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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
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,
nRIU
300000
nRIU
Indicator peak
of Syrup
20000
250000
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.
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
671
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
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.
672
nRIU
nRIU
(a)
16000
(b)
100000
Fig.3(b)
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
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
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.
673
nRIU
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
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
20
40
60
80
100
120
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.
674