse et al., 2014). Composition of honey Plastic bottles were used to handle the honey
is tightly associated to its botanical source and also to samples. Beakers and volumetric flasks were used to
the geographical area from where it originated, because handle chemical and reagents during solution
soil and weather determine melliferous flora preparations. Pipette were used to suck solutions
(Kaskoniene et al., 2010). Its properties and while Pipette tips were t ovoid contamination of
composition also depend on bee species, season, mode samples and reagents. Filter paper was used to filter
of storage, and even harvest technology and conditions residuals and cuvettes of 10 mm path length were
(Everaldo et al., 2017). However, honeys produced used to handle the final prepared samples solutions
from different floral sources may have distinctly during recording of absorption spectra with UV-Vis
different aromas and tastes due to differences in spectrophotometer (Jenway 6705) at maximum
volatile composition which in turn is dependent on the absorbance at wave length of 284nm.
extraction methods and also on the botanical and
geographical origins (Gebreegziabhere et al., 2013). Reagents and chemicals
Carrez solution I (Potassium Ferro cyanide (K4Fe
Nowadays the quality of honey is determined by the (CN)6∙3H2O)) and Carrez solution II (zinc acetate (Zn
concentration of hydroxyl methyl furfural (HMF). (CH3CO2)2∙2H2O) were used to precipitate protein from
HMF occurs naturally in most honeys and increases honey while deionized water was used to prepare
rapidly with heat treatment of honey (Moralles et al., solutions and to washing different materials which are
2009). Therefore, it can be used as an indicator of set in to the experiment. Sodium bisulfite (NaHSO3)
heating and storage time at elevated temperatures. solution was used to make a reference solution. HMF
Good quality honey has lower amount of HMF (Kooh, standard was used to make spiked solution.
2009). Even though HMF is not a harmful substance,
many countries restrict the maximum allowable Sample preparation method

amount of HMF in honey (Tadesse et al., 2014; Experimental method in this work followed the

Shapla et al., 2018; Nicole et al., 2010). method established in University of Malaya Honey
Research Lab, Department of Molecular Medicine
In West Gojjam Zone, Ethioipa, particularly at (Machado De-Melo et al., 2017). This method was
Dembecha Woreda honey is harvested abundantly in used to determine the concentration of HMF in
all corridors of the Woreda but the quality and purity honey. Three solutions were prepared
of the honey has been evaluated ordinarily by tasting simultaneously. The firs solution is a 100ml of Carrez
and observing color of the honey. This paper was solution I was made by dissolving 15g of Potassium
intended to measure concentration of HMF in honey Ferro Cyanide (K4Fe(CN)6∙3H2O) and the second

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3 Int. J. Biomol. Biomed.

solution is a 100ml of Carrez solution II was also LOQ was obtained from triplicate measurement of the
made by dissolving 30g of Zinc Acetate blank solution as:
Zn(CH3CO2)2∙2H2O)) in water and make up to a
volume of 100ml. The e third solution is a 100ml of LOQ= Standard deviation of blank×10 ……………. (2)
0.2% Sodium Bisulphite solution made by dissolving
0.20g of solid hydrogen sulfite, NaHSO3, in water. Recovery test
Recoveries were determined by spiking the sample
From each honey sample 5.0g was completely dissolved with the known concentration of HMF by preparing
in 25.0ml of distilled water. The mixed solution was then
on the same procedure of the sample in triplicate and
transferred into a 50ml volumetric flask and 0.5ml of
measured its value for recovery and accuracy test. The
Carrez solution I and Carrez solution II were again well
method can be an accurate if the recovery test can be
mixed by vortex. This mixture was then filtered using a
in the range 80% _ 120% (Ololo and Goroya, 2018).
filter paper. From the total mixed solution in the
The recovery test in this work was determined as
volumetric flask, the first 10ml of filtered solution was
𝑀. 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑠𝑝𝑖𝑘𝑒𝑑 −𝑚. 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑢𝑛 𝑠𝑝𝑖𝑘𝑒𝑑
rejected while the remaining solution after filtration was %𝑅𝑇 = × 100 … (3)
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑎𝑑𝑑𝑒𝑑 𝑡𝑜 𝑠𝑝𝑖𝑘𝑒

collected. A volume of 1.0ml of the filtrated solution was

pipetted into two separate test tubes with the volume of Precision
1.0ml each. Oneml of water was added in one of the test
The precision of the experiment was cheeked by
tube and mixed well. This solution is named as sample
triplicate measurement of the sample and determines
solution. Also 1.0ml of 0.2% sodium Bisulphite solution
the percentage relative standard deviation (RSD).
was added to the second test tube and mixed well. This
solution is named as reference solution.
standard deviation
%𝑅𝑆𝐷 = × 100……………..…… (4)
𝑚𝑒𝑎𝑛 𝑣𝑎𝑙𝑢𝑒

Validation methods
Method validation is used to confirm the analytical Statistical data analysis methods
procedure employed for specific test is suitable for its All analyses were performed in triplicates and data was
intended use. Results from method validation can be presented as mean and standard deviation. Differences
used to judge the quality, reliability and consistency of between individual/group of honey samples were
the result. In order to validate the analytical method, the
analyzed using analysis of variance one way ANOVA,
following method validation parameters such as
SPSS Statistics (IBM) corporation version 20.
recovery test, instrument detection limit, method
detection limit, limit of quantification, precision and
Results
accuracy and linearity studies were carried out.
Calibration Curve
To determine the concentration of HMF out of the
Limit of detection (LOD)
The limit of detection (LOD) is the lowest quantity taken in the measurement, the known

concentration of the measurement that can be amount of concentration of the HMF standard

detected at a specified level of confidence. In this solution were used. These known concentration and
work, LOD was obtained from triplicate measurement absorbance of the reading enables to plot the
of the blank solution as: calibration graph displayed in Fig. 1.
LOD = Standard deviation of blank×3 ……….……… (1)
Table 1. Absorbance of the HMF standard solution.
Limit of quantification (LOQ)
Concentration
Limit of Quantification is the lowest concentration of 6 12 18 24 30
(mg/kg)
the measurement that can be measured with an
Absorbance 0.132 0.206 0.251 0.319 0.335
acceptable uncertainty. Similar to limit of detection,

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4 Int. J. Biomol. Biomed.

Table 3. Mean HMF concentration of honey in this

work (n = 3).
Mean Absorbance Concentration of the
Sample
at 284nm samples (mg/kg)
A 0.201 11.051± 0.513
B 0.311 25.154 ± 1.049
C 0.564 57.547 ± 0.909
A, B and C are Honey samples of Anjenie, Yemehel,
and Enewond

Fig. 1. Absorbance versus the concentration of the

Discussion
HMF standard solution. The most commonly monitored parameters for the
determination of honey quality and freshness is the level
The calibration curve produced correlation relation of HMF concentration of the honey (Bogdnov and
coefficient (R2) of 0.9998. This value indicates that Martine, 2002). Naturally, in fresh honey HMF is found
absorbance and concentration are directly in a very small amount from 0.06 - 0.2mg/kg (Ghazal et
proportional so Beer Lambert’s law is valid to al., 2008). As can be seen from Table 3, the HMF level of
determine the concentration of HMF in the sample. this work is 11.051 ± 0.0513mg/kg, 25.154±1.049 and
27.547 ±0. 099mg/kg for honey samples which were
Method Validations Result collected from temperate, sub-tropical and tropical,
Results of methods of validations and spectral lines respectively. One way ANOVA analysis shows that there
used in this experiments are displayed in Table 2. is statistical significance difference between the group at
95% confidence level p<0.05.
Table 2. Spectral lines and method validation results
Except the valued obtained in tropical area, HMF
in this work.
concentrations are less than 40mg/kg which is the
Wave length LOD LOQ Recovery Precision
(nm) (mg/kg) (mg/kg) test (%) (%) maximum limit of HMF for none tropical areas as
88.63- declared by EU council directive (2001), the
284 0.00458 0.1528 0.528
109.85
international honey quality regulatory commission
The triplicate measurement of the blank solution (Bogdnov et al., 1999). The Honey which were collected
gives the value of LOD and LOQ as 0.00458mg/kg from the tropical area has an HMF concentration of
and 0.01528mg/kg by applying equations (1) and (2), 57.547 ± 0.909mg/kg. This value is also fall in the range
respectively. Accuracy of the experiment was cheeked of the accepted values of HMF level for tropical areas.

by spiking the stoke solution with the sample as it The EU council directives 2001, Korean food code,

described in the methodology part. The recovery test International honey quality regulatory commission,
National food regulatory institution, limits the
ranges from 88.63% to 109.85%. This result shows
maximum concentration of HMF in honey for tropical
that the method was accurate because the recovery
climatic zone to be less than 80mg/kg.
test falls in the accepted range 80% - 120% (Ololo and
Goroya, 2018). Measurement of the experiment in Nuru (1999) observed that Ethiopian honey contains
this work gives% RSD of 0.528. This value of%RSD mean HMF concentration of 32.4mg/kg while Jose et
shows the measurements were precise, because RSD al. (2009) analyzed honey samples and found its HMF
value falls in the accepted range of less than 15% content ranged from 0.9 to 22.8mg/ kg. Similarly,
(Rehman et al., 2008). Tadesse and Gebremedihn (2014) reported that the
HMF concentration of their honey sample ranges from
Concentration HMF 8.32mg/kg to 45.26mg/kg. Results of current work are
The mean HMF concentration in the honey samples in good agreement with these previous findings. Table 4
collected from the three different temperature zones were shows short summery and comparisons of this work’s
determined and displayed in Table 3. Maximum of 57.547 result to the maximum limits of standards of HMF and
± 0.909 and minimum of 11.051± 0.513 was obtained. the result of other researchers.

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5 Int. J. Biomol. Biomed.

Table 4. HMF concentration of this work with maximum limits of standards.

Current Nuru, Gebiru et al. Tadesse G/Egzeabher, Daniel Ethiopian EU IHC
Results
work (1999) (2015) (2014) (2013) (2018) standard standard standard
HMF 8.32-
11.18-56.94 32.4 14.55-15.13 11.18 30.1-64.5 80 40&80 40&80
mg/kg 45.26

Conclusion European Commission. 2002. Council Directive

In this paper different honey samples collected from 2001/110/EC of 20 December 2001 relating to honey.
different temperature zones were evaluated for their Off. J. Eur. Commun., L 10, 47-52.
quality by the assessment of their HMF concentration by
using UV visible spectroscopy. The HMF concentration Everaldo A, Adrian B. 2017. Physicochemical

is found to be in the range of value of 11.1776 ± Characterization of Maltese Honey. J. INTECH 8,

171-191. DOI: http://dx.doi.org/10.5772/66330
0.0511.051± 0.513mg/kg to 56.9359 ± 0. 57.547 ±
0.909/kg with lower side corresponding the temperate
Gebiru B, Birhanu A, Hayal M. 2015.
and the upper values corresponding to tropical
Physicochemical Characterization of Honey from
temperature zones. The honeys which are produced in
Debre-Nazret Kebele of Tigray Region, Ethiopia.
colder area have low concentration of HMF. Moreover, it
World Appl. Sci. J 33, 1806-1814.
can be said that all the honey samples analyzed in this
DOI: 10.5829/idosi.wasj.2015.33.12.10229
work are in a good quality as their values found in the
acceptable range for the tested parameter of the
Gebreegziabhere G, Gebrehiwot T, Etsay K.
international and national standards. 2013. Physiochemical characteristics of honey
obtained from traditional and modern hive
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