Article

[Link]/JAFC

2‑Acetylfuran-3-Glucopyranoside as a Novel Marker for the

Detection of Honey Adulterated with Rice Syrup
Xiaofeng Xue,†,‡,⊥ Qiang Wang,†,∥ Yi Li,‡,∥ Liming Wu,‡ Lanzhen Chen,§ Jing Zhao,§ and Fengmao Liu⊥,*
†
Institute of Apiculture Research, Chinese Academy of Agricultural Sciences, Beijing 100093, China
‡
Risk Assessment Laboratory for Bee Products Quality and Safety of Ministry of Agriculture, Beijing 100093, China
§
Bee Product Quality Supervision and Testing Center, Ministry of Agriculture, Beijing 102202, China
⊥
Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, 100193, China

ABSTRACT: The determination of honey authenticity is of importance to ensure its quality and safety. There is an urgent need
of effective methods to detect adulterated honey. A simple, rapid, and effective HPLC−DAD method was developed to detect
honey adulteration by rice syrup, using a characteristic compound from rice syrup, which is presently difficult to detect by current
analytical methods. The characteristic compound was identified as 2-acetylfuran-3-glucopyranoside (AFGP) by MS and NMR.
Based on HPLC analyses, the average concentration of AFGP was 92 ± 60 mg/kg in rice syrup. However, AFGP was not
detected in any of the natural honey samples, so it could be used as a marker for the detection of honey adulteration by rice
syrup. The developed method enabled a rapid detection of honey samples adulterated with 10% rice syrup. Using the developed
method, 16 out of 186 honey samples from some markets were found to be adulterated with rice syrup.
KEYWORDS: 2-acetylfuran-3-glucopyranoside, rice syrup, HPLC-DAD, adulterated honey

■ INTRODUCTION
Honey is a natural sweet substance produced by honey bees
adulterated with syrups that contain low levels of oligosacchar-
ides, difructose anhydrides, and polysaccharides. In many
from flower nectar or plant secretions. Honey bees combine countries, SCIRA has been used as an official analytical method
these plant compounds with specific substances and store them to detect honey adulteration by HFCS. However, this analytical
in honeycombs.1 Honey is widely consumed throughout the method is limited for the detection of syrups from C4 plants
world due to its nutritional and therapeutic properties. In (e.g., corn and sugar cane). It is difficult to detect syrups from
comparison with other sweeteners, honey is highly expensive C3 plants because of their similarities in the isotope
which makes it more prone to adulteration. Authentication of composition of natural honey.8 IR and NMR combined with
honey is of utmost importance for both consumers and the chemometrics constitute the simplest and most rapid screening
food industry. Efficient quality control and assurance of honey methods for the detection of adulterated honey and also they
authentication is required to ensure its quality and safety. rely on simple extraction and sample preparation methods. But,
Generally, honey is adulterated with inexpensive sweeteners IR and NMR do have certain limitations: they require a large
such as corn syrup (CS), invert sugar syrup (IS), and high amount of samples and arduous data analyses.
fructose corn syrup (HFCS). This type of adulteration is Honey adulterated with rice syrup has recently emerged in
difficult to detect due to normal natural variations in honey the honey market. Rice syrup, which is obtained from rice, is
carbohydrates and also similarities in the sugar composition very difficult to detect by the current analytical methods. It is
between these syrups and natural honey.2,3 Despite these impossible to detect this syrup by common SCIRA, as it is from
limitations, several analytical methods are available for the C3 plants and thus follows a similar Calvin cycle of
detection of honey adulteration including thin-layer chroma- photosynthesis as natural honey. Additionally, the production
tography (TLC),2,4 gas chromatography (GC), or gas of rice syrup involves the hydrolysis of polysaccharides and
chromatography−mass spectrometry (GC−MS),5−7 high-per- oligosaccharides, making it difficult to detect the presence of
formance anion exchange chromatography with pulsed rice syrup by TLC and HPAEC−PAD. Currently, honey
amperometric detection (HPAEC−PAD),8−12 stable carbon adulterated with rice syrup has become a serious problem that
isotopic ratio analysis (SCIRA),13−17 infrared spectroscopy affects its quality and safety. Therefore, there is an urgent need
(IR),18−20 and nuclear magnetic resonance (NMR).21 These for rapid, accurate, and reliable methods for the detection of
analytical methods are both reliable and valid for the detection honey adulterated with rice syrup. To the best of our
of honey adulteration by sugar syrups but they have some knowledge, no studies have been conducted about this type
limitations. TLC, GC, GC−MS, and HPAEC−PAD are very of adulteration.
effective for the detection of honey adulterated with HFCS and
use oligosaccharides, polysaccharides, and difructose anhydrides Received: May 2, 2013
as adulteration markers. HPAEC−PAD, which was developed Revised: July 11, 2013
by Mehdi,12 have been used in many testing laboratories in Accepted: July 11, 2013
China. However, HPAEC−PAD is not valid for honey Published: July 11, 2013

© 2013 American Chemical Society 7488 [Link]/10.1021/jf401912u | J. Agric. Food Chem. 2013, 61, 7488−7493
Journal of Agricultural and Food Chemistry Article

Figure 1. Chromatograms of rice syrup and natural honey. Arrow indicates the characteristic compound of rice syrup.

The aims of this study were (i) to find a specific compound Fisher Chemicals (FairLawn, NJ). Deionized water was prepared using
in rice syrup that can be used as a marker for the detection of a Millipore Milli-Q Plus system (Millipore, Bedford, MA).
honey adulteration, and to use this marker in the development Sample Preparation. For sample preparation, 5 g of sample and 5
mL of deionized water were added to a 30 mL centrifuge tube, mixed
of a simple, rapid, and effective analytical method based on high
in a vortex for 3 min, and centrifuged at 10 000 rpm for 10 min. The
performance liquid chromatography with diode array detection resulting supernatant was filtered through a 0.45 μm PVDF
(HPLC−DAD); and (ii) to apply the developed analytical membrane. The filtrate was taken up for further analysis.
method to investigate the presence of adulterated honey in the HPLC Analysis. HPLC analysis was performed using a Dionex
market with rice syrup. Ultimate 3000 HPLC system (Blaine, MN) equipped with a P680

■
pump, an ASI-100 auto injector, a TCC-100 column oven, a DAD 100
MATERIALS AND METHODS detector, and an Agilent ZORBAX AqC18 (100 × 2.1 mm, 3.5 μm)
column. Instrument control and data acquisition were performed using
Sample Collection. One hundred and sixty honey samples were as Chromeleon software. The mobile phase gradient consisted of 99%
follows: Acacia honey (25, labeled A1−A25), jujube honey (20, water (solvent A) and 1% acetonitrile (solvent B) for the first 10 min.
labeled J1−J20), rape honey (30, labeled R1−R30), linden honey (15, Solvent B increased to 10% acetonitrile over 5 min and held for 1 min.
labeled L1−L15), litchi honey (20, labeled LZ1−LZ20), clover honey Solvent B was then adjusted to 90% and held for 6 min. Finally,
(25, labeled C1−C25), and multifloral honey (25, labeled M1−M25) solvent B was reduced to 1% over 1 min and held for 10 min. The flow
were obtained from 34 beekeepers located in Beijing, Zhejiang rate was 0.2 mL/min and the injection volume was 20 μL. The column
province; Hubei, Sichuan, Yunnan, Shannxi, Liaoning, Xinjiang and temperature was maintained at 30 °C. The detection wavelength was
Shandong Province, China, respectively. The honey samples were set to 280 nm, the maximum absorption of the standard compound.
selected according to strict criteria with the quality charter ensuring PHPLC. PHPLC (Agilent, Waldbronn, Germany) consisted of a
their authenticity. 1362A preparative pump equipped with a G1365D multiple
Thirty two representative rice syrup samples (labeled RS1−RS32) wavelength detector (MWD) and a preparative column (Prep C18,
were purchased from some markets in China. Syrup RS26 was added 150 × 21.2 mm, 5 μm, Agilent, Santa Clara, CA). The flow rate was set
in different proportions (10, 20, or 50%, w/w) to an authentic honey to 15 mL/min, the injection volume was 5.0 mL, and the column
sample to intentionally simulate honey adulteration. temperature was maintained at 30 °C. The mobile phase, elution
One hundred and eighty six commercial honey samples (labeled conditions, and detection wavelength were same to those used in
sam1−sam186) were randomly purchased from some markets in HPLC. The sample was added to the column and the elute containing
Beijing, Hebei, Henan, Jiangsu, Zhejiang, and Shandong Province, the desired compound was added several times to the column until the
China. compound was purified. Purified compounds were freeze-dried and
Reagents and Standards. The compound 2-acetylfuran-3- analyzed by both liquid chromatography−quadrupole−time of flight
glucopyranoside (≥95% purity determined by HPLC) was obtained mass spectrometry (LC−Q−TOF MS) and NMR.
by preparative high performance liquid chromatography (PHPLC) LC−Q−TOF MS. LC was performed in an Agilent 1100 Series
and lyophilization. Acetonitrile of HPLC grade was purchased from HPLC (Agilent, Palo Alto, CA, USA) equipped with an autoinjector

7489 [Link]/10.1021/jf401912u | J. Agric. Food Chem. 2013, 61, 7488−7493

Journal of Agricultural and Food Chemistry Article

Figure 2. Mass spectrum of the marker compound. Peak assignment is highlighted.

and a quaternary HPLC pump. The separation was performed on an rice syrup and natural honey, an unknown compound at about
Agilent ZORBAX AqC18 (100 × 2.1 mm, 3.5 μm) column. The 12.5 retention time was detected in rice syrup. This compound
injection volume was 5 μL. The mobile phase consisted of acetonitrile was present in all rice syrup samples, was not seen in the
(solvent A) and water (solvent B) in a 99:1 (v/v) ratio. Total run time
natural honey samples. Therefore this compound was selected
was 15 min with flow rate of 0.2 mL/min.
MS was performed in an Agilent 6510 ESI−Q−TOF. The as a suitable marker.
optimized conditions consisted of a capillary voltage of 4.0 kV in Isolation and Purification of the Marker Compound.
positive ionization mode, a fragmentor voltage of 125 V, and a To obtain more information on the marker compound, we
skimmer voltage of 65 V. Gas temperature was 350 °C, drying gas flow followed a thorough analytical procedure. The rice syrup
rate was 9 L/min, and nebulizer pressure was 45 psi. Nitrogen was sample labeled RS6 was selected as the preparative material due
used as the collision gas. MS spectra were acquired within the range of to its relatively higher content of marker. RS6 (200 g) was
100−1,000 m/z using an extended dynamic range and a scan rate of dissolved in 200 mL of hot water (at approximately 50 °C) and
1.4 spectra/s by varying collision energy with mass. The Mass Hunter
filtered through cotton wool to remove solid particles. The
Workstation software (Version B.01.03) was used. A reference mass
solution containing reference ions (121.0508 and 922.0097) was used filtrate was passed through a chromatography column (400 mm
to maintain mass accuracy during the run time. ×12 mm, C18, 10 μm), which was washed with 50 mL of water
NMR Spectroscopy. 1H NMR (600 MHz), 13C NMR (150 to remove sugars and other polar compounds present in honey.
MHz), and 1H−1H correlation spectroscopy (COSY) were recorded The adsorbed compounds were eluted with acetonitrile (50
at 25 °C using a Varian 600 MHz spectrometer in DMSO-d6 and mL). The eluted solution was concentrated at 40 °C under
DMSO-d6+D2O. Tetramethylsilane (TMS) was used as an internal reduced pressure using a rotary evaporator. The residue was
standard for the determination of chemical shifts. dissolved in 10 mL of water. The final solution was filtered

■ RESULTS AND DISCUSSION

Selection of a Marker Compound in Rice Syrup.
through a 0.45 μm membrane filter and injected into PHPLC.
PHPLC analyses were performed as previously described. The
purified compounds were lyophilized, yielding approximately
Selection of an adulteration marker is crucial for the detection 20 mg of marker compound. Based on HPLC analysis, the
of adulterated honey. Suitable markers can be selected from the purity of the marker compound was >95%. The purity and
adulterants (i.e., foreign additives) or from natural honey. In amount of the marker compound met NMR requirements.
addition to glucose and fructose, natural honey contains organic Chemical Identification of the Marker Compound.
acids, proteins, amino acids, phenolic acids, and flavonoids. Mass determination was performed using LC−Q−TOF MS.
These compounds could be used as markers for the detection Background noise and irrelevant ions were excluded from the
of adulterated honey. For example, honey amino acids and results using molecular feature extraction (MFE) data files, a
proteins have been used to detect adulterated honey.22,23 function of the Mass Hunter Workstation software. Mass values
However, the concentrations of amino acids and proteins vary were obtained within an error of <5 ppm, which allowed us to
considerably depending on the type of honey, making it rapidly generate possible molecular formulas. The mass
difficult to detect adulterated honey when low levels of spectrum of the marker compound is shown in Figure 2. The
adulterants have been added. mass spectrum had an m/z of 289.0920 [M+H]+, calculated for
Ideally, markers for the detection of adulterated honey C12H17O8 (deviation: +0.7 ppm); an m/z of 311.0738 [M
should be selected from the adulterants. Oligosaccharides, +Na]+, calculated for C12H16O8Na (deviation: −0.3 ppm); an
polysaccharides, and difructose anhydrides are suitable markers m/z of 599.1584 [2M+Na]+, calculated for C24H32O16Na
for the detection of honey adulteration by corn-based syrups (deviation: +0.2 ppm) and an fragmentation m/z 127.0391
and sugar cane syrup. Researchers have used SCIRA, TLC, calculated for C6H7O3 (deviation +0.8 ppm). Based on these
HPLC−PAD, and GC to detect honey adulteration using these mass data, the Mass Hunter Workstation software revealed that
markers. In this study, a suitable marker was found from rice a possible molecular formula for the marker compound was
syrup to detect honey adulteration. C12H16O8.
1
In this study, 32 rice syrup samples and 160 natural honey H NMR (Figure 3) and 13C NMR of the marker compound,
samples were analyzed by HPLC−DAD. A representative with an arbitrary numbering of the carbon atoms, refer to the
HPLC−DAD chromatogram is shown in Figure 1. By structure shown in Figure 4. 1H NMR results (600 MHz,
comparing the HPLC−DAD chromatograms obtained from DMSO-d6, δ, ppm, J/Hz) were 7.81 (1H, d, J = 2.1, H-6); 6.78
7490 [Link]/10.1021/jf401912u | J. Agric. Food Chem. 2013, 61, 7488−7493
Journal of Agricultural and Food Chemistry Article

Analyses of AFGP in Rice Syrup and Natural Honey

Samples. The presence of AFGP in all rice syrup and natural
honey samples was analyzed using the developed method.
Representative HPLC chromatograms of AFGP standard, rice
syrup, natural honey and positive sample are shown in Figure
5a−d, respectively. A total of 32 rice syrup samples and 160

Figure 3. 1H NMR spectrum of the marker compound.

Figure 4. Chemical structure of the marker compound.

Figure 5. Representative chromatograms of AFGP standard (a), rice
syrup (b), natural honey(c), and a positive sample (d).
(1H, d, J = 2.1, H-4); 5.50 (1H, d, J = 2.4, H-1′); 5.25 (1H, d, J natural honey samples were analyzed. As expected, AFGP was
= 5.4, OH); 5.06 (1H, d, J = 4.8, OH); 5.05 (1H, d, J = 6.0, detected in all rice syrup samples; its concentration ranged
OH); 4.51 (1H, t, J = 6.0, OH); 3.59−3.17 (6H, m, H2′−H6′); from 32 to 152 mg/kg. On the other hand, AFGP was not
and 2.37 (3H, s, H-1). 13C NMR results (150 MHz, DMSO-d6, detected in any of the natural honey samples.
δ) were 183.2 (C-2); 152.4 (C-4); 149.2 (C-6); 137.4 (C-3); Analyses of AFGP in Adulterated Honey with Rice
104.8 (C-5); 99.3 (C-1′); 74.6 (C-5′); 73.0 (C-3′); 71.1 (C-
Syrup and in Honey Market Samples. Honey samples,
2′); 69.6 (C-4′); 60.6 (C-6′); and 27.3 (C-1). These results
adulterated with 10%, 20%, and 50% rice syrup, were analyzed
were confirmed by distortionless enhancement polarization
using the developed method; the resulting AFGP concen-
transfer (DEPT) and 1H−1H COSY.
trations were 3.3, 6.4, and 15.9 mg/kg, respectively. As shown
Combining the data obtained from NMR and MS analyses,
the marker compound was identified as 1-[3-(3,4,5-trihydroxy- in Figure 6, AFGP was easily detected and quantified at the
6-methoxy-tetrahydro-2H-pyran-2-yloxy) furan-2-yl]-ethanone. 10% adulteration level. Below the 10% adulteration level, AFGP
Using ChemDraw Ultra 7.0(CambridgeSoft Corporation), the was easily detected due to good S/N rates present at the 10%
structure of the marker compound is shown in Figure 4. The adulteration level (26:1). However, this low level of
marker compound was abbreviated as 2-acetylfuran-3-glucopyr- adulteration was not a concern in this study because there is
anoside (i.e., AFGP). little profit to earn when adulteration is at a lower level from
Determination of AFGP by HPLC−DAD. A simple and economics/commerce perspective.
fast HPLC−DAD method was developed for the determination Using the developed HPLC−DAD method, 186 honey
of AFGP. The optimum chromatography conditions were samples from different origins were analyzed. The results
described in the HPLC Analysis section. Six AFGP standard revealed that AFGP was detected in 16 samples with its
solutions (0.5, 1, 10, 50, 100, and 200 mg/L) were prepared by concentration ranged from 21.5 to 145.6 mg/kg. The results are
serial dilution with water and analyzed by HPLC. The AFGP summarized in Table 1. By comparing the results to the AFGP
standard curve resulted in a linear relationship described by y = concentration present in rice syrup, it was concluded that the
0.6354x − 0.0364, where y and x represent the peak area and 16 honey market samples almost consisted of 100% rice syrup.
concentration of the standard solution, respectively. The AFGP Comparison of Results with Traditional Analytical
standard curve had a good linearity in the range of 0.5−200 Methods. To validate the developed method for the detection
mg/L (r = 0.9997). Accuracy, determined at three concen- of adulterated honey with rice syrup, the 16 positive samples
trations (1, 5, and 10 mg/kg) was satisfactory (recovery rates were simultaneously analyzed by two traditional analytical
99.2−101.4% with RSD <2.7%). Based on signal-to-noise rate methods: TLC2 and SCIRA14. The results are summarized in
(S/N) of 3 and 10, the limits of detection (LOD) and Table 1. According to the results, SCIRA was not suitable for
quantification (LOQ) were determined by HPLC−DAD using the detection of honey adulteration by rice syrup because it
the AFGP standard solutions analyzed by HPLC. The resulting failed to detect any positive samples. Only four adulterated
LOD and LOQ were 0.15 mg/kg and 0.35 mg/kg, respectively. samples were detected using TLC method. Thus, TLC was
7491 [Link]/10.1021/jf401912u | J. Agric. Food Chem. 2013, 61, 7488−7493
Journal of Agricultural and Food Chemistry Article

Figure 6. HPLC analysis of natural honey (A), honey adulterated with 10% rice syrup (B), honey adulterated with 20% rice syrup (C), and honey
adulterated 50% rice syrup (D).

Table 1. Detection of 16 Positive Honey Samples by AFGP, with different levels of rice syrup, the developed method
TLC, and SCIRAa enabled a rapid detection of honey adulterated with 10% rice
syrup. In comparison with two traditional methods, the
sample no. AFGP content (mg/kg) TLC SCIRA
developed method was more accurate and effective for the
sam9 132.2 − <7% detection of rice syrup as an adulterant. Using this new method,
sam21 125.4 − <7% 16 out of 186 honey market samples were found to be
sam22 121.0 − <7% adulterated with rice syrup. This result indicates that
sam55 90.4 + <7% adulteration by rice syrup is currently very serious in the
sam73 130.2 − <7% honey market. According to the AFGP structure, this
sam74 128.4 − <7% compound is a furan derivative, thus it may have some toxicity
sam75 132.2 − <7% effects. Therefore, future study is needed to evaluate the risks
sam97 21.5 + <7% associated with AFGP consumption.

■
sam112 60.6 − <7%
sam116 70.4 − <7%
sam119 90.6 + <7%
AUTHOR INFORMATION
sam126 145.6 − <7% Corresponding Author
sam143 140.7 − <7% *Phone/fax: +86 10 62733620; e-mail: lfm2000@[Link]
sam149 139.2 − <7% (F.M.L.).
sam163 120.8 + <7%
Author Contributions
sam177 86.4 − <7% ∥
Q.W. and Y.L.: These authors contributed equally to this
a
“+” detected, “−” not detected. “< 7%” addition of syrup is less than
work.
7%, which is a negative sample (SCIRA method).
Notes
partly effective for detecting adulterated honey with rice syrup. The authors declare no competing financial interest.
Compared with the two traditional methods, the developed
method was very effective in detecting adulterated honey with
rice syrup.
■ ACKNOWLEDGMENTS
The study was jointly supported by National Natural Science
In this study, a characteristic compound was detected in rice Foundation of China (No. 31001038), Technical collaboration
syrup and used as a marker of honey adulteration by rice syrup. on rapid detection, traceability and monitoring for quality and
Newly adulterated honey samples in the market are difficult to safety of agro-products (No. 2012DFA31140) and a special
detect using the available and traditional methods. The fund (NYCYTX-43) from the Apicultural Industry Technology
characteristic compound in rice syrup was 1-[3-(3,4,5- System. Special thanks to Professor Hongwei Li and Zhehui
trihydroxy-6-methoxy-tetrahydro-2H-pyran-2-yloxy)furan-2-yl]- Zhao from NMR center of Peking University for helpful
ethanone, which was abbreviated as 2-acetylfuran-3-glucopyr- discussions in structural identification.

■
anoside or AFGP. The results reveal that AFGP meets the
requirements of an adulteration marker: (a) it is not present in
natural honey, (b) it is easily detected by HPLC, (c) it is ABBREVIATIONS
specifically present in rice syrup, and (d) it is present at a HPLC, high performance liquid chromatography; PHPLC,
certain concentration in rice syrup. The presence of AFGP in preparative high performance liquid chromatography; LC-Q-
honey sample is a clear indication of the addition of rice syrup. TOF MS, liquid chromatography-Quadrupole-time of flight
Using AFGP as a marker, a simple, fast, and effective HPLC mass spectrometry; NMR, nuclear magnetic resonance; COSY,
method was developed for detecting adulterated honey with correlation spectroscopy; DEPT, distortionless enhancement
rice syrup. By analyzing intentionally adulterated honey samples polarization transfer; AFGP, 2-acetylfuran-3-glucopyranoside
7492 [Link]/10.1021/jf401912u | J. Agric. Food Chem. 2013, 61, 7488−7493
Journal of Agricultural and Food Chemistry

■
Article

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