European Food Research and Technology (2022) 248:2995–3002

https://doi.org/10.1007/s00217-022-04106-8

ORIGINAL PAPER

Usage of color measurements obtained by modified Seliwanoff test

to determine hydroxymethylfurfural
Aysegul Besir1 · Fehmi Yazici1 · Mehmet Serhat Odabas2

Received: 13 March 2022 / Revised: 30 July 2022 / Accepted: 1 August 2022 / Published online: 13 August 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
In this study, colorimetric parameters of the red color emerging as a result of the novel modified Seliwanoff test were used
for the quantitative determination of HMF in some syrup samples. Three different evaluation method (color space-based
method, absorbance method and artificial neural network (ANN)) were used to determine HMF amounts of samples. While
“da*” and absorbance value of red color were used to obtain a linear relationship with HMF concentration, L*, a*, b*, dL*,
da*, db*, dE* were used as input variables in artificial neural network evaluation. Also HPLC method was carried out as
a reference method. In addition to the absorbance value, ‘da*’ value also showed a linear relationship with the HMF con-
centration (da* = 0.0425 + 0.0612 × HMF concentration, ppm); correlation coefficient: 0.9980; p value of lack of fit: 0.579).
When HMF amounts obtained by each method were compared with that by reference method HPLC, a strong correlation was
found (color space-based method-HPLC, r: 0.996; absorbance method-HPLC, r: 0.997 and ANN-HPLC; r: 0.997). Among
the methods, ANN evaluation method was more prominent in detecting low HMF concentrations (< 4 mg HMF/kg sample).

Keywords HMF · Seliwanoff test · Artificial neural network · ANN · Color

Introduction (HCl) solution containing resorcinol (benzenediol (m-dihy-

5-(Hydroxymethyl) furfural (HMF) is considered a chemi-
cal indicator that should be examined in food quality and
food safety. It is a furanic compound with a cyclic aldehyde
structure. It consists of an aromatic alcohol, aromatic alde-
hyde and furan ring. HMF is formed as a result of two main
reactions known as the non-enzymatic browning reaction:
(1) Maillard reaction; (2) dehydration (caramelization) of
hexoses in an acidic medium [1].
The mechanism of the Seliwanoff test is based on a quali-
tative color reaction known as the Seliwanoff reaction, which
occurs when ketoses are heated with a hydrochloric acid
* Aysegul Besir
aysegul.besir@omu.edu.tr; aysegulbesir@gmail.com
Fehmi Yazici
yazicifehmi@gmail.com
Mehmet Serhat Odabas
mserhat@omu.edu.tr
1
Faculty of Engineering, Department of Food Engineering,
Ondokuz Mayis University, Samsun, Turkey
2
Bafra Vocational School, Ondokuz Mayis University,
Bafra, Samsun, Turkey
droxybenzene)). The reaction begins when three molecules
of water are separated from the structure of monosaccha-
rides, that is, they undergo dehydration and turn into HMF
(ketohexoses) or furfurals (ketopentoses). Finally, HMF and
furfural react with resorcinol in the presence of acid with
the effect of heat to give red color and bluish-green color,
respectively [2, 3]. In a previous study [4], Seliwanoff test
was modified (called modified Seliwanoff test) to determine
the amount of HMF quantitatively and was applied to honey
samples. With that study, a novel spectrophotometric method
was proposed using the quantitative relationship between
the HMF concentration and the red color that is released
in the chemical reaction of the HMF and Seliwanoff rea-
gent. Seliwanoff test, a well-known qualitative color reaction
for ketoses, was modified into a quantitative method to the
determination of HMF concentration.
Some quantitative analysis results have been used as input
in the artificial neural network (ANN) system. Cabrera and
Prieto [5] were used artificial neural networks to the predic-
tion of the antioxidant activity of essential oils. The DPPH-
free radical scavenging activity of different tea extract
samples was reported both using a spectrophotometer and
machine learning by Musa, Abdullah [6]. Buciński, and

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Zieliński [7] were confirmed that the benefits of learnt ANN
to predict the TAEC value with respect to the contents of
total phenolic compounds, inositol hexaphosphate, glucosi-
nolates, soluble proteins, ascorbic acid, and total tocophe-
rols. ANN was proven to be efficient tool both for predicting
the antioxidant activity potential of the tea samples and for
their classification by Cimpoiu, Cristea [8]. Guiné, Barroca
[9] showed that antioxidant activity and phenolic compounds
can be predicted accurately using ANN from the input vari-
ables: banana variety, dryness state and type and order of
extract. ANN has also used determination of some qual-
ity parameters: sugar concentration [10], stability of honey
crystallization [11], cholesterol quantification in egg [12],
predict L. monocytogenes population on fresh-cut tomato
and carrot [13], detection of cold injury in peaches [14]
and prediction of the antibacterial activity of garlic extract
on E. coli, S. aureus and B. Subtilis [15].
Seliwanoff test, a well-known qualitative color reaction
for ketoses, had modified into a quantitative method to the
determination of HMF concentration in the previous study
(modified Seliwanoff test) [4]. In this study, HMF determi-
nation was performed by that modified Seliwanoff test and
the results were evaluated with different methods. The red
color measurements revealed as a result of the modified Seli-
wanoff test were used for the quantitative determination of
HMF according to three different evaluation methods based
on: (1) “a” value; (2) absorbance and (3) artificial neural net-
work. Also HPLC method was used as a reference method
and the amounts of HMF found according to each evaluation
method were compared.
Material and methods
Materials
Sugar syrups were provided by two different companies in
Adana and Samsun. While syrups coded with F1, F2, F3
were fructose syrups, G1, G2, G3, G4 were glucose syr-
ups. E (concentrated apple juice), M (maltose syrup), and S
(sucralose) were also other samples. Which sugars in what
proportions are in the samples were given in the Supplemen-
tary material (S1). 5-Hydroxymethyl furfural (≥ 99%), HCl
(36.5–38%) and acetonitrile (≥ 99.9%) were purchased from
Sigma-Aldrich (St. Louis, MO, USA) and resorcinol from
Merck (Darmstadt, Germany) was used analysis.
Sample preparation
Samples were diluted twofold (except for sample S) and
0.5 ml of Carez-1 (15% of potassium hexacyanoferrate, w/v)
and Carez-2 (30% of zinc acetate dehydrate, w/v) solutions
were added, respectively. It was mixed and centrifuged at
13
European Food Research and Technology (2022) 248:2995–3002
5000 rpm for 15 min. The obtained supernatant was used
for HMF analysis.
Determination of HMF amount
Modified Seliwanoff method
Novel modified Seliwanoff test carried out by Besir, Yazici
[4] was used to determination of HMF in samples. Briefly,
samples were mixed with Seliwanoff reagent (0.1% resor-
cinol in 12% HCl). Sample: Seliwaoff reagent ratio was 1:2.
After 30 min at 25 °C incubation, absorbance and L*, a*, b*,
dL*, da*, db*, dE* values of red color obtained by applying
modified Seliwanoff test were measured with the apparatus
given in the following methods.
Absorbance‑based method The absorbance values were
measured with UV–Vis spectrophotometer (Agilent Tech-
nologies, Cary 60, Victoria, Australia) at 485 nm using a
1-cm quartz cuvette light path. Seliwanoff reagent (0.1%
resorcinol in 12% HCl) was used as blank.
Colour space‑based method (CIE lab) L*, a*, b*, dL*, da*,
db*, dE* values were measured by Bench-Top Spectropho-
tometer CM-5 (Konica Minolta) using transmission mode
with 12.5 mm standard glass cell. Seliwanoff reagent (0.1%
resorcinol in 12% HCl) was used as a target.
Artificial neural network‑based method Artificial neural
network (ANN) is one of the powerful and versatile tools
powered by artificial intelligence [16]. It has been used by
engineers for many years in countless applications [17].
Artificial neural network is a tool that allows computation
of the minimal mathematical model of neural functions.
Artificial neural network will consist of three layers [18].
Input layer is the first layer and is the part where data entry
is made. Hidden layer learns the relationship between input
and output values and ensures that the algorithm can func-
tion in accordance with its purpose. The Output layer is the
part of the ANN system that produces the results according
to the given inputs (Fig. 1).
In this research, the artificial neural network has seven
inputs, two hidden layers, and one output. The basic struc-
ture of the system includes neurons and connections, weights,
learning rate, and loss function. Neurons are the basic unit of
artificial neural networks. Each neuron carries a certain value
and produces output to be transmitted to the neurons in the
next layer. This transmission provides connections between
neurons. All links in the system have a weight value associated
with them. Weights are an essential element of the learning
process. The training process of ANN is about the optimiza-
tion of these values. Learning rate is the speed of the optimiza-
tion process. The loss function is the function that expresses
European Food Research and Technology (2022) 248:2995–3002 2997

Fig. 1  Schema of artificial

neural network. 17 × 7 mm
(600 × 600 DPI)

Table 1  Characterization of calibration curve obtained HMF concen-

tration (ppm) versus “da” values

Calibration curve equation da* = 0.0425 + 0.0612 × HMF

concent.(ppm)

SSƐ 0.345
SSlof 0.034
σ2lof 0.0085
σ2Ɛ 0.0115
LOD 1.40 ppm
LOQ 4.66 ppm
Fisher ratio, F 0.7391
Correlation coefficient 0.9980
p value of lack of fit 0.579

SSƐ Pure experimental error sum of squares, SSloff Lack of fit error
sum of squares, σ2lof = ­SSlof/(number of concentration level-2),
σ2Ɛ = ­SSƐ /(number of concentration level (replicate number of each
concentration-1))

networks increases thanks to the updated weight values. The

system repeats this cycle over and over for all data for the
best results. Thus, an artificial neural network is obtained that
works with the least error, suitable for the purpose wanted.

Reference method

HPLC–UV system (Agilent 1260, USA) equipped with

a C18 column, 5 μm and 4.6 mm × 250 mm was used as
a reference method to determine HMF levels of samples.
Fig. 2  Regression line of the calibration curve (a) and residual plots HPLC–UV conditions were as follows: mobil phase, 95%
(b). 18 × 28 mm (300 × 300 DPI)
acetic acid solution (1%) plus 5% acetonitrile; flow rate,
1 ml/min; injection volume, 20 μl; column temperature,
the incorrectness of the system's predictions after the training. 25 °C. The wavelength of the UV detector was 284 nm.
At the beginning of the neural network, the weight values are Method validation parameters were carried out in the previ-
randomly determined. Therefore, the initial estimates of the ous study [4].
system are different from what they should be. After this pro-
cess, we train the system to increase the predictive power of
the network. Weight values are updated according to the data
entered into the system during the training process. At the
end of all processes, the predictive power of artificial neural

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2998 European Food Research and Technology (2022) 248:2995–3002

Statistical analysis curve drawn by considering the change in “a” values

Minitab® 17.1.0 was used to determine the linearity of the
curve plots and correlation coefficient between two different
methods. Other data were evaluated by analysis of variance
(ANOVA) with SPSS program (SPSS 22.0). Multiple com-
parison tests were evaluated with the Duncan test based on
the 95% confidence interval.
Results and discussions
The calibration curve characterization obtained for the
quantitative relationship between absorbance and HMF
concentration was demonstrated in the previous work [4].
In this study, the calibration curve and its characterization
were also investigated to describe the relationship between
the change in the "da*" value in the color space and the
HMF concentration.
Linear relationship between ‘da*’ value and HMF
concentration
against ppm HMF concentration is given in Fig. 2a and
also calibration curve characterization parameters are
given in Table 1.
To prove the linearity of the calibration curve, F
table > F-calculated, p value of lack-of-fit should be insig-
nificant and the correlation coefficient close to 1 [19].
According to Table 1, at 4 and 34 degrees of freedom,
the F table value (2.6499) was higher than F calculated
(0.7391) and the lack-of-fit value is insignificant accord-
ing to the 95% confidence interval (0.579 > 0.05), it can
be said that the calibration graph was linear.
The residual graphs obtained to investigate the linearity
of the calibration curve are shown in Fig. 2b. To decide
whether a regression model is a good fit, the analysis of
residuals, a mathematical method, provides a sophisti-
cated approach. The residuals appeared to be normally
distributed (probability and histogram plots are shown on
the left) and generally random (right-hand plots showing
residuals against their fitted values and in their observa-
tion order).
Determination of HMF in syrup samples

The amounts of HMF calculated according to different eval-

Since the standard deviation in the total color change (dE)
uation method are given in Table 2.
obtained in the same sample measurements was higher
Results obtained by the modified Seliwanoff method
than that of obtained from "da*" values and the resulting
(based on a value) were slightly higher than those deter-
color is red, the calibration curve was determined by taking
mined by HPLC (Table 2). Nevertheless, linear regression
into account the change in the "− a… + a" scale. The meas-
results (Fig. 2a) demonstrated that there is a linear functional
urement of the color emerging as a result of the new modi-
relationship between the HMF concentrations (mg k­ g−1)
fied Seliwanoff test applied to standard HMF solutions at
determined by the two methods (Freg = 796.36; p = 0.000;
different concentrations (5–10–25–50–75–100 ppm) was
correlation coefficient, r = 0.9960) (Fig. 3a). There was a
carried out with a Konica Minolta CM-5 device. Accord-
good linear relationship between the modified Seliwan-
ing to the results obtained, the graph of the calibration
off (absorbance based) method and the HPLC method
Table 2  Comparison of the Samples HMF amounts, mg/kg sample
amounts of HMF detected using
different methods Modified Seliwanoff test Reference
method
Color space based Absorbance based method Artificial neural (HPLC)
method (da) network

G1 21.92 ± 0.99a 21.32 ± 0.15a 16 15.04 ± 0.06b

G2 24.76 ± 1.31a 21.21 ± 0.11c 18 17.59 ± 0.88b
G3 35.06 ± 1.41a 34.0 ± 0.19a 25 24.69 ± 0.37b
G4 123.66 ± 3.34a 116.50 ± 1.11a 99 98.48 ± 0.47b
F1 52.42 ± 1.68a 46.21 ± 0.11c 42 34.99 ± 1.83b
F2 53.51 ± 1.43a 51.21 ± 0.3a 46 45.97 ± 1.56b
F3 46.87 ± 0.82a 45.0 ± 0.20a 37 37.17 ± 1.76b
E 4.82 ± 0.01 N.D 4 3.05 ± 0.47
M 4.17 ± 0.01 N.D 5 2.94 ± 0.03
S N.D N.D 1 1.14 ± 0.05

N.D. Not detected. Values are means ± standard deviation. (a−b) Different letters on the same line show
significant differences (p < 0.05) between methods

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European Food Research and Technology (2022) 248:2995–3002 2999

(Freg = 739.73; p = 0.000; correlation coefficient, r = 0.9970)

(Fig. 3b). When we compare ANN and reference method
(HPLC), a good relationship was obtained between the
methods (Freg = 1477.96; p = 0.000; correlation coefficient,
r = 0.997) (Fig. 3c). Similarly de Andrade, de Andrade [20]
reported a good linear relationship (correlation coefficient,
r = 1.000) in the HMF concentrations obtained by differ-
ence spectrophotometry methods and HPLC in corn syrups.
Another study comparing two official methods (White and
HPLC) was carried out by Truzzi, Annibaldi [21]. A rela-
tively strong relationship (r = 0.9891) was found between the
methods in that study.
Consequently, each evaluation method can be proposed
to calculate HMF concentration. According to the modifed
Seliwanoff method (based on absorbance) because of the
fact that LOQ value was 8.97 mg HMF/kg sample [4], HMF
concentration in E, M and S coded samples was not able to
determine (Table 2). Also HMF concentration in S sample
could not be calculated using the ‘a’ value since ‘a’ value
of the color obtained as a result of the modified Seliwanoff
test was not different from the target value. However, ANN
evaluation method could be proposed as a useful tool for
the determination of the amount of HMF even at low HMF
concentration values (< 4 mg HMF/kg sample).

The result of the artificial neural network (ANN)

In this research, there are two hidden layers in the neural

network. The value of the performance function against the
number of iterations is shown in (Fig. 4a). Each iteration of
the complete training set is called an epoch. In each epoch,
the network adjusts the weights in the direction that reduces
the error. It stops training of the neural network when gen-
eralization stops, as indicated by an increase in the MSE
value of the validation data. A low MSE value means that
the network contains few errors. The error histogram is the
histogram of the errors between actual values and predicted
values after training a feed-forward neural network (Fig. 4b).
Y-axis represents the number of samples obtained from the
dataset which lies in a particular bin.
The neural network toolbox included in the MATLAB
Fig. 3  Comparison of reference method HPLC with a based on ‘da’
software was used to find the best model for the result, and value, b based on absorbance and c ANN method by linear regression
a graph of the best-performing network based on average at 95% confidence level. 9 × 19 mm (600 × 600 DPI)
training performance and the number of training periods and
test errors are shown in Fig. 5a.
It is seen that the results are 99% accurate between the 7.65767e−1, respectively (Table 3). These criteria were cal-
actual values and the values predicted by the ANN. The culated using Eqs. 1 and 2:
reliability of the data used in ANN is important in obtain-
ing this high accuracy value (Fig. 5b). As a result of the N �
∑ �2
yai − ypi
artificial neural network, MSE values of training, validation i=1 (1)
and test values were found as 2.99209e−1, 1.30030e−1, and MSE =
N−1

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3000 European Food Research and Technology (2022) 248:2995–3002

Fig. 4  The learning curve (a); The error histogram for the training of the data using the Levenberg–Marquardt (b). 23 × 8 mm (600 × 600 DPI)

Fig. 5  The plot for the training state parameters (a); the results for the regression between the output data and the targets for the LM (b).
20 × 8 mm (600 × 600 DPI)

Table 3  The mean square error (MSE) and R values where yi and ypi are measured and predicted values.
MSE R It has been shown that the model calculated after ANN,
training and testing of the data will give high accuracy
Training 2.99209e−1 9.99870e−1 results. ANN is a non-linear, data-driven, self-adaptive
Validation 1.30030e−1 9.99076e−1 system that can learn and identify correlated patterns
Testing 7.65767e−1 9.99815e−1 between input datasets and corresponding target values,
even if the data relationship is unknown. From this point
�
N �
� of view, it is widely used today as a method that provides
high accuracy.
∑ �� �
yai − ya ypi − yp
i=1
R2 = (2)
N � �2 � �2
∑
yai − ya ypi − yp
i=1

13
European Food Research and Technology (2022) 248:2995–3002
Conclusion
The red color, the output of the modified Seliwanoff test,
provided data for the quantitative detection of HMF. A sig-
nificant correlation (r: 0.9980) was observed between the
HMF concentration and ‘da’ value in the color space. This
relationship was defined with a graph showing linear proper-
ties and was used for the quantitative determination of HMF.
In addition, the artificial neural network was chosen as one
of the methods used to estimate the amount of HMF using
the parameters of the color space (L*, a*, b*, dL*, da*, db*,
dE*). Thus, the amount of HMF in syrup samples was deter-
mined by three different evaluation methods and compared
with the reference method HPLC. When the amounts of
HMF determined by each evaluation method were compared
with those determined by the reference method HPLC, the
regression lines obtained had high R2 values (color space-
based method-HPLC, R2: 99.1%; absorbance method-HPLC,
R2: 99.3% and artificial neural network-HPLC, R2: 99.5%).
The artificial neural network method was more useful than
other methods in detecting low HMF amount. With this
study, it has been shown that the red color resulting from
the modified Seliwanoff test provides appropriate data for
different evaluation methods in the determination of HMF.
Acknowledgements Experiments was supported (excluding publica-
tion funding) by Ondokuz Mayis University of Samsun, Turkey [Pro-
ject number: PYO.MUH.1904.18.007]. The authors also thank to Tat
Nişasta for providing some syrup samples.
Author contributions AB: methodology, investigation, formal analysis,
Writing – original draft. FY: supervision. MSO: Writing – review &
editing.
Declarations
Conflict of interest There are no conflicts of interest.
Compliance with ethics requirements This article does not contain
any studies with human and animal subjects.
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