Journal of Chromatography A, 1272 (2013) 132135

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Application of a newly developed and validated high-performance thin-layer

chromatographic method to control honey adulteration
Anitta Puscas, Anamaria Hosu, Claudia Cimpoiu
Babes-Bolyai University, Faculty of Chemistry and Chemical Engineering, 11 Arany Janos, 400028 Cluj-Napoca, Romania

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

Article history: Honey is a saturated solution of sugars, used for a long time as a natural source of sugars and is an
Received 14 September 2012 important ingredient in traditional medicine due to its antimicrobial, anti-inammatory and antioxi-
Received in revised form 23 October 2012 dant effects. Therefore, methods for quality control of honey and detection of its adulteration are very
Accepted 25 November 2012
important. For this reason, the aim of this study is to develop and validate a new, simple and economical
Available online 30 November 2012
analytical method for detecting the adulteration of some Romanian honeys based on high-performance
thin-layer chromatography (HPTLC) combined with image analysis. The proposed method involved the
Keywords:
chromatographic separations of glucose, fructose and sucrose on silica gel HPTLC plates, developed twice
Romanian honeys
HPTLC validated method
with ethyl acetatepyridinewateracetic acid, 6:3:1:0.5 (v/v/v/v), followed by dipping in an immersion
Glucose solution. The documentation of plates was performed using TLC visualization device and the images of
Fructose plates were processed using a digital processor. The developed HPTLC method was validated for selectiv-
Sucrose ity, linearity and range, LOD and LOQ, precision, robustness and accuracy. The method was then applied
Adulteration for quantitative determination of glucose, fructose and sucrose from different types of Romanian honeys,
commercially available.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Honey is a supersaturated natural solution of sugars, in which
the monosaccharide fructose and glucose are the most abundant
(typically about 8085% of the solids in the honey) [1]. Other
disaccharides (i.e. trehalose, isomaltose, etc.) and higher sugars
(trisaccharides and oligosaccharides) are also present, although
in quite small quantities [2]. It also contains a wide range of
minor constituents, such as phenolic compounds, minerals, pro-
teins, organic acids (gluconic acid, acetic acid), free amino acids,
enzymes (invertase, glucose oxidase, catalase, phosphatases) and
vitamins (ascorbic acid, niacin, pyridoxine, etc.) [3,4]. The compo-
sition of honey is rather variable and depends primarily on its oral
and geographical source, but certain external factors, such as sea-
sons, processing, packaging and storage conditions, also play an
important role [3,5].
Honey is a sweet and avorful natural product which has been
consumed for its high nutritive value and its contribution in human
health for a long time [6]. Studies have shown that honey has
both antimicrobial, anti-inammatory and antioxidant properties,
useful in stimulation of wounds, burns healing and gastric ulcers
Corresponding author. Tel.: +40 264 583833; fax: +40 264 590818.
E-mail addresses: ccimpoiu@chem.ubbcluj.ro, ccimpoiu@yahoo.com
(C. Cimpoiu).
treatment [5,7]. The limited availability and the increased price of
honey have provided major incentives for falsication with other
carbohydrate materials [1]. Therefore, methods for quality control
of honey and detection of its adulteration are very important.
The most common methods of honey adulteration are by adding
sucrose, which cannot be more than 1% of its dried mass, or by
overfeeding of bees with sugar and other types of sucrose [8,9].
Other methods for adulteration, met in honey industry, involve the
adding of fructose or industrial glucose and, therefore, changing the
fructose/glucose ratio, which has to be 11.2 in pure honey. When
the ratio differs from this value it can be concluded that the honey
has been adulterated. Different analytical methods were used to
evaluate some properties of honey which can enable to distinguish
between adulterated honey with various sugars and pure honey
[913].
Sugars can be determined by different analytical methods, such
as ow injection analysis [14], electrochemical analysis [15], enzy-
matic methods [16], gas-chromatography [17], high-performance
liquid chromatography [18,19], anion-exchange liquid chromatog-
raphy [20,21] and thin-layer chromatography [22,23].
The aim of this study was to develop a new, modern and simple
analytical high-performance thin-layer chromatographic (HPTLC)
method for detecting the adulteration of some Romanian honeys
on the basis of fructose/glucose ratio and sucrose content. This
goal has been also selected because EU Commission is encourag-
ing the development of harmonized analytical methods to permit

0021-9673/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2012.11.064
 A. Puscas et al. / J. Chromatogr. A 1272 (2013) 132135
the verication of compliance with the quality specication for the
different honeys [8].
2. Materials and methods
2.1. Reagents and chemicals
All solvents were of analytical grade and were obtained
from Microchim (Bucharest, Romania). The standard compounds
(glucose, fructose and sucrose), diphenylamine and aniline
hydrochloride, were purchased from Merck (Darmstadt, Germany).
Fifteen commercial honeys from three types of owers: lime (L),
polyoral (PF) and acacia (A) made by different producers (15)
were obtained from the local market.
The dipping solution was prepared as follows: 2 mg of dipheny-
lamine and 2 mg of aniline hydrochloride were dissolved in 80 mL
acetone and 15 mL phosphoric acid and then the solution was
diluted to 100 mL with acetone.
2.2. Standard solution and samples preparation
Standard solutions containing 1 mg/mL of fructose, glucose
and sucrose were prepared in methanol. The sample solutions
(1 mg/mL) were prepared in methanol after the homogenization
of honeys in a Elmasonic E60H bath at 38 C for 30 min.
2.3. Chromatographic separation
Different volumes of each standard solution were applied
as 8 mm bands at 1.2 cm from the low edge with a rate of
30 nL/s using a semi-automatic application device (Linomat 5
Camag). The chromatographic analysis was performed on silica gel
HPTLC aluminum sheets (20 cm 10 cm, Merck, Germany) devel-
oped twice with a mixture of ethyl acetatepyridinewateracetic
acid, 6:3:1:0.5 (v/v/v/v) as mobile phase. The plate was devel-
oped to a distance of 80 mm at room temperature in normal
chromatographic twin trough chamber (Camag) pre-saturated for
30 min with mobile phase. After development, the dried plate was
immersed for 3 s in the dipping solution and heated at 110 C
for 15 min. The dipping of the plates was performed using an
immersion device (Camag) with a vertical speed of 35 mm/s. The
documentation of the plates was done in visible light using a TLC
visualization device (Digistore 2, Camag) and the digital processing
of chromatographic images was performed with TLC Videoscan
software (Camag). All instruments were controlled by WinCats soft-
ware.
2.4. Method validation
2.4.1. Selectivity
The selectivity, dened as the ability to distinguish the analyte
from other substances, was conrmed by comparing the Rf of fruc-
tose, glucose and sucrose in samples with the Rf of the reference
standards. Additional indications of the selectivity are peak res-
olutions. On the other hand, other compounds did not appear as
individual spots in any chromatographic systems tested.
2.4.2. Linearity and range
The calibration curves were generated using six data points. Vol-
umes of 6, 8, 10, 12, 14 and 16 L from each standard solution were
analyzed by proposed method in triplicate. Linear calibration plots
were obtained over the calibration range belonging to the 75125%
range from target value for each compound.
133
2.4.3. Limit of detection (LOD) and limit of quantication (LOQ)
The LOD and LOQ were obtained graphically by plotting the
regression line together with the condence intervals and were
calculated using ordinary least squares method.
2.4.4. Precision
The precision was tested as repeatability (intra-assay precision)
and intermediate precision (inter-assay precision). Intra-assay pre-
cision and inter-assay precision was expressed as relative standard
deviation (RSD) of six individual spots of 100% of the test concen-
tration for each compound on the same day and six repeated assay
of reference compounds at 100% of the test concentration over a
period of 6 days, respectively.
2.4.5. Accuracy
The accuracy of the method was tested by performing recov-
ery studies in terms of standard addition technique. Known
quantities of each reference standard compounds were added to
pre-quantied sample solution in the range of low (75%), medium
(100%) and high (125%) nominal analytical concentrations. Each
experiment was performed in triplicate and the accuracy was cal-
culated as recovery and RSD of the compound in the sample.
2.4.6. Robustness
As is specied in ICH guideline [24], the robustness of an analyt-
ical procedure is a measure of its capacity to remain unaffected by
small, but deliberate variations in method parameters and provides
an indication of its reliability during normal usage. Minor changes
in chromatographic conditions were made in mobile phase com-
position (2%), chamber saturation period, development distance
and the effect of these changes on retention factor was measured.
2.5. Honeys analysis
In order to quantify the fructose, glucose and sucrose in honey,
20 L of each honey solutions was applied as 8 mm bands on HPTLC
aluminum plates and was analyzed by the validated method. The
amounts of each sugar were calculated on the basis of calibration
curves.
3. Results and discussion
3.1. Chromatographic separation
Several mobile phases were tested (Table 1) in order to obtain
a good chromatographic separation. The mobile phase 8 (ethyl
acetatepyridinewateracetic acid, 6:3:1:0.5 (v/v/v/v)) was found
to be the most suitable mobile phase. The results obtained using the
other mobile phases were unsatisfactory regarding the separation
of target compounds After two developments of the chromato-
graphic plate with the mobile phase 8 differences between Rf values
of fructose, glucose and sucrose were obtained (Table 2), making
easy the discrimination of these compounds from the samples.
Table 1
The composition of mobile phase tested.
No. Mobile phase Composition (v/v/v)
1 Ethyl acetateisopropanolwater 20:10:2 and 6:3:3
2 Ethyl acetateethanolwater 65:30:5 and 26:2:12
3 Ethyl acetatemethanolacetic acidwater 16:8:8:4
4 Ethyl acetatemethanolwater 26:12:2
5 Acetonitrilewater 17:3 and 70:10
6 Acetonitrilewaterformic acid 34:6:0.5
7 Acetonitrileethanolwater 26:12:2
8 Ethyl acetatepyridinewateracetic acid 6:3:1:0.5
134 A. Puscas et al. / J. Chromatogr. A 1272 (2013) 132135

Table 2
The chromatographic and calibration parameters, LOD and LOQ.

Compound Rf Regression equation R2 RSD (%) LOD (ng/L) LOQ (ng/L)

Glucose 0.46 y = 3413.4x 0.9985 1.14 14 33

Fructose 0.52 y = 7634.4x + 23,952 0.9934 1.32 31 63
Sucrose 0.39 y = 6414.5x + 16,415 0.9982 1.13 22 43

with correlation coefcients (R2 ) greater than 0.99 and the relative
standard deviation (RSD) less than 1.32% (Table 2).
The LOD and LOQ are the points where the upper con-
dence interval, respectively the lower condence interval intersect
the y-axis horizontally until reaching the regression curve, then
proceeding vertically to the x-axis [25]. The LOD and LOQ values
(Table 2) revealed that the method shows a high sensitivity for the
investigated compounds. The lowest LOD and LOQ are obtained for
glucose.
The results of repeatability and intermediate precision (RSD (%),
n = 6) are: 1.25% for glucose, 0.28 for fructose, 1.08 for sucrose and
0.14 for glucose, 1.32 for fructose, 1.13 for sucrose, respectively.
The obtained values demonstrate the precision of the developed
method.
The results obtained for accuracy were presented in Table 3.
The recoveries and RSD for each studied compound showed that
the method is highly accurate and suitable for the future use.
The results obtained on the robustness study showed that slight
variations in the chromatographic conditions had no signicant
changes in the Rf values of analytes.
The validation parameters were found within the recommended
limits highlighted that the proposed method is suitable for the
quantitative determination of glucose, fructose and sucrose in
honey samples.

3.3. Quantitative determination of sugars from Romanian honeys

Fig. 1. The HPTLC separation of standards and honeys. Tracks: 1 glucose; 2
fructose; 3 sucrose; 4 1L; 5 1PF; 6 1A.
3.2. Method validation
The proposed HPTLC method was validated according to ICH
guideline [24] for the following parameters: selectivity, linearity
and range, LOD and LOQ, precision, accuracy and robustness.
The selectivity of the method was conrmed by comparing
the Rf of analytes and standards and it has been observed the
absence of interfering or overlapping bands in the analyzed sam-
ples (Fig. 1). The selectivity of the method was also conrmed
by the value of resolution (RS > 1.5) between the adjacent bands
(RS sucroseglucose = 3.60; RS glucosefructose = 1.81).
The linearity of the calibration curves was studied on the work-
ing range of 616 g/bands for all three compounds. The linear
responses for the peak area against concentration were obtained
The applicability of the validated method was checked by ana-
lyzing the glucose, fructose and sucrose in commercially available
Romanian honeys in order to detect the adulteration.
The values of fructose/glucose ratio (Table 4) showed that seven
honeys are adulterated with fructose from other sources than nat-
ural ones. The results reveal that one honey (5PF) was adulterated
with industrial glucose because the fructose/glucose ratio is bel-
low 1 (0.88). Also, sucrose is determined in three honeys, but the
values are below the admitted limit. The presence of the sucrose
could be an indication of adulteration by bees overfeeding with
sucrose syrup. It is interesting to note that in almost all cases of
acacia honeys the fructose/glucose ratio exceed the admitted value.
This situation could be explained by the fact that acacia honey is
generally not too sweet and the producers raise the sensory prop-
erties of honey by fructose adding. Therefore, it can be concluded
that acacia honeys are most often adulterated.

Table 3
Accuracy of the proposed method.

Compound Amount in sample (g/mLa ) Level (%) Amount added (g/mL) Amount founded (g/mL) Recovery (%) RSD (%)

75 260 613 101.16 1.16

Glucose 346 100 346 694 100.29 0.29
125 433 785 100.77 0.77

75 287 680 101.49 1.49

Fructose 383 100 383 775 101.17 1.17
125 479 871 101.04 1.04

75 89 210 101.45 1.43

Sucrose 118 100 118 241 102.12 2.07
125 148 270 101.50 1.48
a
g/mL of honey solution.
 A. Puscas et al. / J. Chromatogr. A 1272 (2013) 132135 135

Table 4
The amount of analyzed sugars in different types of honeys.

Honey Fructose Glucose Sucrose Fructose/glucose ratio

mg/ga SD RSD (%) mg/g SD RSD (%) mg/g SD RSD (%)

1L 488 11 2.25 439 21 4.78 ndb 1.11

2L 383 6 1.57 346 10 2.89 118 3 2.54 1.11
3L 406 14 3.45 406 8 1.97 81 2 2.47 1.00
4L 472 8 1.69 394 13 3.30 nd 1.28
5L 378 13 3.44 302 9 2.98 nd 1.25
1PF 453 16 3.53 415 13 3.13 nd 1.09
2PF 456 11 2.41 425 14 3.29 nd 1.07
3PF 448 9 2.01 389 11 2.83 nd 1.15
4PF 446 9 2.02 290 8 2.76 nd 1.54
5PF 343 7 2.04 390 6 1.54 nd 0.88
1A 487 10 2.05 339 8 2.36 124 4 3.23 1.44
2A 459 11 2.40 308 9 2.92 nd 1.49
3A 402 7 1.74 295 7 2.37 nd 1.36
4A 332 7 2.11 203 11 5.42 nd 1.64
5A 380 14 3.68 328 9 2.74 nd 1.16
a
mg/g of honey.
b
nd not detected.

Moreover, experimental results showed that the average fruc-
tose content is 42% and the average glucose content is 35%, in
accordance with those obtained by other authors for Romanian
honeys [4] and the total content of inverted sugars is over 77 g/100 g
of honey, according to the EC Directive 110/2001.
4. Conclusions
In this study a simple, sensitive and accurate HPTLC method
for simultaneous determination of fructose, glucose and sucrose
in commercial honey was developed. The presented method is a
useful tool to identify the adulteration of Romanian honeys on
the basis of fructose/glucose ratio and sugar content. The validated
method is straightforward being faster and more economical than
the commonly other methods.
This study provides a reliable set of information regarding adul-
teration of honey. Moreover, the information related to Romanian
honeys is highly interesting for consumers and producers all over
the world. The fructose/glucose ratio and amount of sucrose deter-
mined in commercial honey samples can be used in quality control
and R&D laboratories for similar products.
Acknowledgement
This research is supported by performance scholarship for stu-
dents no. 30068/15/19.01.2012.
References
[1] E. Anklam, Food Chem. 63 (1998) 549.
[2] M. Zamora, C. Chirife, Food Control 17 (2006) 59.
[3] J.M. Alvarez-Suarez, A.M. Gonzales-Paramas, C. Santos-Buelga, M. Battino, J.
Agric. Food Chem. 58 (2010) 9817.
[4] L.A. Marghitas, D. Dezmirean, A. Moise, O. Bobis, L. Laslo, S. Bogdanov, Food
Chem. 112 (2009) 863.
[5] C. Cimpoiu, A. Hosu, V. Miclaus, A. Puscas, Spectrochim. Acta A (2012),
http://dx.doi.org/10.1016/j.saa.2012.04.008.
[6] U. Kropf, M. Korosec, J. Bertoncelj, N. Ogrinc, M. Necemer, P. Kump, T. Golop,
Food Chem. 121 (2010) 839.
[7] S. Ouchemoukh, H. Louaileche, P. Schweitzer, Food Control 18 (2007) 52.
[8] A. Guler, A. Bakan, C. Nisbet, O. Yavuz, Food Chem. 105 (2007) 1119.
[9] J.F. Cotte, H. Casabianca, S. Chardon, J. Lheritier, M.F. Grenier-Loustalot, J. Chro-
matogr. A 1021 (2003) 145.
[10] S. Bogdanov, C. Lullmann, P. Martin, W.V.D. Ohe, H. Russmann, G. Vor-
wohl, Swiss Bee Research Centre, FAM, Liebefeld, Switzerland, 1999.
http://www.agroscope.admin.ch/data/publikationen/1315917764 374.pdf
[11] Codex Alimentarius Commission Standards, CODEX STAN 12-1981, 2001.
[12] J.W. White Jr., K. Winters, P. Martin, A. Rossmann, J. AOAC Int. 81 (1998)
610.
[13] G.J. Padovan, D. De Jong, L.P. Rodrigues, J.S. Marchini, Food Chem. 82 (2003)
633.
[14] M. Peris-Tortajada, R. Puchades, A. Maquieira, Food Chem. 43 (1992) 65.
[15] P. Gritzapis, M. Timotheou-Potamia, Anal. Chim. Acta 218 (1989) 37.
[16] J.-H. Le Marec, G. Lesgards, Ann. Falsif. Lexpert. Chim. Toxicol. 83 (1990)
393.
[17] N.G. Carlsson, H. Karlsson, A.S. Sandberg, J. Agric. Food Chem. 40 (1992)
2404.
[18] E. Bugner, M. Feinberg, J. AOAC Int. 75 (1992) 443.
[19] M. Antosova, M. Polakovic, V. Bales, Biotechnol. Tech. 13 (1999) 889.
[20] K.W. Swallow, N.H. Low, J. AOAC Int. 77 (1994) 695.
[21] I. Goodall, J.J. Dennis, I. Parker, M. Sharman, J. Chromatogr. A 706 (1995)
353.
[22] M. Pukl, M. Prosek, J. Planar Chromatogr. Mod. TLC 3 (1990) 173.
[23] K. Reiffova, R. Nemcova, J. Chromatogr. A 1110 (2006) 214.
[24] International Conferences on Harmonization, Note for Guidance on Validation
of Analytical Methods, Text and Methodology. http://www.ich.org/leadmin/
Public Web Site/ICH Products/Guidelines/Quality/Q2 R1/Step4/Q2 R1
Guideline.pdf
[25] C. Cimpoiu, A. Hosu, L. Seserman, M. Sandru, V. Miclaus, J. Sep. Sci. 33 (2010)
3794.