Food Control 78 (2017) 183e186

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Short communication

Determination and occurrence of 5-hydroxymethyl-2-furaldehyde in

white and brown sugar by high performance liquid chromatography

, Peter Simko
Michaela Polovkova *

Institute of Food Science and Nutrition, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinsk
eho 9, 812 37 Bratislava, Slovak
Republic

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

Article history: 5-hydroxymethyl-2-furaldehyde (HMF) is not only indicator of food freshness and quality, but also
Received 20 January 2017 contaminant forming during Maillard reaction, or by dehydratation of saccharides, respectively. While
Received in revised form data about presence of HMF in white and brown sugar are scarce, 13 kinds of white sugar and 25 kinds of
24 February 2017
brown sugar were analysed. Sugar was dissolved in deionised water, clarified with Carrez solutions,
Accepted 25 February 2017
Available online 28 March 2017
filtered and content of HMF was determined using high performance liquid chromatography with diode
array detector (HPLC-DAD) with detection at 284 nm when separation run on Poroshel 120 EC C18 at
32
C. Elution was performed under isocratic conditions using 95:5 water/acetonitrile mobile phase at a
Keywords:
5-Hydroxymethyl-2-furaldehyde
flow rate of 0.9 mL/min and analysis run 5 min. Method was validated in in house regime and its pa-
HPLC rameters such as limit of detection e (LOD ¼ 0.05 mg/kg), limit of quantification (LOQ ¼ 0.15 mg/kg),
White sugar specificity, repeatability and recovery enabled its application for sugar analysis. While white sugar was
Brown sugar free of HMF, all kinds of brown sugar exhibited presence of HMF, when content in 15 kinds varied be-
Food safety tween 0.17 and 6.45 mg/kg, content in other 10 kinds was under LOQ. On the base of obtained results was
postulated that brown sugar contains HMF either due to absence of refining processes, or it is re-
Chemical compound studied in this article:
5-Hydroxymethyl-2-furaldehyde (PubChem
contaminated by treacle adding to white sugar during production of brown sugar.
CID: 237332) © 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Sugar is being produced almost exclusively from sugar cane or
sugar beet. As a member of saccharide group, it belongs to dis-
acharides, it means that it is composed of two reducing mono-
saccharide e hexoses such as glucose and fructose. Sometimes,
sugar is also named as sucrose, or saccharose, respectively. By The
International Union of Pure and Applied Chemistry (IUPAC) nomen-
clature, it is (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-
bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-
3,4,5-triol and a more frequent “chemical” name is O-a-D-gluco-
pyranosyl-(1 / 2)-b-D-fructofuranoside. In its pure state it is nor-
mally available as white crystals (Manley, 2011). Whether extracted
from cane or sugar-beet, sugar manufacture uses only very simple
purification and extraction processes, without using any additives
or synthetic products (Linden & Lorient, 1999).
As mentioned above, sugar can be extracted either from sugar
beet or sugar cane, respectively. Normally, these two sources are
* Corresponding author.

E-mail address: qsimko@stuba.sk (P. Simko).
equivalent even though some trace impurities are different. How-
ever, there is one area where the two sources are not equivalent and
that is regarding brown sugars. Cane sugar that has not been
completely purified has a pleasant taste and can be used either as
an ingredient for food production, or direct consumption. Beet
sugar, however, is not acceptable unless it is completely white due
to unpleasant “beety” sub flavour of non-refined sugar. In some
products, brown sugar or even molasses are used to add colour and
flavour to them. Alternatively, in some products a less than
completely white product, brown sugar is used simply to save
money (Edwards, 2000). Molasses is the product left when no more
sugar can be obtained from thick syrup. Beet sugar molasses is
unpleasant in taste and flavour and therefore it is not normally used
for human food. However, cane sugar molasses does have some
direct food use, normally in the form of treacle, what is clarified
molasses. Treacle is normally stored at 50
C to maintain its
liquidity (Edwards, 2007). In final, beet sugar refiners do produc-
tion of brown sugar by addition of cane sugar molasses to refined e
white beet sugar (Edwards, 2000).
HMF is spontaneously formed compound by the Maillard reac-
tion (the nonenzymatic browning) or during dehydration of

http://dx.doi.org/10.1016/j.foodcont.2017.02.059
0956-7135/© 2017 Elsevier Ltd. All rights reserved.
184 , P. Simko / Food Control 78 (2017) 183e186
M. Polovkova
reducing saccharides (fructose and glucose) under acidic conditions
and, in general, its concentration tends to rise as a result of heating
processes or long-term storage, respectively. Therefore, HMF is
found in fruit, vegetable, cereal food products containing reducing
monosaccharides, infant formulas and sugar (Demirhan et al., 2015;
Zhang, Wei, Liu, Lin, & Yuan, 2014), and in other heat-processed
food such as juices (Lee, Sakai, Manaf, Rodhi, & Saad, 2014) and
treacle (Edris, Murkovic, & Siegmund, 2007), sugar cane molasses
(Ruiz-Matute, Soria, Sanz, & Martínez-Castro, 2010), or honey
(Castoldi, Milani, Rossini, Pezza, & Pezza, 2016).
Apart from criterion quality, HMF is also frequently discussed as
the compound with adverse effects to living organisms. As found,
HMF can be metabolically transformed to 5-sulfooxymethylfurfural
which may play role as an ultimate electrophilic metabolite in
toxification of the parent compound in vivo (Lee, Shlyankevich,
Jeong, Douglas, & Surh, 1995; Surh, Lie c, Miller, & Tannenbaum,
1994). Toxicity and risk assessment aspects were lately summar-
ised and comprehensively discussed by Capuano and Fogliano
(2011).
For determination of HMF, HPLC coupled with DAD operating in
UV spectra is frequently used as a reference method for its analysis
in various food matrix such as bakery, fruit and vegetable products
(Zhang et al., 2014), honey (Truzzi et al., 2012; Zappala , Fallico,
Arena, & Verzera, 2005; Risner, Kiser, & Dube, 2006), coffee, soft
drinks (Xu, Liu,Yu, Yu, & Zhao, 2015), vinegars and cereal based
baby foods (Bignardi, Cavazza, & Corradini, 2014), or even royal jelly
(Ciulu et al., 2013).
Up to present time, there is no comprehensive information
about determination of HMF and any survey about its content in
sugar in literature. Therefore, the aim of this work was elaboration
and in house validation of HPLC-UV method for determination of
HMF in white and brown sugar and find real situation regarding its
content in both kind of sugar available on Slovakian market.
2. Materials and methods
2.1. Samples
All available kinds of crystal sugar (mesh size 0.8e1 mm) were
bought in local markets in territory of Bratislava, capital city of
Slovak Republic during the spring of 2016. Samples were packed in
1 kg, or 5 g paper package, respectively. Samples were stored in dry
and dark room at ambient temperature until analysed.
2.2. Chemicals
HMF (as analytical standard) was purchased from Sigma-
Aldrich, Darmstad, Germany. The standard was used to prepare
the working standard solutions (4.0e60.0 mg/L) dissolving in
deionised water. Acetonitrile (HPLC grade) was purchased from
Mikrochem Ltd., Pezinok, Slovak Republic, potassium ferrocyanide
K4[Fe(CH)6] and zinc sulphate ZnSO4, both of analytical grade from
Lachema Brno, Czech Republic. Membrane disc filters (0.2 mm) were
obtained from Hermes Lab Systems Ltd., Bratislava, Slovak Republic.
2.3. Sample preparation
4 g of sugar were dissolved in 8 mL of deionised water using a
magnetic stirrer. Then, solution was transferred into 10 mL volu-
metric flask and 0.3 mL of Carrez I (15% aqueous solution of
K4[Fe(CH)6]) and 0.3 mL Carrez II (20% aqueous solution of ZnSO4)
solutions were added. After thorough stirring, the volume of sam-
ple solution was adjusted to 10 mL with deionised water, filtered
using a disc filter, placed in a vial and analysed by HPLC.
2.4. HPLC analysis
HPLC analysis were performed employing Agilent 1260 Infinity
HPLC system (Agilent Technologies, Palo Alto, CA, USA), equipped
with autosampler and a diode array detector set at 284 nm. Sam-
ples aliquots of 100 mL were injected onto pre-separation column
Agilent UHPLC Guard 3PK SB-C18 (4.6  2.1 mm, 1.8 mm) coupled
with separation column Agilent Poroshel 120 EC C18 (4.6  50 mm,
2.7 mm) maintained at 32
C. Elution was performed under isocratic
conditions using 95:5 (v/v) water/acetonitrile mixture as the mo-
bile phase at a flow rate of 0.9 mL/min. At these conditions, time of
analysis was set to 5 min, while HMF eluted in 2.5 min of analysis.
Confirmation of HMF identity was made by comparison of retention
time and comparison of scanned UV spectrum of peaks and stan-
dard solution of HMF. All analysis were done in triplicate.
3. Results and discussion
3.1. Sample treatment
From the analytical point of view, sugar is relative simple matrix
and therefore it does not need time consuming and complicated
pre-treatment. However, some protein based impurities, especially
in brown sugar could be present and therefore Carrez solutions
were preventively applied. Mobile phase composition was adopted
from Ramírez-Jime nez, Guerra-Herna ndez, and García-Villanova
(2003).
3.2. In-house validation of the method
European Commission Regulation No. 836/2011 EC (2011) de-
fines performance criteria for methods of analysis for contaminants
in foods. So, in accordance with the regulation, parameters such as
specificity, repeatability, reproducibility, recovery, limit of detec-
tion (LOD), limit of quantification (LOQ) are required. Instead of
collaborative trial validation, in-house validation may also be used
when it fulfils set out performance criteria (Suranova , Semanova,
Sklarsov 
a, & Simko, 2015).
3.2.1. LOD, LOQ, specificity and linearity
LOD and LOQ values were calculated from the calibration curve
as 3 s/slope and 10 s/slope, respectively, where s is the standard
deviation of the signal obtained from five independent measure-
ments (International Conference on Harmonisation, 2005). So,
while LOD was equal to 0.05 mg/kg, LOQ was equal to 0.15 mg/kg.
Specificity was tested for eventual co-eluting impurities and
spectral interferences during HPLC analysis by comparison of
scanned UV spectrum of the HMF standard and HMF in samples,
identified by external standard addition procedure. As follows from
Fig. 1, both spectra exhibit very high similarity, what confirms
sufficient quality of separation of HMF from other interferences in
analysed sugar. Linearity of detector response to HMF amounts was
followed by analysis of standard solutions of HMF in the range of
4.0e60.0 mg/L, loaded on the column. As confirmed value of
regression coefficient (R2 ¼ 0.99995), there was a sufficient linear
accordance between amount of HMF and intensity of absorbance
measured during analysis.
3.2.2. Repeatability and recovery
Repeatability and recovery are other criteria of the European
Commission regulation (2011). Repeatability, expressed by HOR-
RATr value, has to be less than value 2 (Horwitz & Albert, 2006).
HORRATr value calculated for two concentration levels of 0.5 and
5 mg/L were 0.27 and 0.22 what indicates good repeatability. Ac-
cording to the regulation, recovery sets should be in the range of
 , P. Simko / Food Control 78 (2017) 183e186
M. Polovkova 185

Fig. 2. Chromatographic record of brown sugar (green) and record of the same sample
with external addition of HMF standard (blue). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 1. Spectra of HMF scanned during HPLC analysis e red curve is spectrum of HMF
standard while blue curve is the spectrum of HMF scanned during analyses of brown
sugar. (For interpretation of the references to colour in this figure legend, the reader is sample of dark brown sugar. Authors compared results of three
referred to the web version of this article.) analytical methods to each other and they reported about HMF
content in light brown sugar in the range 11.9e16.4 mg/kg, while in
dark brown sugar in 12.3e23.3 mg/kg Risner et al. (2006).
50e120%; recovery for concentration of 0.5 mg/L was 94 ± 6%,
while for 5 mg/L reached 99 ± 2%, which is in close accordance with
4. Conclusions
the regulation.
Summarising the results and findings obtained in this study, the
3.3. HMF content in sugar
following conclusions can be postulated as follows: For determi-
nation of HMF in brown and white sugar, a simple and rapid
During experiments, 25 kinds of brown sugar, available on
method using HPLC was elaborated. The method passed through in
Slovakian market were analysed. As follows from Table 1, HMF was
house validation criteria and it is suitable as operative method for
observed in all kinds, however, 10 kinds contained less content than
control of HMF content in sugar. On the base of results obtained it
was LOQ. Typical chromatographic record of brown sugar alone and
was found that brown sugar shows in the majority of cases the HMF
identification of HMF by addition of HMF as external standard is
content between 0.17 and 6.45 mg/kg due to absence of refining
shown in Fig. 2. On the contrary, all 13 kinds of white sugar were
processes, or it is re-contaminated by HMF contained in treacle
free of HMF probably due to completion of refining process. On the
adding to white sugar during production of brown sugar. On the
other hand, addition of molasses, or treacle to white sugar to obtain
other hand, white sugar is free of HMF with regard to complete
sugar brown causes its re-contamination by HMF. Although there is
refining process. These information could be useful for complexion
limited information about HMF content in treacle, some data are
of data about HMF in foods, what is important for calculation of risk
available, e.g. Edris et al. (2007) analysed three kinds of treacle and
assessment as a crucial tool for real situation in food safety relating
found 66.1, 92.4, and 179 mg/kg of HMF in them. Similarly, Ruiz-
to this topic.
Matute et al. (2010) reported about presence of HMF in sugarcane
molasses (labelled as Black Treacle) at the level of 100 mg/kg. Risner
Compliance with ethics requirements
et al. (2006) analysed 4 molasses and found that HMF content
varied from 5.7 to 18.3 mg/kg. As mentioned above, data about
This article does not contain any studies with human or animal
presence of HMF in sugar are scarce. It was found the only infor-
subjects.
mation about analysis of one sample of light brown sugar and one
Acknowledgement
Table 1
HMF content in brown sugar. This contribution is the result of a project funded by the Slovak
Sample No. Average ± SD [mg/kg] RSDra [%] PRSDRb [%] HorRat value Scientific Grant Agency VEGA No. 1/0487/16.

1. 0.31 ± 0.02 7.4 18.9 0.39

2. 0.17 ± 0.02 10.4 20.7 0.50
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