Food Chemistry 172 (2015) 669674

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Detection of honey adulteration with starch syrup by high performance

liquid chromatography
Shaoqing Wang , Qilei Guo, Linlin Wang, Li Lin, Hailiang Shi, Hong Cao, Baosen Cao
China National Food Quality & Safety Supervision and Inspection Center, No. 17 Fengde East Road, Yongfeng Industrial Base, Haidian District, Beijing 100094, China

a r t i c l e

i n f o

Article history:
Received 26 April 2013
Received in revised form 11 June 2014
Accepted 10 September 2014
Available online 17 September 2014
Keywords:
Honey
Starch syrup
Honey adulteration
High fructose syrups (HFS)
High performance liquid chromatogram
(HPLC)

a b s t r a c t
According to saccharide prole comparison between starch syrups and pure honeys analysed through
high performance liquid chromatography (HPLC), a characteristic peak was found at 15.25 min retention
time in HPLC chromatogram of syrup, but no peak was observed at the same retention time in chromatogram of pure honeys. This characteristic peak for syrup was identied as an overlapping peak of oligosaccharides with more than 5 degree of polymerisation (DP) based on HPLC chromatogram comparison
between starch syrup and a series of standard mono-, di- and oligosaccharides of 37 DP. Additionally
syrup content correlated linearly with the height of the characteristic peak of syrup under different slope
in two ranges 2.57.5% and 10100%, respectively. Therefore, the characteristic peak at 15.25 min retention time can serve as a syrup indicator in HPLC analysis of the adulterated honeys. This new HPLC
method for honey adulteration detection was further applied in an authenticity inspection on more than
100 commercial honeys. In addition to the improved accuracy of honey adulteration detection, the proposed HPLC method was simple, low cost and easy practice for honey product quality control by government department considering the popularity of HPLC device and technology.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
With rapid growth in honey production, Chinas honey has an
increasing share of the world honey trade (Wei, Huang, & Yang,
2012). Due to its high cost and worldwide popularity, honey is
always the main target of food adulteration. This has attracted
the attention of many researchers on food authenticity control. In
order to assure Chinese honey product quality, Chinese government has invested a lot of money to develop the new technology
for honey adulteration detection in addition to the common tests
for honey product quality control. In the past several decades,
researchers developed several methods to disclose the honey falsication, such as water, sucrose and 5-hydroxylmethyl-2-furaldehyde (HMF) content analysis and stable carbon isotope ratio
analysis (SCIRA) method (AOAC, 2005; White, 1978; White &
Winters, 1989). Water content analysis was mainly used to control
honey quality to eliminate some immature honey products from
the market and sucrose content analysis was mainly used to monitor honey adulteration with commercial sucrose because authentic honey contains only about 5% sucrose (Guo, Zhu, Liu, & Zhuang,
2010; Wang & Li, 2011). As the byproduct of sucrose acidication,

Corresponding author. Tel.: +86 10 82479325; fax: +86 10 62348045.

E-mail address: wwwshq@hotmail.com (S. Wang).
http://dx.doi.org/10.1016/j.foodchem.2014.09.044
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

HMF concentration was monitored to control honey adulteration

with reducing sugar syrup produced by sucrose acidication.
Recently, this method was doubted because HMF concentration
increases spontaneously when honey is stored in a warm environment (Ajlouni & Sujirapinyokul, 2010). Based on SCIRA method, the
addition of high fructose corn syrup (HFCS) in honey would be
detected when the adulteration is more than 7% (White et al.,
1998; Simsek, Bilsel, & Goren, 2012). However, it is difcult using
this method to disclose the honey adulteration with other high
fructose syrup (HFS) from C3 plant (C3 HFS), such as rice, beet
and cassava etc, because the difference of d13C between C3 HFS
and honey is too small to be used as a standard to prove the adulteration in honey (Krueger & Reesman, 1982). Recently, more
researches have been focused on the carbohydrate prole of honey
again, which is usually applied to control the botanical and geographical origin of honey (Anklam, 1998; Consonni, Cagliani, &
Cogliati, 2013; Cotte, Casabianca, Chardon, Lheritier, & GrenierLoustalot, 2004).
It was well know that honey carbohydrate mainly includes a
complex mixture of 70% monosaccharides (glucose and fructose),
10% disaccharides, and small amount of trisaccharides and tetrasaccharides. No oligosaccharides of more than 5 degree of polymerisation (DP) was found in honey. But a large amount of these high
oligosaccharides was present in starch syrups as the intermediate

S. Wang et al. / Food Chemistry 172 (2015) 669674

product of syrup producing process, enzymolysis of starch (Low,

1998; White, 1978). Therefore these high oligosaccharides may be
taken as an indicator of starch syrups in honey adulteration detection (Morales, Corzo, & Sanz, 2008).
A ngerprint prole of honey oligosaccharides can be obtained
through high performance anion-exchange chromatographypulsed amperometric detection (HPAEC-PAD) system (Morales
et al., 2008; Ouchemoukh, Schweitzer, Bachir Bey, Djoudad-Kadji,
& Louaileche, 2010), gas chromatography (GC) analysis (RuizMatute, Brokl, Soria, & Martnez-Castro, 2010) or Raman spectrum
(zbalci, Boyaci, Topcu, Kadlar, & Tamer, 2013). Before HPAECPAD analysis, the oligosaccharides in honey must be fractionated
by passing the sample through a gel permeation chromatography
(GPC) column or being treated with activated charcoal. In analysis,
a gradient elution solution was used with different concentration
of sodium hydroxide. GCMS provides better resolution for honey
oligosaccharide analysis (disaccharides, trisaccharides and tetrasaccharides). But derivatization, which is an essential step in carbohydrates analysis using GCMS, may result in very complex
chromatograms because of many carbohydrate isomers in nal
reaction
solution
(Ruiz-Matute,
Hernndez-Hernndez,
Rodrguez-Snchez, Sanz, & Martnez-Castro, 2011).
However, so much detail information of oligosaccharides is not
necessary for the detection of honey adulteration. In fact, if only a
certain amount of the oligosaccharides were detected in honeys,
these honey samples could be directly considered being adulterated with starch syrup. Therefore, taking the oligosaccharides peak
at 15.25 min retention time as syrup indicator, a simple, low cost,
environmental and precise method was found for the detection of
honey adulteration with starch syrup through high performance
liquid chromatography (HPLC) equipped with common refractive
index detector (RID). During the whole analysis process, no preliminary treatment and no any organic solvent were needed.
2. Materials and methods
2.1. Materials
2.1.1. Chemical materials and standards
MilliQ water was used in the whole research work in lab;
Glucose, fructose, sucrose were obtained from Beijing chemical
industry group Co. Ltd. (Beijing, China). Maltose, maltotriose,
maltotetraose, maltopentaose, maltohexaose and maltoheptaose
standards were purchased from Tokyo Chemical Industry Co. Ltd.
(Tokyo, Japan). All chemicals used in honey protein purication
were also obtained from Beijing Chemical Ltd. (Beijing, China).

nRIU
300000

nRIU

Indicator peak
of Syrup

20000

250000

Auto signal of RID

670

H15

15000

S2

10000

200000

5000

150000

Inset
figure

100000
S2

50000

14

14.5

15

15.5

16

16.5

min

H15

0
0

10

20

30

40

min

Retention time
Fig. 1. HPLC chromatogram comparison between an authentic chaste honey
sample, H15 and a rice starch HFS sample, S2.

The experimental consumables used in d13C analysis were

obtained from Elemental Microanalysis Ltd. (Okehampton, UK).
2.1.2. Honey and syrup samples collection
The pure honey samples from different nectar sources were
provided by locate Bee farmer in various province of China. Detail
information for these samples was summarised in Table 1. The
commercial honey samples were purchased from the supermarkets located in different provinces in China. The collected syrup
samples included high fructose syrup (HFS) of F55 type: S1, S3
S7 from corn starch, S2 from rice starch and S8 from cavassa
starch; HFS of F42 type: S10S13 from corn starch and S9 from rice
starch; oligoisomaltose syrup: S14S16 from corn starch; oxyl-oligosaccharide syrup: S17 from corn stalk. All the collected syrup
samples were mainly provided by different producers located in
different province in China (Sn was the denoted syrup sample
number and F55 or F42 was the type of high fructose syrup
sample).
2.2. Preparation of articial fraud honey
The series of articial fraud honey samples were prepared by
mixing one authentic acacia honey, H9 with 2.5%, 5%, 7.5%, 10%,
30%, 50%, 75% and 100% (w/w) of rice HFS, S2. The sum mass of
honey and syrup was 1 g in one articial fraud honey sample. Then
the mixture was solved in 99 g pure water. All the mixed sample

Table 1
Geographic origin and nectar source of pure honey samples.
Sample No.

Nectar source

Geographic origin

H1-H14
H15-H33
H34-H41
H42-H44
H45-H49
H50-H52
H53-H56
H57-H61
H62-H63
H64-H65
H66-H68
H69-H70
H71-H72
H73
H74
H75
H76

Acacia
Chaste
Wildower
Rape
Jujube
Citrus
Longan
Lychee
Loquat
Eucalypt
Linden
Osmanthus
Motherwort
Clover
Winter
Buckwheat
Apple

Beijing Miyun, Hebei Xingtai, Liaoning Jinzhou, Shandong Yantai/Linyi/Qingdao, Shanxi Changzhi/Yangquan
Beijing Miyun, Liaoning Jinzhou, Shandong Linyi, Shanxi Niangziguan/Yangquan
Beijing Miyun, Gansu Gannan, Shandong Linyi, Shanxi Yangquan
Gansu Gannan, Jiangsu Wuxi/Nantong
Henan Luoyang, Liaoning Jinzhou, Shandong Taian/Yantai
Fujian Quanzhou, Hunan Changde
Fujian Quanzhou, Guangdong Conghua, Guangxi Guiping, Hainan Haikou
Fujian Quanzhou, Guangdong Conghua, Guangxi Guiping, Hainan Haikou
Fujian Quanzhou, Guangxi Guiping
Hainan Haikou, Guangdong Conghua
Heilongjiang Yichun/Yabuli, Jilin Tonghua
Fujian Quanzhou, Hunan Changde
Hubei Wuhan, Liaoning Jinzhou
Hunan Changde
Guangdong Conghua
Inner Mongolia Chifeng
Liaoning Gaizhou

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S. Wang et al. / Food Chemistry 172 (2015) 669674

solutions were stored overnight at room temperature to further

homogenise the components of the mixture before analysis.
2.3. HPLC analysis
For HPLC analysis, samples were prepared by dissolving 1.0 g
honey or syrup in 100 ml of MilliQ water and homogenised for
10 min in an ultrasound water bath or overnight at room temperature. The sample solution was ltered through 0.45 lm membrane into auto sampler vials for HPLC analysis.
All HPLC analyses were accomplished with an Agilent 1200
liquid chromatography system (Agilent Technologies Deutschland,
Waldbronn, Germany), equipped with a vacuum degasser, a quaternary solvent delivery pump, a thermo-stated column compartment and a refractive index detector (RID). All HPLC analyses
were carried on a Carbomix Ca-NP5: 8% column (7.8  300 mm,
5 lm) at 80 C. Pure water was used as mobile phase in elution.
The ow-rate was 0.3 ml/min. 30 ll of sample solution was

injected for each HPLC analysis. For the analysis of authentic honey
and syrup samples, each sample was analysed twice in triplicate.
For the determination of linearity of peak height, six replicate analyses at each content level of syrup were performed. Finally, for the
commercial samples inspection, all samples were analysed in triplicate at certain concentration.
2.4. Commercial honey samples analysis using SCIRA method
All d13C determination were performed on Continue-Flowing
isotopic ratio mass spectrometer (CF-IRMS), 20-20H from Sercon
(Cheshire, UK). The whole procedure for SCIRA analysis was the
same as that of AOAC998.12 method. In brief, 2 lL of honey or
syrup, or 2.8 mg of protein was sealed into 6  4 mm tin capsules
for d13C determination according to one standard olive oil
(1.5 lL in one capsule, d13Cstd = 28.51 0.16). After nishing the analysis of one batch of samples, d13C value for each sample was calculated and printed out automatically.

Syrup indicator
peak

nRIU
nRIU

(a)

(b)

1200

Auto signal of RID

20000
800

15000
10000

400

Fig.2(b)
5000

0
10

20

30

40

min

nRIU

nRIU

(c)
Auto signal of RID

14

15

15.5

16

min

(d)

1500

Syrup indicator
peak

15000
1000

10000

Fig.2(d)

5000

500

0
0

nRIU
Auto signal of RID

14.5

30000

10

20

30

40

14

min

nRIU

(e)

16000

Syrup indicator
peak

12000

14.5

15

15.5

16

20

30

40

min

(f)
Syrup indicator
peak

20000
8000
10000

4000

0
0

10

20

30

Retention time

40

min

10

min

Retention time

Fig. 2. HPLC chromatograms of the collected syrup samples in this work: (a, b) HFS of F55 type: S1, S3S7 from corn starch, S2 from rice starch and S8 from cavassa starch; (c,
d) HFS of F42 type: S10S13 from corn starch and S9 from rice starch; (e) oligoisomaltose syrup: S14S16 from corn starch; (f) oxyl-oligosaccharide syrup: S17 from corn
stalk.

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S. Wang et al. / Food Chemistry 172 (2015) 669674

nRIU

nRIU

(a)

16000

(b)

100000

Fig.3(b)

Auto signal of RID

Auto signal of RID

120000

80000
60000
H49
H67
H60
H56
H25
H36
H4
H3
H43

40000
20000
0
0

10

20

30

40

50

12000

No Syrup
indicator
peak

8000

4000

min

14

14.5

15

Retention time

15.5

16.5 min

16

Retention time

Fig. 3. HPLC chromatograms of 12 pure honey samples from different nectar source and geographical origin in China, including H43, H3, H4, H36, H25, H56, H60, H67 and
H49. Detail information about the 12 pure honey samples refer to Table 1.

nRIU
10000

nRIU

98 7

(a)

(b)

11200

Auto signal of RID

Auto signal of RID

11000
8000

Fig.4(b)
6000
4000

10800
10600
10400

2000
10200
0
0

10

20

30

40

15

min

15.5

16

Retention time

nRIU
16000

16.5

17

17.5

18 min

Retention time
nRIU
3500

(c)

(d)

12000

Auto signal of RID

Auto signal of RID

3000

Fig.4(d)
8000

4000

2500
2000
1500
1000
500

0
0

10

20

30

40

min

Retention time

15

15.5

16

16.5

17

17.5

min

Retention time

Fig. 4. HPLC chromatogram comparison between (a, b) a series of standard saccharides (1) fructose, (2) glucose, (3) sucrose, (4) D-(+)-maltose, (5) D-(+)-maltotriose, (6)
maltotetraose, (7) maltopentaose, (8) maltohexaose, (9) maltoheptaose and (c, d) the rice starch HFS, S2.

3. Results and discussion

3.1. HPLC chromatogram comparison between honey and starch syrup
For the detection of food falsication in food quality control, the
key is to nd a notable distinction between the adulterant and
authentic food. Furthermore the notable distinction must originate
from the adulterant, but do not being contained in the authentic
food. Therefore, the HPLC chromatograms of one authentic honey
and one rice HFS were compared in Fig. 1 by overlaying signals
to see ne difference between them. Most of the two chromatograms were essentially coincident except one small peak at

15.25 min retention time on the investigated HFS chromatogram.

This small peak can be seen clearly in the enlarged inset gure.
In contrast, no swelling slope was observed on the base line of
honey chromatogram at the same retention time. Whether this
small peak of the HFS chromatogram can be taken as a notable distinction between honey and syrup must be validated through a
detail inspection on a lot of authentic honey from various nectar
sources or geographical origin and various kinds of syrup from different producers. Fig. 2 showed HPLC chromatogram of HFS from
corn, rice or cavassa starch (Fig. 2(a)(d)), oligoisomaltose
(Fig. 2(e)) and oxyl-oligosaccharide syrup (Fig. 2(f)) produced by
different producers in China. All the inspected HFS chromatograms

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S. Wang et al. / Food Chemistry 172 (2015) 669674

showed a small peak at 15.25 min retention time clearly. In the

case of oligoisomaltose and oxyl-oligosaccharide syrup, a very high
peak was also observed at the same retention time on HPLC chromatogram. However, no small peak was observed at the same
retention time on the chromatogram of 12 pure honey samples
from different nectar sources and geographical origin (Fig. 3).
Totally, 76 pure honey samples (Table 1) were checked through
HPLC in the present work (Data was not all shown here). Almost
all the authentic honey samples showed a at base line at
15.25 min retention time, except 3 honey samples showed a negligible swelling slope at 15.25 min retention time on their HPLC
chromatograms. Being compared to the peak of articial fraud
honey sample of 2.5% syrup content (Fig. 5), the biggest swelling
slope of these 3 honey samples was corresponding to about 1%
(w/w) of syrup content, which is statistically negligible. That is to
say, the peak at 15.25 min retention time was a notable distinction
point between honey and syrup samples inspected in this work
and it can be taken as a syrup indicator for honey adulteration
detection in the present new method.
3.2. HPLC chromatogram comparison between syrup and standard
oligosaccharide of various DP

of different DP may remained in the nal syrup product (Reeve,

1992), which may be detected out in the HPLC or GCMS chromatogram of syrup (Low, 1998). In Fig. 4, HPLC chromatogram of
a rice syrup, S2, was compared with that of a series of standard saccharides. As expected, the monosaccharide (both glucose and fructose) peaks of syrup S2 (Fig. 4(c)) appeared at the same retention
time, respectively, as the correspond standard (Fig. 4(a)). In the
case of disaccharides (sucrose and maltose) (Fig. 4(b)), their peaks
overlapped into one peak at about 17.1 min retention time for
syrup S2 (Fig. 4(d)). For tri- and tetrasaccharide (Fig. 4(b)), the
overlapping peak was included in the peak at 16.1 min retention
time for syrup S2 (Fig. 4(d)). Finally, the abutting peaks of maltopentaose, maltohexaose and maltoheptaose were contained in
the peak at 15.25 min retention time for syrup S2, which may contain some other oligosaccharides of higher DP additionally
(Fig. 4(d)). So far, no literatures reported that any maltopentaose,
maltohexaose or maltoheptaose was found in the carbohydrate
prole of honey. Therefore, the indicator peak of syrup at
15.25 min retention time should be corresponding to the oligosaccharides of higher DP than 4.
3.3. Characterisation of the present HPLC method for honey
adulteration detection

In order to indentify the material represented by the indicating

HPLC peak of starch syrup, an HPLC chromatogram comparison
was performed between starch syrup and several standard oligosaccharides of various DP. Usually, syrup was produced through
enzymatic conversion of starch, during which starch was rstly
converted into polysaccharide segments of different DP. In the following, these polysaccharides were hydrolysed into oligosaccharides and nally into monosaccharide. However, during this
enzymatic conversion process some intermediate oligosaccharides

nRIU

As discussed above, the adulterated honey samples with syrup

can be detected according to the presence of this characteristic
peak of syrup at 15.25 min retention time. Here, this method was
characterised on linearity. A series fraud honey samples were prepared in laboratory by intermingling one authentic acacia honey
and one rice HFS sample in different mass proportions, 2.5100%.
HPLC chromatograms of these articial honey samples were shown
by overlaying signals in Fig. 5(a) and (b). Along with the increasing

nRIU

(a)
Auto signal of RID

100%

(b)

250000

16000

200000

75%
12000

150000

50%
8000

Fig.3(b)

100000

30%
4000

50000
0
0

10

20

30

40

min

7.5%
5%
2.5%

10%

14

14.5

15

Retention time

15.5

16

min

Retention time

Height of syrup characteristic peak (nRIU)

2500

(d)

16000

(c)
2000

12000
1500
y = 148.96x + 1168.4
R2 = 0.9952

1000

8000

4000

500

y = 120.09x + 4277.9
R2 = 0.9835

HFS content (m%)

20

40

60

80

100

120

HFS content (m%)

Fig. 5. (a, b) HPLC chromatogram of a series of articial fraud honey samples with different proportion of rice HFS content, 2.5%, 5%, 7.5%, 10%, 30%, 50%, 75% and 100% (w/w);
(c, d) linear regression between syrup indicator peak height and syrup with different slope coefcient in two ranges of syrup content 2.57.5% and 10100% (w/w),
respectively for the above fraud honey samples. Preparation of the series of articial fraud honey samples refer to the part of Section 2.

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S. Wang et al. / Food Chemistry 172 (2015) 669674

amount of syrup, the original at baseline at 15.25 min retention

time changed into a small swelling slope rstly, and then increased
into a higher peak gradually (Fig. 5(b)). The height of the growing
peaks correlated linearly with the adulterated amount of rice HFS,
S2 in two ranges 2.57.5% and 10100% (w/w), respectively
(Fig. 5(c) and (d)). According to Fig. 5(c), as low as 2.5% of HFS in
the adulterated honey samples could still be detected using the
present HPLC method.
In principle, the linear regression equations in Fig. 5(c) and (d)
can be used to calculate syrup content in fraud honey when the
analysis and operation conditions are the same. But, according to
Fig. 2, the inspected various syrup samples had different content
of oligosaccharides of higher DP. Thus, when the equations in
Fig. 5(c) and (d) was used to calculate the added amount of syrup
in fraud honeys, a positive or negative deviation will be found
when the used syrup possesses a higher or lower peak at
15.25 min retention time on HPLC chromatogram. In fact, once a
certain amount of syrup was detected in honey sample, this honey
can be considered fraud no matter how much syrup was used in
the falsication.
3.4. Commercial honey samples inspection by the present HPLC
method
To check the validity of the present HPLC method, an inspection
was carried out on more than 100 commercial honey samples from
different nectar sources and producers. All these honey samples
were examined rstly by AOAC998.12 method. According to the
result of AOAC998.12 analysis, all the inspected samples can be
divided into two groups, the pure honey samples 1 > X > 1
and the adulterated honey samples X > 1 or X < 1
(X = d13CHoney d13CProtein). As expected, syrup was detected
in most of the adulterated honey samples, X > 1 or X < 1.
Contrarily, in some authentic honey samples, 1 > X > 1, a high
proportion of syrup was detected by the proposed HPLC method.
That was conrmed with another starch syrup detection method,
thin-layer chromatography (TLC) method (AOAC, 1988), which
was only valid when the starch syrup content was higher than
10% (w/w). These misjudged fraud honeys may be adulterated with
mixture of C4 and C3 syrups according to a certain ratio.
4. Conclusion
In the present work, an indicator peak of starch syrup on HPLC
chromatograms was found valid for honey adulteration detection
with a detectable syrup content near 2.5% (w/w), which is lower
than that of both SCIRA method 7% (AOAC, 2005) and TLC method
10% (AOAC, 1988). According to the height of this syrup indicator
peak, syrup content in the adulterated honeys can be calculated
out approximately. Especially, the proposed HPLC method can
detect both C4 and C3 starch syrup in honey. However, SCIRA

method was only valid for the detection of C4 starch syrup in

honey. In addition to the increased accuracy for honey adulteration
detection, the proposed new HPLC method was simple, low cost
and easy practice for honey product quality control by government
departments considering the popularity of HPLC device and
technology.
Acknowledgements
This work was supported by the research project 2010QK010
funded by AQSIQ (General Administration of Quality Supervision,
Inspection and Quarantine of the Peoples Republic of China).
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