Detection of honey adulteration with starch syrup by high performance liquid chromatography

Accepted Manuscript

Analytical Methods

Detection of honey adulteration with starch syrup by high performance liquidchromatography

Shaoqing Wang, Qilei Guo, Linlin Wang, Li Lin, Tongna Mu, Hong Cao,Baosen Cao

PII: S0308-8146(14)01411-3DOI: http://dx.doi.org/10.1016/j.foodchem.2014.09.044Reference: FOCH 16400

To appear in: Food Chemistry

Received Date: 26 April 2013Revised Date: 11 June 2014Accepted Date: 10 September 2014

Please cite this article as: Wang, S., Guo, Q., Wang, L., Lin, L., Mu, T., Cao, H., Cao, B., Detection of honeyadulteration with starch syrup by high performance liquid chromatography, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.09.044

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http://dx.doi.org/10.1016/j.foodchem.2014.09.044

http://dx.doi.org/http://dx.doi.org/10.1016/j.foodchem.2014.09.044

http://dx.doi.org/http://dx.doi.org/10.1016/j.foodchem.2014.09.044

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Detection of honey adulteration with starch syrup by high performance liquid chromatography 1

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Shaoqing Wang*, Qilei Guo, Linlin Wang, Li Lin, Tongna Mu, Hong Cao, Baosen Cao 7

China National Food Quality & Safety Supervision and Inspection Center, No.17 Fengde East Road, 8

Yongfeng Industrial Base, Haidian District, Beijing, China 100094 9

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*Corresponding author, Tel: +86-10-82479325, Fax: +86-10-62348045, 18

E-mail address: [email protected]. 19

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Abstract 23

According to saccharide profile comparison between starch syrups and pure honeys analyzed through high 24

performance liquid chromatography (HPLC), a characteristic peak was found at 15.25min retention time in 25

HPLC chromatogram of syrup, but no peak was observed at the same retention time in chromatogram of 26

pure honeys. This characteristic peak for syrup was identified as an overlapping peak of oligosaccharides 27

with more than 5 DP based on HPLC chromatogram comparison between starch syrup with a series of 28

standard mono-, di- and oligosaccharides of 3-7 degree of polymerization (DP). Additionally syrup content 29

correlated linearly with the height of the characteristic peak of syrup under different slope in two ranges 30

2.5%~7.5% and 10%~100%, respectively. Therefore, the characteristic peak at 15.25min retention time can 31

serve as a syrup indicator in HPLC analysis of the adulterated honeys. This new HPLC method for honey 32

adulteration detection was further applied in an authenticity inspection on more than 100 commercial 33

honeys. In addition to the improved accuracy of honey adulteration detection, the proposed HPLC method 34

was simple, low cost and easy practice for honey product quality control by government department 35

considering the popularity of HPLC device and technology. 36

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Keywords: honey, starch syrup, honey adulteration, high fructose syrups (HFS), high performance liquid 38

chromatogram (HPLC) 39

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1. Introduction 41

With rapid growth in honey production, China’s honey has an increasing share of the world honey trade 42

(Wei, Huang ＆ Yang, 2012). Due to its high cost and worldwide popularity, honey is always the main 43

target of food adulteration. This has attracted the attention of many researchers on food authenticity control. 44

In order to assure Chinese honey product quality, Chinese government has invested a lot of money to 45

develop the new technology for honey adulteration detection in addition to the common tests for honey 46

product quality control. In the past several decades, researchers developed several methods to disclose the 47

honey falsification, such as water, sucrose and 5-hydroxylmethyl-2-furaldehyde (HMF) content analysis 48

and stable carbon isotope ratio analysis (SCIRA) method (White, & Winters, 1989; White, 1978; AOAC, 49

2005). Water content analysis was mainly used to control honey quality to eliminate some immature honey 50

products from the market and sucrose content analysis was mainly used to monitor honey adulteration with 51

commercial sucrose because authentic honey contains only about 5% sucrose (Guo, et al., 2010; Wang, ＆ 52

Li, 2011). As the byproduct of sucrose acidification, HMF concentration was monitored to control honey 53

adulteration with reducing sugar syrup produced by sucrose acidification. Recently, this method was 54

doubted because HMF concentration increases spontaneously when honey is stored in a warm environment 55

(Ajlouni, ＆ Sujirapinyokul, 2010). Based on SCIRA method, the addition of high fructose corn syrup 56

(HFCS) in honey would be detected when the adulteration is more than 7% (White, et al., 1998; Smsek, 57

Bilsel, ＆Goren, 2012). However, it is difficult using this method to disclose the honey adulteration with 58

other high fructose syrup (HFS) from C3 plant (C3 HFS), such as rice, beet and cassava etc, because the 59

difference of δ13C‰ between C3 HFS and honey is too small to be used as a standard to prove the 60

adulteration in honey (Krueger, & Reesman, 1982). Recently, more researches have been focused on the 61

carbohydrate profile of honey again, which is usually applied to control the botanical and geographical 62

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origin of honey (Anklam, 1998; Cotte, et al., 2004; Consonni, Cagliani, & Cogliati, 2013). 63

It was well know that honey carbohydrate mainly includes a complex mixture of 70% 64

monosaccharides (glucose and fructose), 10% disaccharides, and small amount of trisaccharides and 65

tetrasaccharides. No oligosaccharides of more than 5 degree of polymerization (DP) was found in honey. 66

But a large amount of these high oligosaccharides was present in starch syrups as the intermediate product 67

of syrup producing process, enzymolysis of starch (White Jr., 1978; Low, 1998). Therefore these high 68

oligosaccharides may be taken as an indicator of starch syrups in honey adulteration detection (Morales, 69

Corzo, & Sanz, 2008). 70

A fingerprint profile of honey oligosaccharides can be obtained through high performance 71

anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) system (Morales, Corzo, 72

& Sanz, 2008; Ouchemoukh, et al., 2010), gas chromatography (GC) analysis (Ruiz-Matute, et al., 2010) or 73

Raman spectrum (Özbalci, et al., 2013). Before HPAEC-PAD analysis, the oligosaccharides in honey must 74

be fractionated by passing the sample through a gel permeation chromatography (GPC) column or being 75

treated with activated charcoal. In analysis, a gradient elution solution was used with different 76

concentration of sodium hydroxide. GC-MS provides better resolution for honey oligosaccharide analysis 77

(disaccharides, trisaccharides and tetrasaccharides). But derivatization, which is an essential step in 78

carbohydrates analysis using GC-MS, may result in very complex chromatograms because of many 79

carbohydrate isomers in final reaction solution (Ruiz-Matute, et al., 2011). 80

However, so much detail information of oligosaccharides is not necessary for the detection of honey 81

adulteration. In fact, if only a certain amount of the oligosaccharides were detected in honeys, these honey 82

samples could be directly considered being adulterated with starch syrup. Therefore, taking the 83

oligosaccharides peak at 15.25min retention time as syrup indicator, a simple, low cost, environmental and 84

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precise method was found for the detection of honey adulteration with starch syrup through high 85

performance liquid chromatography (HPLC) equipped with common refractive index detector (RID). 86

During the whole analysis process, no preliminary treatment and no any organic solvent were needed. 87

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2. Materials and methods 89

2.1. Materials 90

2.1.1 Chemial materials and standards 91

MilliQ water was used in the whole research work in lab; Glucose, fructose, sucrose were obtained from 92

Beijing chemical industry group Co., Ltd. (Beijing, China). Maltose, maltotriose, maltotetraose, 93

maltopentaose, maltohexaose and maltoheptaose standards were purchased from Tokyo Chemical Industry 94

Co., Ltd. (Tokyo, Japan). All chemicals used in honey protein purification were also obtained from Beijing 95

Chemical Ltd. (Beijing, China). The experimental consumables used in δ13C‰ analysis were obtained 96

from Elemental Microanalysis Ltd. (Okehampton, UK). 97

2.1.2 honey and syrup samples collection 98

The pure honey samples from different nectar sources were provided by locate Bee farmer in various 99

province of China. Detail information for these samples was summarized in Table 1. The commercial honey 100

samples were purchased from the supermarkets located in different provinces in China. The collected syrup 101

samples included high fructose syrup (HFS) of F55 type: S1, S3~S7 from corn starch, S2 from rice starch 102

and S8 from cavassa starch; HFS of F42 type: S10~ S13 from corn starch and S9 from rice starch; 103

oligoisomaltose syrup: S14~S16 from corn starch; oxyl-oligosaccharide syrup: S17 from corn stalk. All the 104

collected syrup samples were mainly provided by different producers located in different province in China 105

(Sn was the denoted syrup sample number and F55 or F42 was the type of high fructose syrup sample). 106

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2.2. Preparation of artificial fraud honey 107

The series of artificial fraud honey samples were prepared by mixing one authentic acacia honey, H9 with 108

2.5%, 5%, 7.5%, 10%, 30%, 50%, 75% and 100%(w/w) of rice HFS, S2. The sum mass of honey and syrup 109

was 1g in one artificial fraud honey sample. Then the mixture was solved in 99g pure water. All the mixed 110

sample solutions were stored overnight at room temperature to further homogenize the components of the 111

mixture before analysis. 112

2.3. HPLC analysis 113

For HPLC analysis, samples were prepared by dissolving 1.0 g honey or syrup in 100ml of MilliQ water 114

and homogenized for 10 min in an ultrasound water bath or overnight at room temperature. The sample 115

solution was filtered through 0.45µm membrane into auto sampler vials for HPLC analysis. 116

All HPLC analyses were accomplished with an Agilent 1200 liquid chromatography system (Agilent 117

Technologies Deutschland, Waldbronn, Germany), equipped with a vacuum degasser, a quaternary solvent 118

delivery pump, a thermo-stated column compartment and a refractive index detector (RID). All HPLC 119

analyses were carried on a Carbomix Ca-NP5:8% column (7.8×300mm, 5µm) at 80°C. Pure water was 120

used as mobile phase in elution. The flow-rate was 0.3ml/min. 30µl of sample solution was injected for 121

each HPLC analysis. For the analysis of authentic honey and syrup samples, each sample was analysed 122

twice in triplicate. For the determination of linearity of peak height, six replicate analyses at each content 123

level of syrup were performed. Finally, for the commercial samples inspection, all samples were analysed 124

in triplicate at certain concentration. 125

2.4. Commercial honey samples analysis using SCIRA method 126

All δ13C‰ determination were performed on Continue-Flowing isotopic ratio mass spectrometer 127

(CF-IRMS), 20-20H from Sercon (Cheshire, UK). The whole procedure for SCIRA analysis was the same 128

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as that of AOAC998.12 method. In brief, 2 µL of honey or syrup, or 2.8mg of protein was sealed into 6 × 4 129

mm tin capsules for δ13C‰ determination according to one standard olive oil (1.5µL in one capsule, 130

δ13C‰std = -28.51‰±0.16‰ ). After finishing the analysis of one batch of samples, δ13C‰ value for each 131

sample was calculated and printed out automatically. 132

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3. Results and discussion 134

3.1 HPLC chromatogram comparison between honey and starch syrup 135

For the detection of food falsification in food quality control, the key is to find a notable distinction 136

between the adulterant and authentic food. Furthermore the notable distinction must originate from the 137

adulterant, but do not being contained in the authentic food. Therefore, the HPLC chromatograms of one 138

authentic honey and one rice HFS were compared in Fig. 1 by overlaying signals to see fine difference 139

between them. Most of the two chromatograms were essentially coincident except one small peak at 15.25 140

min retention time on the investigated HFS chromatogram. This small peak can be seen clearly in the 141

enlarged inset figure. In contrast, no swelling slope was observed on the base line of honey chromatogram 142

at the same retention time. Whether this small peak of the HFS chromatogram can be taken as a notable 143

distinction between honey and syrup must be validated through a detail inspection on a lot of authentic 144

honey from various nectar sources or geographical origin and various kinds of syrup from different 145

producers. Fig.2 showed HPLC chromatogram of HFS from corn, rice or cavassa starch (Fig.2 (a~d)), 146

oligoisomaltose (Fig.2 (e)) and oxyl-oligosaccharide syrup (Fig.2 (f)) produced by different producers in 147

China. All the inspected HFS chromatograms showed a small peak at 15.25 min retention time clearly. In 148

the case of oligoisomaltose and oxyl-oligosaccharide syrup, a very high peak was also observed at the same 149

retention time on HPLC chromatogram. However, no small peak was observed at the same retention time 150

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on the chromatogram of 12 pure honey samples from different nectar sources and geographical origin (Fig. 151

3). Totally, 76 pure honey samples (Table 1) were checked through HPLC in the present work (Data was 152

not all shown here). Almost all the authentic honey samples showed a flat base line at 15.25 min retention 153

time, except 3 honey samples showed a negligible swelling slope at 15.25 min retention time on their 154

HPLC chromatograms. Being compared to the peak of artificial fraud honey sample of 2.5% syrup content 155

(Fig.5), the biggest swelling slope of these 3 honey samples was corresponding to about 1%(w/w) of syrup 156

content, which is statistically negligible. That is to say, the peak at 15.25 min retention time was a notable 157

distinction point between honey and syrup samples inspected in this work and it can be taken as a syrup 158

indicator for honey adulteration detection in the present new method. 159

3.2 HPLC chromatogram comparison between syrup and standard oligosaccharide of various DP 160

In order to indentify the material represented by the indicating HPLC peak of starch syrup, an HPLC 161

chromatogram comparison was performed between starch syrup and several standard oligosaccharides of 162

various DP. Usually, syrup was produced through enzymatic conversion of starch, during which starch was 163

firstly converted into polysaccharide segments of different DP. In the following, these polysaccharides were 164

hydrolysed into oligosaccharides and finally into monosaccharide. However, during this enzymatic 165

conversion process some intermediate oligosaccharides of different DP may remained in the final syrup 166

product (Reeve, 1992), which may be detected out in the HPLC or GC-MS chromatogram of syrup (Low, 167

1998). In Fig. 4, HPLC chromatogram of a rice syrup, S2, was compared with that of a series of standard 168

saccharides. As expected, the monosaccharide (both glucose and fructose) peaks of syrup S2 (Fig. 4(c)) 169

appeared at the same retention time, respectively, as the correspond standard (Fig. 4(a)). In the case of 170

disaccharides (sucrose and maltose) (Fig. 4(b)), their peaks overlapped into one peak at about 17.1min 171

retention time for syrup S2 (Fig. 4(d)). For tri- and tetrasaccharide (Fig. 4(b)), the overlapping peak was 172

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included in the peak at 16.1min retention time for syrup S2 (Fig. 4(d)). Finally, the abutting peaks of 173

maltopentaose, maltohexaose and maltoheptaose were contained in the peak at 15.25 min retention time for 174

syrup S2, which may contain some other oligosaccharides of higher DP additionally (Fig. 4(d)). So far, no 175

literatures reported that any maltopentaose, maltohexaose or maltoheptaose was found in the carbohydrate 176

profile of honey. Therefore, the indicator peak of syrup at 15.25 min retention time should be 177

corresponding to the oligosaccharides of higher DP than 4. 178

3.3 Characterization of the present HPLC method for honey adulteration detection 179

As discussed above, the adulterated honey samples with syrup can be detected according to the presence of 180

this characteristic peak of syrup at 15.25 min retention time. Here, this method was characterized on 181

linearity. A series fraud honey samples were prepared in laboratory by intermingling one authentic acacia 182

honey and one rice HFS sample in different mass proportions, 2.5%~100%. HPLC chromatograms of these 183

artificial honey samples were shown by overlaying signals in Fig. 5(a, b). Along with the increasing amount 184

of syrup, the original flat baseline at 15.25min retention time changed into a small swelling slope firstly, 185

and then increased into a higher peak gradually (Fig. 5(b)). The height of the growing peaks correlated 186

linearly with the adulterated amount of rice HFS, S2 in two ranges 2.5%~7.5% and 10%~100% (w/w), 187

respectively (Fig. 5(c,d)). According to Fig. 5(c), as low as 2.5% of HFS in the adulterated honey samples 188

could still be detected using the present HPLC method. 189

In principle, the linear regression equations in Fig. 5 (c, d) can be used to calculate syrup content in 190

fraud honey when the analysis and operation conditions are the same. But, according to Fig. 2, the 191

inspected various syrup samples had different content of oligosaccharides of higher DP. Thus, when the 192

equations in Fig. 5(c, d) was used to calculate the added amount of syrup in fraud honeys, a positive or 193

negative deviation will be found when the used syrup possesses a higher or lower peak at 15.25min 194

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retention time on HPLC chromatogram. In fact, once a certain amount of syrup was detected in honey 195

sample, this honey can be considered fraud no matter how much syrup was used in the falsification. 196

3.4 Commercial honey samples inspection by the present HPLC method 197

To check the validity of the present HPLC method, an inspection was carried out on more than 100 198

commercial honey samples from different nectar sources and producers. All these honey samples were 199

examined firstly by AOAC998.12 method. According to the result of AOAC998.12 analysis, all the 200

inspected samples can be divided into two groups, the pure honey samples 1>X‰>-1 and the adulterated 201

honey samples X‰>1 or X‰<-1(X‰ = δ13CHoney‰ - δ13CProtein‰). As expected, syrup was detected in 202

most of the adulterated honey samples，X‰>1 or X‰<-1. Contrarily, in some “authentic” honey samples, 203

1>X‰>-1, a high proportion of syrup was detected by the proposed HPLC method. That was confirmed 204

with another starch syrup detection method, thin-layer chromatography (TLC) method (AOAC, 1988), 205

which was only valid when the starch syrup content was higher than 10% (w/w). These misjudged fraud 206

honeys may be adulterated with mixture of C4 and C3 syrups according to a certain ratio. 207

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Conclusion 209

In the present work, an indicator peak of starch syrup on HPLC chromatograms was found valid for honey 210

adulteration detection with a detectable syrup content near 2.5% (w/w), which is lower that of both SCIRA 211

method 7% (AOAC, 2005) and TLC method 10% (AOAC, 1988). According to the height of this syrup 212

indicator peak, syrup content in the adulterated honeys can be calculated out approximately. Especially, the 213

proposed HPLC method can detect both C4 and C3 starch syrup in honey. However, SCIRA method was 214

only valid for the detection of C4 starch syrup in honey. In addition to the increased accuracy for honey 215

adulteration detection, the proposed new HPLC method was simple, low cost and easy practice for honey 216

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product quality control by government departments considering the popularity of HPLC device and 217

technology. 218

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Reference 220

Ajlouni, S., ＆ Sujirapinyokul, P. (2010). Hydroxymethylfurfuraldehyde and amylase contents in 221

Australian honey. Food Chemistry, 119, 1000-1005. 222

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of honey. Food Chemistry, 63(4), 549-562. 224

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of Analysis, (979.22), 71, 88. 228

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analysis. Food Control, 32, 543~548. 230

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analysis of sugars applied to the characterisation of monofloral honey. Analytical and Bioanalytical 232

Chemistry, 380(4), 698-705. 233

Guo, W., Zhu, X., Liu, Y., ＆ Zhuang, H. (2010). Sugar and water contents of honey with dielectric 234

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Low, N. H. (1998). Oligosaccharides analysis. In P. R. Ashurst & M. J. Dennis (Eds.), Analytical methods 238

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on food authentication (la Ed., pp. 97-136). London: Blackie Academic and Professional Ed. 239

Morales, V., Corzo, N., & Sanz, M. L. (2008). HPAEC-PAD oligosaccharide analysis to detect 240

adulaterations of honey with sugar syrups. Food Chemistry, 107, 922-928. 241

Ouchemoukh, S., Schweitzer, P., Bachir Bey, M., Djoudad-Kadji, H., & Louaileche, H. (2010). HPLC 242

sugar profiles of Algerian honeys. Food Chemistry, 121, 561-568. 243

Özbalci, B., Boyaci, İ. H., Topcu A., Kadılar, C., & Tamer, U. (2013). Rapid analysis of sugars in honey by 244

processing Raman spectrum using chemometric methods and artificial neural networks. Food 245

Chemistry, 136, 1444-1452. 246

Reeve, A. (1992). Starch hydrolysis: Process and equipment. In F. W. Schenk & R. F. Hebeda (Eds.), Starch 247

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Ruiz-Matute, A. I., Brokl, M., Soria, A. C., & Martínez-Castro, I. (2010). Gas chromatographic-mass 249

spectrometric characterization of tri- and tetrasaccharides in honey. Food chemistry, 120, 637-642. 250

Ruiz-Matute, A. I., Hernández-Hernández, O., Rodríguez-Sánchez, S., Sanz, M. L., Martínez-Castro, I. 251

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White Jr, J. W. (1978). Honey. Advances in Food Research, 24, 287-374. 261

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907-911. 264

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Figure Captions: 266

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Figure 1 HPLC chromatogram comparison between an authentic acacia honey sample, H9 and a rice starch 268

HFS sample, S2. 269

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Figure 2 HPLC chromatograms of the collected syrup samples in this work: (a, b) HFS of F55 type: S1, 271

S3~S7 from corn starch, S2 from rice starch and S8 from cavassa starch; (c, d) HFS of F42 type: S10~ S13 272

from corn starch and S9 from rice starch; (e) oligoisomaltose syrup: S14~S16 from corn starch; (f) 273

oxyl-oligosaccharide syrup: S17 from corn stalk. 274

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Figure 3 HPLC chromatograms of 12 pure honey samples from different nectar source and geographical 276

origin in China, including H43, H3, H4, H36, H25, H56, H60, H67 and H49. Detail information about the 277

12 pure honey samples refer to Table 1. 278

279

Figure 4 HPLC chromatogram comparison between (a, b) a series of standard saccharides 1 Fructose, 2 280

Glucose, 3 Sucrose, 4 D-(+)-Maltose, 5 D-(+)-Maltotriose, 6 Maltotetraose, 7 Maltopentaose, 8 281

Maltohexaose, 9 Maltoheptaose and (c, d) the rice starch HFS, S2. 282

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Figure 5 (a, b) HPLC chromatogram of a series of artificial fraud honey samples with different proportion 284

of rice HFS content, 2.5%, 5%, 7.5%, 10%, 30%, 50%, 75% and 100% (w/w); (c, d) Linear regression 285

between syrup indicator peak height and syrup with different slope coefficient in two ranges of syrup 286

content 2.5%~7.5% and 10%~100% (w/w), respectively for the above fraud honey samples. Preparation of 287

the series of artificial fraud honey samples refer to the part of 2 Materials and methods. 288

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S. Wang et al. Detection of honey adulteration with starch syrups by high performance liquid 291

chromatography 292

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Table 1 Geographic origin and nectar source of pure honey samples， 294

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Sample No. Nectar Source Geographic Origin

H1-H14 Acacia Beijing Miyun, Hebei Xingtai, Liaoning Jinzhou, Shandong

Yantai/Linyi/Qingdao, Shanxi Changzhi/Yangquan

H15-H33 Chaste Beijing Miyun, Liaoning Jinzhou, Shandong Linyi, Shanxi

Niangziguan/Yangquan,

H34-H41 Wildflower Beijing Miyun, Gansu Gannan, Shandong Linyi, Shanxi Yangquan,

H42-H44 Rape Gansu Gannan, Jiangsu Wuxi/Nantong,

H45-H49 Jujube Henan Luoyang, Liaoning Jinzhou, Shandong Taian/Yantai,

H50-H52 Citrus Fujian Quanzhou, Hunan Changde

H53-H56 Longan Fujian Quanzhou, Guangdong Conghua, Guangxi Guiping, Hainan Haikou,

H57-H61 Lychee Fujian Quanzhou, Guangdong Conghua, Guangxi Guiping, Hainan Haikou,

H62-H63 Loquat Fujian Quanzhou, Guangxi Guiping

H64-H65 Eucalypt Hainan Haikou, Guangdong Conghua

H66-H68 Linden Heilongjiang Yichun/Yabuli, Jilin Tonghua

H69-H70 Osmanthus Fujian Quanzhou, Hunan Changde

H71-H72 Motherwort Hubei Wuhan, Liaoning Jinzhou

H73 Clover Hunan Changde

H74 Winter Guangdong Conghua

H75 Buckwheat Inner Mongolia Chifeng

H76 Apple Liaoning Gaizhou

296

297

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nRIU

0

400

800

1200

min14 14.5 15 15.5 16 min14 14.5 15 15.5 16

nRIU

0

400

800

1200

nRIU

0

400

800

1200

(d)


min14 14.5 15 15.5 16

nRIU

0

500

1000

1500

(d)


min14 14.5 15 15.5 16

nRIU

0

500

1000

1500

min14 14.5 15 15.5 16 min14 14.5 15 15.5 16

nRIU

0

500

1000

1500

min0 10 20 30 40

nRIU

0

5000

10000

15000

20000

(a)

Aut

o s

igna

l of

RID

Fig.2(b)

min0 10 20 30 40

nRIU

0

5000

10000

15000

20000

min0 10 20 30 40 min0 10 20 30 40

nRIU

0

5000

10000

15000

20000

nRIU

0

5000

10000

15000

20000

(a)

Aut

o s

igna

l of

RID

Fig.2(b)

Aut

o si

gnal

of

RID

(c)

min0 10 20 30 40

nRIU

0

5000

10000

15000

Fig.2(d)Aut

o si

gnal

of

RID

(c)

min0 10 20 30 40

nRIU

0

5000

10000

15000

min0 10 20 30 40 min0 10 20 30 40

nRIU

0

5000

10000

15000

nRIU

0

5000

10000

15000

Fig.2(d)

Retention time

Aut

o s

ign

al o

f R

ID

(e)

min0 10 20 30 40

nRIU

0

10000

20000


peak

Retention time

Aut

o s

ign

al o

f R

ID

(e)

min0 10 20 30 40

nRIU

0

10000

20000

30000

min0 10 20 30 40

nRIU

0

10000

20000


peak

Retention time

(f)

min0 10 20 30 40

nRIU

0

4000

8000

12000

16000


Retention time

(f)

min0 10 20 30 40

nRIU

0

4000

8000

12000

16000

min0 10 20 30 40 min0 10 20 30 40

nRIU

0

4000

8000

12000

16000

nRIU

0

4000

8000

12000

16000


18


chromatography 314

315

316

317

Figure 3 318

319

320

321

No Syrup indicator

peak

min14 14.5 15 15.5 16 16.5

0

nRIU

4000

8000

12000

16000

Retention time

Aut

o si

gnal

of

RID

(b)

Fig.3(b)

(a)

min0 10 20 30 40 50

nRIU

0

20000

40000

60000

80000

100000

120000

Retention time

Aut

o si

gnal

of

RID

H49H67

H60H56

H4H3

H25H36

H43

No Syrup indicator

peak

min14 14.5 15 15.5 16 16.5

0

nRIU

4000

8000

12000

16000

Retention time

Aut

o si

gnal

of

RID

(b)

No Syrup indicator

peak

min14 14.5 15 15.5 16 16.5

0

nRIU

4000

8000

12000

16000

Retention time

Aut

o si

gnal

of

RID

(b)

Fig.3(b)

(a)

min0 10 20 30 40 50

nRIU

0

20000

40000

60000

80000

100000

120000

Retention time

Aut

o si

gnal

of

RID

H49H67

H60H56

H4H3

H25H36

H43

Fig.3(b)

(a)

min0 10 20 30 40 50

nRIU

0

20000

40000

60000

80000

100000

120000

min0 10 20 30 40 50 min0 10 20 30 40 50

nRIU

0

20000

40000

60000

80000

100000

120000

nRIU

0

20000

40000

60000

80000

100000

120000

Retention time

Aut

o si

gnal

of

RID

H49H67

H60H56

H4H3

H25H36

H43

19


chromatography 323

324

325

Figure 4 326

327

328

329

min15 15.5 16 16.5 17 17.5 18

nRIU

10200

10400

10600

10800

11000

11200

Aut

o si

gnal

of

RID

Retention time

(b)

min15 15.5 16 16.5 17 17.5

nRIU

0

500

1000

1500

2000

2500

3000

3500

Retention time

Aut

o si

gnal

of

RID

(d)

3456789

Aut

o si

gnal

of

RID

Retention time

(a)

Fig.4(b)

12

min0 10 20 30 40

nRIU

0

2000

4000

6000

8000

10000

Retention time

Aut

o si

gnal

of

RID

(c)

Fig.4(d)

min10 20 30 400

nRIU

0

4000

8000

12000

16000

min15 15.5 16 16.5 17 17.5 18 min15 15.5 16 16.5 17 17.5 18

nRIU

10200

10400

10600

10800

11000

11200

nRIU

10200

10400

10600

10800

11000

11200

Aut

o si

gnal

of

RID

Retention time

(b)

min15 15.5 16 16.5 17 17.5

nRIU

0

500

1000

1500

2000

2500

3000

3500

Retention time

Aut

o si

gnal

of

RID

(d)

min15 15.5 16 16.5 17 17.5

nRIU

0

500

1000

1500

2000

2500

3000

3500

min15 15.5 16 16.5 17 17.5

nRIU

0

500

1000

1500

2000

2500

3000

3500

Retention time

Aut

o si

gnal

of

RID

(d)

3456789

Aut

o si

gnal

of

RID

Retention time

(a)

Fig.4(b)

12

min0 10 20 30 40

nRIU

0

2000

4000

6000

8000

10000

Aut

o si

gnal

of

RID

Retention time

(a)

Fig.4(b)

12

min0 10 20 30 40

nRIU

0

2000

4000

6000

8000

10000

min0 10 20 30 40 min0 10 20 30 40

nRIU

0

2000

4000

6000

8000

10000

nRIU

0

2000

4000

6000

8000

10000

Retention time

Aut

o si

gnal

of

RID

(c)

Fig.4(d)

min10 20 30 400

nRIU

0

4000

8000

12000

16000

Retention time

Aut

o si

gnal

of

RID

(c)

Fig.4(d)

min10 20 30 400

nRIU

0

4000

8000

12000

16000

min10 20 30 400 min10 20 30 400

nRIU

0

4000

8000

12000

16000

nRIU

0

4000

8000

12000

16000

20


chromatography 331

332

333

334

335

Figure 5 336

337

338

339

340

HFS content（m%)

y = 148.96x + 1168.4

R2 = 0.9952

0

500

1000

1500

2000

2500

0 2 4 6 8

Hei

ght o

f sy

rup

char

acte

rist

ic p

eak（

nRIU

）

y = 120.09x + 4277.9

R2 = 0.9835

0

4000

8000

12000

16000

0 20 40 60 80 100 120

HFS content（m%)

(c) (d)

Retention time

min14 14.5 15 15.5 16

nRIU

0

4000

8000

12000

16000

100%

75%

50%

30%

10% 7.5%5%2.5%

(b)

Fig.3(b)

Retention time

Aut

o s

igna

l of

RID

(a)

min0 10 20 30 40

nRIU

0

50000

100000

150000

200000

250000

HFS content（m%)

y = 148.96x + 1168.4

R2 = 0.9952

0

500

1000

1500

2000

2500

0 2 4 6 8

Hei

ght o

f sy

rup

char

acte

rist

ic p

eak（

nRIU

）

y = 120.09x + 4277.9

R2 = 0.9835

0

4000

8000

12000

16000

0 20 40 60 80 100 120

HFS content（m%)

(c) (d)

HFS content（m%)

y = 148.96x + 1168.4

R2 = 0.9952

0

500

1000

1500

2000

2500

0 2 4 6 8

Hei

ght o

f sy

rup

char

acte

rist

ic p

eak（

nRIU

）

y = 120.09x + 4277.9

R2 = 0.9835

0

4000

8000

12000

16000

0 20 40 60 80 100 120

HFS content（m%)HFS content（m%)

y = 148.96x + 1168.4

R2 = 0.9952

0

500

1000

1500

2000

2500

0 2 4 6 8

Hei

ght o

f sy

rup

char

acte

rist

ic p

eak（

nRIU

）

y = 148.96x + 1168.4

R2 = 0.9952

0

500

1000

1500

2000

2500

0 2 4 6 8

y = 148.96x + 1168.4

R2 = 0.9952

0

500

1000

1500

2000

2500

0 2 4 6 8

Hei

ght o

f sy

rup

char

acte

rist

ic p

eak（

nRIU

）

y = 120.09x + 4277.9

R2 = 0.9835

0

4000

8000

12000

16000

0 20 40 60 80 100 120

y = 120.09x + 4277.9

R2 = 0.9835

0

4000

8000

12000

16000

0 20 40 60 80 100 120

HFS content（m%)

(c) (d)

Retention time

min14 14.5 15 15.5 16

nRIU

0

4000

8000

12000

16000

100%

75%

50%

30%

10% 7.5%5%2.5%

(b)

Fig.3(b)

Retention time

Aut

o s

igna

l of

RID

(a)

min0 10 20 30 40

nRIU

0

50000

100000

150000

200000

250000

Retention time

min14 14.5 15 15.5 16

nRIU

0

4000

8000

12000

16000

100%

75%

50%

30%

10% 7.5%5%2.5%

(b)

Retention time

min14 14.5 15 15.5 16

nRIU

0

4000

8000

12000

16000

100%

75%

50%

30%

10% 7.5%5%2.5%

(b)

min14 14.5 15 15.5 16

nRIU

0

4000

8000

12000

16000

100%

75%

50%

30%

10% 7.5%5%2.5%

(b)

Fig.3(b)

Retention time

Aut

o s

igna

l of

RID

(a)

min0 10 20 30 40

nRIU

0

50000

100000

150000

200000

250000

Fig.3(b)

Retention time

Aut

o s

igna

l of

RID

(a)

min0 10 20 30 40

nRIU

0

50000

100000

150000

200000

250000

(a)

min0 10 20 30 40 min0 10 20 30 40

nRIU

0

50000

100000

150000

200000

250000

21


chromatography 342

343

Research Highlights 344

345

A syrup indicator peak was found on HPLC chromatogram of starch syrup in comparison with pure 346

honeys. 347

The syrup indicator peak was identified as oligosaccharides with more than 5 degree of polymerization. 348

The syrup indicator peak height correlated linearly with syrup content in the adulterated honey samples. 349

This HPLC method was used to examine some commercial honey samples. 350

The present HPLC method was a simple, fast, low cost and easy practice method for the detection of honey 351

adulteration with starch syrup. 352

353

354

355

356

357

358

359

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361

Documents

Detection of honey adulteration with starch syrup by high performance liquid chromatography