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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2019 Nov 29;57(4):1216–1232. doi: 10.1007/s13197-019-04153-2

A comprehensive analysis of 13C isotope ratios data of authentic honey types produced in China using the EA-IRMS and LC-IRMS

JinZhong Xu 1,✉, Xiuhong Liu 2, Bin Wu 3, YanZhong Cao 4
PMCID: PMC7054487  PMID: 32180618

Abstract

In the current study, we have comprehensively analyzed different kinds of pure honey which was produced in various areas in China according to δ13C-EA -IRMS (AOAC method 998.12) and δ13C-LC-IRMS (proposed by the Intertek laboratory in Europe) methods. As for the δ13C-EA -IRMS method, the study was confirmed that the C4 sugar of all authentic honey samples was qualified. Further inter-laboratory comparison experiments using the δ13C-LC-IRMS method found that all authentic honey samples had Δδ13C (‰) values within the naturally occurring range of ± 1‰ for Δδ13C (‰) fru-glu. However, about 70% samples had Δδ13C (‰) values outside the range of ± 2.1‰ for Δδ13C (‰) max., indicating that a large proportion of pure honey in China can’t pass the δ13C-LC-IRMS test, although these honeys were extracted from unadulterated sources. Based on the present findings, we consider that the δ13C-LC-IRMS method is not appropriate to reliably detect adulterated honeys with C3 sugars in China.

Keywords: Elemental analyzer-isotope ratio mass spectrometry (EA-IRMS), Liquid chromatography-isotope ratio mass spectrometry (LC-IRMS), Pure honey, Adulterated honey, C4 sugar, C3 sugar

Introduction

Honey, originating predominantly from flower nectar or honeydew, is the oldest sweet substance known to people. Due to its large nutritive and high medicinal value, the demand for authentic honey was growing fast in the past decades (Cabanero et al. 2006; Padovan et al. 2003; Ruiz-Matute et al. 2010). The mainly ingredients of honey are carbohydrates such as fructose (30–44%), glucose (22–40%), and sucrose (0.25–7.7%). It was found that honey contains more carbohydrates than any other animal product. Honey authenticity has two different aspects. The first of these is refers to its content, it must be 100 percent real honey and has not been contaminated with any sugar syrup. The second is concerning its geographical and botanical origin. Both aspects, content and origin are required for honey to be authentic. The high demand for authentic honey coupled with poverty results in its scarcity and a large market profit for honey sales. Hence, honey is always a target for adulteration, with acid-inverted sugar syrups, corn syrups, and syrups of natural origin (such as maple, cane sugar, beet sugar, etc.) added into it (Ruiz-Matute et al. 2010; www.nhb.org, 2010; Padovan et al. 2007). It is very difficult to estimate the differences between adulterated honey and real honey for consumers. Laboratory tests are therefore needed to prove the difference.

There are many analytical techniques have been reported to test the adulteration of honey by analyzing different physicochemical parameters, such as using HPLC method to analyze the botanical origin of sugars in honey (Földahzi 1994), developing an effective anionic chromatographic method (HPAEC-PAD) for honey analysis and adulteration detection (Cordella et al. 2005), coupling Fourier transform mid-infrared spectroscopic technique and chemometric methods to detect honey adulterated by various syrups (Kelly et al. 2006), using isotope ratio mass spectrometry (IRMS) couple with chemometric approach to investigate the impact of botanical origin and harvesting period of Greek honey (Karabagias et al. 2016), coupling one-dimensional and two-dimensional high-resolution nuclear magnetic resonance with multivariate statistical analysis to detect adulteration in honey falsified by intentional addition of different concentrations of commercial sugar syrup (Bertelli et al. 2010). In addition, MS was always used to confirm the structure of the markers such as polysaccharides, difructose anhydrides (DFAs), and 2-acetylfuran-3-glucopyranoside (AFGP) that exist in adulterants while not exist in natural honey (Xue et al. 2013). It is very difficult to detect some of these common adulterants simultaneously, therefore, Du et al. (2015) generating a small-scale database containing the specific chromatographic and mass spectrometry information on sugar syrup markers and developing a ultrahigh-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF–MS) method for the detection of adulterated honey. Apart from these, gas chromatography–mass spectrometry (GC–MS) method (Ruiz-Matute et al. 2007) and the Raman spectroscopy were also used to detect honey adulteration (Oroian et al. 2018), and so on. Although these techniques could detect the adulterated honey to some extent, these methods require complex sample preparation and laborious data analyses, highly time-consuming, need to detect many samples to generate database, or involve very expensive equipment that many laboratories can’t afford to buy, some of these methods cannot give satisfactory results in the detection of more sophisticated adulterations with syrups to imitate the sugar profile of honey, such as adulteration with sugar syrups from C4 plants like corn and cane or C3 plants like beet and rice. As for the former, it was easy to detect the addition of C4 sugar because of its average δ13C value (i.e., the 13C/12C isotope ratio related to Vienna Pee Dee Belemnite as reference standard material, expressed in  ‰) of –9.7‰ compared to honey with an average δ13C value of –25.4‰ by involving stable carbon isotope ratio mass spectrometry (SCIRA, EA-IRMS) method (Simsek et al. 2012), which was proposed by JW White (by AOAC 2000). Pang et al. (2006) did a meaningful work to analyze many honey samples from 135 honey-related enterprises in 25 provinces of China by IRMS. The results showed that White’s theory, that was, Δδ13C (‰) protein − honey: ≥ –1.0‰, was applicable to Chinese honeys and honeys from all over the world. However, the origin of all the samples that analyzed by Pang et al. was no guarantee of authenticity. While as for the latter adulteration, Elflein and Raezke (2008) reported for the first time that this problem could be solved by coupling an isotope ratio mass spectrometer both to an elemental analyzer and to a liquid chromatograph (EA/LC-IRMS). In their work they measured the δ13C values of bulk honey, isolated protein, fructose, glucose, disaccharides, and trisaccharides in authentic honeys and calculated the natural occurring differences between these values (Δδ13C values), and it was proposed that the Δδ13C values of authentic honey should be fall in the naturally range of ± 1‰ for Δδ13C (fructose–glucose) and ± 2.1‰ for Δδ13C (‰) max. (maximum difference between all measured δ13C values), respectively. This test method is now used as the main generic C3 sugars adulterated honey detection in the international trade for many years.

In this article, we did an investigation of different kind of pure honey in different areas in China according to δ13C-EA -IRMS and δ13C-LC-IRMS methods. These pure honey samples were collected by the international independent institute (SGS) to keep the purity of these samples. Inter-laboratory comparison experiments from Jiangsu Entry-exit Inspection and Quarantine Bureau Lab and Qinhuangdao Entry-exit Inspection and Quarantine Bureau Lab were conducted.

Materials and methods

Reagents

Ultrapure water was produced from Millipore (18.2 MΩ). Crystalline phosphoric acid, sodium peroxodisulfate, and sodium tungstate dihydrate were purchased from Fluka (puriss. p.a. ≥ 99%).

Standards

For the carbon stable isotope reference standard of LC-IRMS, sucrose IAEA-CH6 (δ13C value: − 10.45‰), was obtained from the IAEA. For the carbon stable isotope reference standard of EA-IRMS, beet sugar IA-R00513C (δ13C value: – 26.03‰), was obtained from Sercon. Quality control sample was D-(+)-melibiose and was purchased from Fluka (puriss. p.a. ≥ 99%,).

Sample Preparation

For the EA-IRMS (determination of δ13C values of honey and protein) analysis, the following procedures were applied: (a) For honey samples, 1 g honey sample were weighted and dissolved with 1 ml ultrapure water in a centrifuged tube, the tube was centrifuged for 5 min at 3500 rpm to remove insoluble impurities, then 3 µL of original honey sample was pipetted into a tin capsule. (b) For honey protein samples, 30–35 g of honey sample was mixed well with 20 mL of water in a 50 mL centrifuge tube. Then 3 mL of 100 g/L sodium tungstate solution and 3 mL of 0.335 mol/L sulfuric acid solution were added and mixed well. The centrifuge tube was subsequently placed in an 80 °C water bath for at least 30 min until a visible protein floc had formed, with the tube being swirled for 20 s at 5–10 min intervals during the heating process. Then the tube was centrifuged for 5 min at 3500 rpm and the supernatant were decanted. A further 40 mL of water was added to the centrifuge tube to wash the precipitated protein, the tube was centrifuged for 5 min at 3500 rpm and the supernatant was decanted; this procedure was repeated three times to get the precipitated protein. 100–200 μg of each protein were weighed in small tin capsules using an MX-5 ultra-microbalance from Mettler-Toledo, Giessen, Germany.

For the LC-IRMS analysis: the honey sample was dissolved with ultrapure water in order to prepare a 1 g/L solution, the solution was then filtrated by using 0.22 μm filter membrane, and the filtrate was used for test.

Instrumentation and Measurement

EA-IRMS: a Thermo-Electron Flash EA 1112 elemental analyzer (Jiangsu CIQ) or SerCon (Qinhuangdao CIQ) was coupled via a Thermo-Finnigan ConFlo III Interface (He pressure: 0.8 bar, CO2 pressure: 2 bar) to a Thermo-Electron Delta V Advantage Isotope Ratio Mass Spectrometer. The oxidation and reduction reactors were heated to 1000 °C and 825 °C, respectively. Oven temperature: 50 °C. Flow rate of He and O2: 120 ml/min and 175 mL/min. Each honey or protein was measured two times. One run took 8 min.

LC-IRMS: a LC system consisting of a bio-compatible Knauer Smart line P1000 Pump, a Knauer Smartline Manager 5000 gradient module, a Merck-Hitachi AS2000A autosampler, a Jetstream 2 Plus column oven (operated at 55 ◦C) and a Carbomix Ca-NP5 (300 mm × 7.8 mm, 5 μm; Sepax Technologies Company) separation column was coupled via a Finnigan LC IsoLink Interface to a Thermo-Finnigan Delta + Advantage Isotope Ratio Mass Spectrometer. The pressure gauges were adjusted to 0.5 bar for He carrier gas, 1.2 bar for helium purge gas and 1.2 bar for CO2 reference gas. 10 μL sample was injected. The eluent was 100% ultrapure water (18.2 MΩ) with a flow rate of 0.3 mL/min. The CO2 reference gas pulse was introduced two times at the beginning and at the end of each run. One run took 45 min.

Honey samples

All honeys used as authentic reference samples were supplied by the international independent institute SGS (Société Générale de Surveillance S.A.) company. As a very reputable company, SGS was entrusted by us to get all these authentic honey samples at bloom time in different areas in China, such as Henan, Sichuan, Hubei, Jiangsu, Jilin, Shanxi, Xinjiang Uygur Autonomous, and Inner Mongolia Autonomous. All honey samples were stored in glass containers, shipped to the laboratory and maintained at 4 °C. An additional confirmative authenticity analysis was conducted (diastase number, HMF content, proline content, high fructose corn syrup detection (GB/T 18932.2-2002 2002), rice syrup marker detection (Xue et al. 2013), beet syrup marker detection (ZL 2013107003474.8, 2013) and beta-fructofuranosidase activity detection (Wang et al. 2011), the statistics were summarized in Table 1.

Table 1.

Statistics of honey samples that delivered by the SGS Company

Honey species Locality of honey Location number Diastase number HMF content (mg/kg) Proline content (mg/kg) High fructose corn syrup detection Rice syrup marker detection Beet syrup marker detection Beta-fructo-furanosidase activity
Acacia honey Hebei Province G001 39.9 ND 430 Negativea NDb NDc NDd
G002 23.6 ND 358 Negative ND ND ND
G003 39.1 ND 384 Negative ND ND ND
G004 41.8 ND 486 Negative ND ND ND
Mean ± SD 36.1 ± 7.3 ND 415 ± 48.7 / / / /
Liaoning Province ‘5–1 12.3 ND 205 Negative ND ND ND
‘5–2 15.8 ND 214 Negative ND ND ND
‘5–3 8.2 ND 183 Negative ND ND ND
‘5–4 17.3 ND 314 Negative ND ND ND
‘5–5 25.9 ND 327 Negative ND ND ND
Mean ± SD 15.9 ± 5.9 ND 249 ± 59.7 / / / /
Shandong Province F001 15.6 ND 421 Negative ND ND ND
F002 12.5 ND 430 Negative ND ND ND
F003 12.6 ND 367 Negative ND ND ND
F004 12.3 ND 372 Negative ND ND ND
F005 9.7 ND 375 Negative ND ND ND
F006 4.0 ND 333 Negative ND ND ND
F007 13.6 ND 379 Negative ND ND ND
F008 18.9 ND 436 Negative ND ND ND
F009 17.5 ND 367 Negative ND ND ND
Mean ± SD 13.0 ± 4.1 ND 387 ± 32.6 / / / /
Henan Province E001 21.4 ND 207 Negative ND ND ND
E002 11.2 ND 169 Negative ND ND ND
E003 36.1 ND 302 Negative ND ND ND
E004 29.2 ND 217 Negative ND ND ND
E005 27.7 ND 301 Negative ND ND ND
E006 31.9 ND 333 Negative ND ND ND
E007 31.1 ND 262 Negative ND ND ND
E008 10.7 ND 267 Negative ND ND ND
E009 18.9 ND 232 Negative ND ND ND
E010 11.4 ND 261 Negative ND ND ND
E011 15.4 ND 192 Negative ND ND ND
Mean ± SD 22.3 ± 8.9 ND 249 ± 48.6 / / / /
Rape honey Sichuan Province A003 22.6 1.4 251 Negative ND ND ND
A004 7.1 1.1 203 Negative ND ND ND
A005 16.7 0.9 190 Negative ND ND ND
A006 1.5 ND 146 Negative ND ND ND
A007 7.7 1.4 183 Negative ND ND ND
A008 11.4 ND 195 Negative ND ND ND
A009 19.8 1 230 Negative ND ND ND
A010 7.5 ND 163 Negative ND ND ND
Mean ± SD 11.8 ± 6.8 0.7 ± 0.2 195 ± 31.7 / / / /
Hubei Province B001 30.0 1.4 250 Negative ND ND ND
B002 11.7 2 151 Negative ND ND ND
B003 13.6 1.7 142 Negative ND ND ND
B004 13.8 2 132 Negative ND ND ND
B005 20.0 2.1 144 Negative ND ND ND
Mean ± SD 17.8 ± 6.7 1.8 ± 0.3 164 ± 43.5 / / / /
Xinjiang Uygur Autonomous Region C001 16.1 ND 130 Negative ND ND ND
C002 37.9 ND 428 Negative ND ND ND
C003 19.2 ND 160 Negative ND ND ND
C004 13.1 ND 149 Negative ND ND ND
C005 17.2 ND 151 Negative ND ND ND
C006 33.4 ND 563 Negative ND ND ND
C007 59.6 ND 586 Negative ND ND ND
C008 16.5 ND 190 Negative ND ND ND
C009 21.3 ND 261 Negative ND ND ND
Mean ± SD 26.0 ± 14.2 ND 291 ± 174.4 / / / /
Jiangsu Province D001 8.6 ND 166 Negative ND ND ND
D002 21.4 ND 209 Negative ND ND ND
D003 25.4 ND 211 Negative ND ND ND
D004 19.2 ND 197 Negative ND ND ND
D005 21.3 ND 227 Negative ND ND ND
D006 14.3 ND 252 Negative ND ND ND
D007 11.1 ND 207 Negative ND ND ND
D008 23.2 ND 215 Negative ND ND ND
D009 37.0 ND 274 Negative ND ND ND
D010 40.4 ND 293 Negative ND ND ND
D011 28.3 1.0 249 Negative ND ND ND
Mean ± SD 22.7 ± 9.4 ND 227 ± 34.9 / / / /
Vitex honey Hubei Province B006 25.0 ND 304 Negative ND ND ND
B007 23.8 ND 433 Negative ND ND ND
Mean ± SD 24.4 ± 0.6 ND 369 ± 64.5 / / / /
Henan Province E015 48.2 ND 498 Negative ND ND ND
E016 36.6 ND 467 Negative ND ND ND
E017 26.8 ND 349 Negative ND ND ND
E018 55.6 ND 497 Negative ND ND ND
E019 30.9 ND 342 Negative ND ND ND
E020 31.0 ND 449 Negative ND ND ND
E021 42.0 ND 533 Negative ND ND ND
E022 65.3 ND 567 Negative ND ND ND
E023 75.6 ND 626 Negative ND ND ND
Mean ± SD 45.8 ± 15.9 ND 481 ± 88.0 / / / /
Cotton honey

Xinjiang

Uygur Autonomous Region

 N003 24.0 ND 243 Negative ND ND ND
N004 15.6 ND 166 Negative ND ND ND
N005 19.2 ND 179 Negative ND ND ND
N006 32.2 ND 178 Negative ND ND ND
N007 32.1 ND 254 Negative ND ND ND
N008 27.9 ND 199 Negative ND ND ND
N009 48.0 ND 255 Negative ND ND ND
N010 54.7 ND 281 Negative ND ND ND
N011 14.5 ND 211 Negative ND ND ND
N012 19.6 ND 279 Negative ND ND ND
Mean ± SD 28.8 ± 12.8 ND 225 ± 41.0 / / / /
Linden honey Jilin Province P001 16.4 ND 140 Negative ND ND ND
P002 8.8 ND 174 Negative ND ND ND
P003 9.5 ND 164 Negative ND ND ND
P004 8.6 ND 177 Negative ND ND ND
Mean ± SD 10.8 ± 3.2 ND 164 ± 14.5 / / / /
Jujube honey Shanxi Province I010 25.4 ND 191 Negative ND ND ND
I011 28.3 ND 328 Negative ND ND ND
I012 24.7 ND 295 Negative ND ND ND
Mean ± SD 26.1 ± 1.6 ND 271 ± 58.4 / / / /
Sunflower honey Inner Mongolia Autonomous Region J001 21.4 ND 257 Negative ND ND ND
J002 26.2 ND 245 Negative ND ND ND
Mean ± SD 23.8 ± 2.4 ND 251 ± 6.0 / / / /
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aLOQ ≥ 5% (v/v) high fructose starch syrup

bLOQ ≥ 0.35 mg/kg

cLOQ ≥ 5% (v/v) beet syrup

dLOQ ≥ 20 U/kg

Results and discussion

A total of 94 pure honey samples are analyzed by Jiangsu CIQ (Jiangsu Entry-exit Inspection and Quarantine Bureau Lab) and Qinhuangdao CIQ (Qinhuangdao Entry-exit Inspection and Quarantine Bureau Lab) labs. The honey δ13C values (δ13CH) and the protein δ13C values (δ13CP) for the botanical origins of these Chinese honey samples were given in Table 2, along with other basic statistics. There were only slight differences in the ranges and mean values, which were always overlapping. Thus, no clear differentiation between the δ13Choney and δ13Cprotein values among these honey types can be indicated. The EA-IRMS and LC-IRMS results of all samples were summarized in Table 3.

Table 2.

Basic statistics of δ13C in Chinese honeys of different botanical origins

Honey species Locality of honey n Test by Qinhuangdao CIQ Test by Jiangsu CIQ
δ13Cprotein (‰) δ13Choney (‰) δ13Cprotein (‰) δ13Choney (‰)
mean SD min max mean SD min max mean SD min max mean SD min max
Acacia honey Hebei Province 4 − 24.48 0.16 − 24.68 − 24.24 − 23.96 0.06 − 24.04 − 23.90 − 24.44 0.50 − 25.31 − 24.10 − 24.34 0.08 − 24.48 − 24.48
Liaoning Province 5 − 23.73 0.30 − 24.11 − 23.20 − 23.63 0.58 − 24.55 − 22.82 − 23.59 0.29 − 23.93 − 23.06 − 24.33 0.71 − 25.35 − 23.21
Shandong Province 9 − 23.68 0.18 − 23.94 − 23.36 − 24.33 0.09 − 24.44 − 24.18 − 23.88 0.12 − 24.06 − 23.73 − 24.81 0.12 − 25.04 − 24.68
Henan Province 11 − 24.11 0.50 − 24.58 − 22.96 − 24.38 0.45 − 24.86 − 23.73 − 24.08 0.48 − 24.51 − 23.16 − 24.64 0.45 − 25.09 − 24.12
Rape honey Sichuan Province 8 − 27.69 0.34 − 28.42 − 27.46 − 29.37 0.49 − 30.40 − 28.84 − 27.56 0.46 − 28.64 − 27.30 − 29.21 0.20 − 29.49 − 28.93
Hubei Province 5 − 27.17 0.20 − 27.39 − 26.82 − 27.68 0.16 − 27.84 − 27.41 − 27.20 0.27 − 27.69 − 26.88 − 27.84 0.19 − 28.06 − 27.53
Xinjiang Uygur Autonomous Region 9 − 26.24 0.62 − 27.37 − 25.30 − 27.44 0.59 − 28.08 − 26.41 − 26.34 0.81 − 27.57 − 25.15 − 27.81 0.59 − 28.40 − 26.84
Jiangsu Province 11 − 26.00 0.51 − 26.84 − 25.20 − 27.80 0.57 − 28.54 − 27.28 − 26.59 0.51 − 27.29 − 25.77 − 28.06 0.44 − 28.69 − 27.25
Vitex honey Hubei Province 2 − 23.94 0.00 − 23.94 − 23.94 − 25.41 0.03 − 25.44 − 25.38 − 23.98 0.14 − 24.11 − 23.84 − 25.90 0.07 − 25.97 − 25.83
Henan Province 9 − 24.04 0.21 − 24.30 − 23.73 − 25.96 0.28 − 26.56 − 26.64 − 24.39 0.43 − 25.15 − 23.81 − 26.35 0.49 − 27.34 − 25.73
Cotton honey

Xinjiang

Uygur Autonomous Region

 10 − 22.68 1.69 − 24.56 − 19.44 − 24.73 0.29 − 25.14 − 24.28 − 22.91 1.81 − 24.90 − 19.72 − 24.80 0.23 − 25.06 − 24.34
Linden honey Jilin Province 4 − 24.05 0.11 − 24.12 − 23.86 − 24.37 0.11 − 24.54 − 24.22 ND ND ND ND − 24.73 0.11 − 24.86 − 24.56
Jujube honey Shanxi Province 3 − 24.41 0.07 − 24.48 − 24.32 − 23.95 0.08 − 24.06 − 23.87 − 24.12 0.25 − 24.46 − 23.87 − 24.14 0.28 − 24.35 − 23.74
Sunflower honey Inner Mongolia Autonomous Region 2 − 23.64 0.14 − 23.78 − 23.50 − 25.03 0.01 − 25.04 − 25.02 − 23.83 0.10 − 23.92 − 23.73 − 25.29 0.08 − 25.37 − 25.21

Xinjiang

Uygur Autonomous Region

 2 − 24.41 0.01 − 24.42 − 24.40 − 25.32 0.05 − 25.37 − 25.26 − 24.44 0.12 − 24.55 − 24.32 − 25.27 0.04 − 25.31 − 25.22
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Table 3.

EA-IRMS and LC-IRMS results of authentic honey samples (n = 94) from Qinhuangdao CIQ and Jiangsu CIQ labs

Honey species Locality of honey Location number Test by Qinhuangdao CIQ
δ13C (‰)
protein (p)		 δ13C (‰)
honey (h)		 δ13C (‰) frua δ13C (‰) grub δ13C (‰) dsc δ13C (‰) tsd fru-gru Δδ13C (‰)e Δδ13C (‰) max. (abs)f
Acacia honey Hebei Province G001 − 24.53 − 24.00 − 24.42 − 24.60 − 21.67 − 23.19 0.18 2.93
G002 − 24.24 − 23.91 − 24.47 − 24.70 − 21.47 − 22.26 0.23 3.23
G003 − 24.48 − 24.04 − 24.65 − 24.98 − 21.65 − 23.44 0.33 3.33
G004 − 24.68 − 23.90 − 24.35 − 24.47 − 22.31 − 23.01 0.12 2.37
Mean ± SD − 24.48 ± 0.16 − 23.96 ± 0.06 − 24.47 ± 0.11 − 24.69 ± 0.19 − 21.78 ± 0.32 − 22.98 ± 0.44 0.22 ± 0.08 2.97 ± 0.37
Liaoning Province ‘5–1 − 23.20 − 22.82 − 23.39 − 23.77 − 19.38 − 21.67 0.38 4.39
‘5–2 − 23.84 − 23.95 − 24.77 − 25.34 − 21.72 − 23.46 0.57 3.62
‘5–3 − 23.84 − 24.55 − 25.42 − 26.09 − 22.09 − 24.15 0.67 4.00
‘5–4 − 24.11 − 23.52 − 24.34 − 24.70 − 21.42 − 22.80 0.36 3.28
‘5–5 − 23.66 − 23.33 − 24.22 − 24.53 − 21.73 − 22.80 0.31 2.80
Mean ± SD − 23.73 ± 0.30 − 23.63 ± 0.58 − 24.43 ± 0.67 − 24.89 ± 0.78 − 21.27 ± 0.97 − 22.98 ± 0.82 0.46 ± 0.14 3.62 ± 0.55
Shandong Province F001 − 23.77 − 24.24 − 24.99 − 25.18 − 22.43 − 23.48 0.19 2.75
F002 − 23.50 − 24.24 − 24.94 − 25.27 − 22.11 − 23.35 0.33 3.16
F003 − 23.50 − 24.18 − 24.99 − 25.30 − 22.07 − 23.34 0.31 3.23
F004 − 23.68 − 24.35 − 24.99 − 25.21 − 22.22 − 23.32 0.22 2.99
F005 − 23.80 − 24.35 − 24.96 − 25.18 − 22.16 − 23.27 0.22 3.02
F006 − 23.68 − 24.44 − 24.95 − 25.27 − 22.16 − 23.49 0.32 3.11
F007 − 23.36 − 24.44 − 24.98 − 25.28 − 22.16 − 23.50 0.30 3.12
F008 − 23.90 − 24.44 − 25.05 − 25.40 − 22.12 − 23.27 0.35 3.28
F009 − 23.94 − 24.31 − 24.99 − 25.27 − 22.13 − 23.36 0.28 3.14
Mean ± SD − 23.68 ± 0.18 − 24.33 ± 0.09 − 24.98 ± 0.03 − 25.26 ± 0.06 − 22.17 ± 0.10 − 23.38 ± 0.09 0.28 ± 0.05 3.09 ± 0.15
Henan Province E001 − 23.37 − 23.73 − 24.12 − 23.75 − 24.71 − 23.29 − 0.37 1.42
E002 − 24.75 − 24.13 − 24.37 − 24.13 − 25.65 − 23.55 − 0.24 2.10
E003 − 24.31 − 24.90 − 25.34 − 25.03 − 25.79 − 23.48 − 0.31 2.31
E004 − 24.38 − 24.86 − 25.30 − 25.02 − 25.55 − 23.55 − 0.28 2.00
E005 − 24.27 − 24.82 − 25.27 − 24.98 − 26.05 − 23.16 − 0.29 2.89
E006 − 24.30 − 24.68 − 25.25 − 24.93 − 25.94 − 23.16 − 0.32 2.78
E007 − 24.58 − 24.70 − 25.18 − 24.75 − 26.10 − 23.51 − 0.43 2.59
E008 − 24.34 − 24.68 − 25.29 − 25.03 − 25.42 − 23.49 − 0.26 1.93
E009 − 23.96 − 23.79 − 24.37 − 24.32 − 24.17 − 22.98 − 0.05 1.39
E010 − 23.96 − 23.90 − 24.35 − 24.13 − 24.48 − 22.81 − 0.22 1.67
E011 − 22.96 − 24.00 − 24.55 − 24.31 − 24.61 − 23.38 − 0.24 1.65
Mean ± SD − 24.11 ± 0.50 − 24.38 ± 0.45 − 24.85 ± 0.47 − 24.58 ± 0.44 − 25.32 ± 0.66 − 23.31 ± 0.24 − 0.27 ± 0.09 2.07 ± 0.50
Rape honey Sichuan Province A003 − 27.52 − 28.95 − 29.26 − 29.46 − 30.99 ND 0.20 3.47
A004 − 27.96 − 28.84 − 28.99 − 29.22 − 30.79 ND 0.23 2.83
A005 − 27.28 − 28.92 − 28.85 − 29.16 − 30.92 ND 0.31 3.64
A006 − 27.78 − 30.40 − 29.79 − 29.88 − 31.84 ND 0.09 4.06
A007 − 27.62 − 29.42 − 29.42 − 29.60 − 31.25 ND 0.18 3.63
A008 − 27.48 − 29.68 − 29.65 − 29.82 − 31.60 ND 0.17 4.12
A009 − 27.46 − 29.14 − 29.30 − 29.49 − 31.04 ND 0.19 3.58
A010 − 28.42 − 29.62 − 29.37 − 29.50 − 31.48 ND 0.13 3.06
Mean ± SD − 27.69 ± 0.34 − 29.37 ± 0.49 − 29.33 ± 0.29 − 29.52 ± 0.24 − 31.24 ± 0.35 ND 0.19 ± 0.06 3.55 ± 0.41
Hubei Province B001 − 26.82 − 27.41 − 27.54 − 27.66 − 29.65 ND 0.12 2.83
B002 − 27.39 − 27.84 − 28.00 − 28.10 − 29.31 ND 0.10 1.92
B003 − 27.08 − 27.72 − 27.89 − 27.92 − 29.26 ND 0.03 2.18
B004 − 27.22 − 27.59 − 27.73 − 27.80 − 29.13 ND 0.07 1.91
B005 − 27.32 − 27.84 − 28.11 − 28.23 − 29.48 ND 0.12 2.16
Mean ± SD − 27.17 ± 0.20 − 27.68 ± 0.16 − 27.85 ± 0.20 − 27.94 ± 0.20 − 29.37 ± 0.18 ND 0.09 ± 0.03 2.20 ± 0.34
Xinjiang Uygur Autonomous Region C001 − 26.36 − 28.08 − 28.43 − 28.57 − 26.08 ND 0.14 2.49
C002 − 25.49 − 27.00 − 27.20 − 27.37 − 26.20 ND 0.17 1.88
C003 − 26.42 − 27.82 − 28.17 − 28.25 − 26.41 ND 0.08 1.84
C004 − 26.38 − 27.80 − 28.29 − 28.48 − 26.13 ND 0.19 2.35
C005 − 27.37 − 27.62 − 27.74 − 27.88 − 26.68 ND 0.14 1.20
C006 − 25.30 − 26.41 − 26.78 − 26.96 − 25.88 ND 0.18 1.66
C007 − 25.62 − 26.55 − 26.69 − 26.82 − 26.37 ND 0.13 1.20
C008 − 26.64 − 27.78 − 28.09 − 28.22 − 27.36 ND 0.13 1.58
C009 − 26.60 − 27.94 − 27.98 − 28.08 − 28.49 ND 0.10 1.89
Mean ± SD − 26.24 ± 0.62 − 27.44 ± 0.59 − 27.71 ± 0.62 − 27.85 ± 0.61 − 26.62 ± 0.78 ND 0.14 ± 0.03 1.79 ± 0.42
Jiangsu Province D001 − 26.34 − 28.44 − 28.40 − 28.54 − 27.22 ND 0.14 2.20
D002 − 26.04 − 28.54 − 28.53 − 28.67 − 27.49 ND 0.14 2.63
D003 − 25.20 − 27.62 − 27.69 − 27.83 − 26.73 ND 0.14 2.63
D004 − 25.38 − 27.77 − 27.76 − 27.85 − 27.33 ND 0.09 2.47
D005 − 26.84 − 27.94 − 27.87 − 28.04 − 28.10 ND 0.17 1.26
D006 − 26.31 − 28.12 − 28.05 − 28.18 − 27.97 ND 0.13 1.87
D007 − 26.74 − 28.05 − 28.07 − 28.20 − 27.67 ND 0.13 1.46
D008 − 26.10 − 28.26 − 28.30 − 28.52 − 27.80 ND 0.22 2.42
D009 − 25.54 − 27.28 − 27.49 − 27.51 − 28.41 ND 0.02 2.87
D010 − 25.75 − 26.54 − 26.75 − 26.77 − 28.51 ND 0.02 2.76
D011 − 25.71 − 27.28 − 27.36 − 27.41 − 28.78 ND 0.05 3.07
Mean ± SD − 26.00 ± 0.51 − 27.80 ± 0.57 − 27.84 ± 0.49 − 27.96 ± 0.54 − 27.82 ± 0.59 ND 0.11 ± 0.06 2.33 ± 0.55
Vitex honey Hubei Province B006 − 23.94 − 25.38 − 25.88 − 26.45 − 22.11 − 24.59 0.57 4.34
B007 − 23.94 − 25.44 − 25.99 − 26.32 − 22.21 − 24.15 0.33 4.11
Mean ± SD − 23.94 ± 0.00 − 25.41 ± 0.03 − 25.94 ± 0.05 − 26.39 ± 0.06 − 22.16 ± 0.05 − 24.37 ± 0.22 0.45 ± 0.12 4.23 ± 0.12
Henan Province E015 − 24.05 − 25.64 − 25.84 − 26.03 − 23.22 − 24.33 0.19 2.81
E016 − 24.26 − 26.02 − 26.11 − 26.33 − 24.02 − 24.16 0.22 2.31
E017 − 23.73 − 25.64 − 25.87 − 26.22 − 22.94 − 24.44 0.35 3.28
E018 − 23.92 − 25.69 − 25.76 − 26.12 − 22.72 − 24.19 0.36 3.40
E019 − 23.82 − 25.86 − 26.14 − 26.50 − 23.04 − 24.67 0.36 3.46
E020 − 23.82 − 26.07 − 26.12 − 26.29 − 24.82 − 25.32 0.17 2.47
E021 − 24.24 − 26.56 − 26.58 − 26.63 − 25.79 − 25.29 0.05 2.39
E022 − 24.21 − 26.18 − 26.02 − 26.01 − 26.89 ND − 0.01 2.68
E023 − 24.30 − 25.94 − 25.89 − 25.83 − 26.73 ND − 0.06 2.43
Mean ± SD − 24.04 ± 0.21 − 25.96 ± 0.28 − 26.04 ± 0.23 − 26.22 ± 0.24 − 24.46 ± 1.56 − 24.63 ± 0.46 0.18 ± 0.15 2.80 ± 0.43
Cotton honey

Xinjiang

Uygur Autonomous Region

 N003 − 20.08 − 24.92 − 25.13 − 25.09 − 23.66 ND − 0.04 5.05
N004 − 23.10 − 24.61 − 24.90 − 24.80 − 24.88 ND − 0.10 1.80
N005 − 22.70 − 24.44 − 24.70 − 24.63 − 23.16 ND − 0.07 2.00
N006 − 22.96 − 24.28 − 24.40 − 24.28 − 24.30 ND − 0.12 1.44
N007 − 23.71 − 25.10 − 24.98 − 25.28 − 24.44 ND 0.30 1.57
N008 − 24.34 − 24.89 − 24.74 − 25.09 − 24.79 ND 0.35 0.75
N009 − 24.27 − 25.14 − 25.01 − 25.26 − 25.21 ND 0.25 0.99
N010 − 24.56 − 24.92 − 24.80 − 25.04 − 26.16 ND 0.24 1.60
N011 − 21.62 − 24.65 − 24.75 − 24.73 − 23.27 ND − 0.02 3.13
N012 − 19.44 − 24.34 − 24.49 − 24.43 − 24.68 ND − 0.06 5.24
Mean ± SD − 22.68 ± 1.69 − 24.73 ± 0.29 − 24.79 ± 0.22 − 24.86 ± 0.33 − 24.46 ± 0.87 ND 0.07 ± 0.18 2.36 ± 1.52
Linden honey Jilin Province P001 − 23.86 − 24.34 − 24.76 − 24.94 − 22.58 − 22.13 0.18 2.81
P002 − 24.12 − 24.36 − 24.94 − 25.19 − 22.73 − 22.87 0.25 2.46
P003 − 24.10 − 24.22 − 24.64 − 24.81 − 22.57 − 22.63 0.17 2.24
P004 − 24.12 − 24.54 − 25.13 − 25.43 − 23.02 − 22.83 0.30 2.60
Mean ± SD − 24.05 ± 0.11 − 24.37 ± 0.11 − 24.87 ± 0.19 − 25.09 ± 0.24 − 22.73 ± 0.18 − 22.62 ± 0.29 0.23 ± 0.05 2.53 ± 0.21
Jujube honey Shanxi Province I010 − 24.42 − 24.06 − 24.26 − 25.03 − 22.52 − 22.56 0.77 2.51
I011 − 24.48 − 23.92 − 24.16 − 24.92 − 22.50 − 22.85 0.76 2.42
I012 − 24.32 − 23.87 − 24.09 − 24.91 − 22.49 − 22.58 0.82 2.42
Mean ± SD − 24.41 ± 0.07 − 23.95 ± 0.08 − 24.17 ± 0.07 − 24.95 ± 0.05 − 22.50 ± 0.01 − 22.66 ± 0.13 0.78 ± 0.03 2.45 ± 0.04
Sunflower honey Inner Mongolia Autonomous Region J001 − 23.78 − 25.02 − 24.98 − 25.32 − 25.05 ND 0.34 1.54
J002 − 23.50 − 25.04 − 24.98 − 25.37 − 25.48 ND 0.39 1.98
Mean ± SD − 23.64 ± 0.14 − 25.03 ± 0.01 − 24.98 ± 0.00 − 25.35 ± 0.03 − 25.27 ± 0.22 ND 0.37 ± 0.03 1.76 ± 0.22

Xinjiang

Uygur Autonomous Region

 N001 − 24.40 − 25.37 − 25.31 − 25.61 − 24.95 ND 0.30 1.21
N002 − 24.42 − 25.26 − 25.15 − 25.46 − 24.14 ND 0.31 1.32
Mean ± SD − 24.41 ± 0.01 − 25.32 ± 0.05 − 25.23 ± 0.08 − 25.54 ± 0.07 − 24.55 ± 0.40 ND 0.31 ± 0.01 1.27 ± 0.06
Honey species Locality of honey Location number Test by Jiangsu CIQ
δ13C (‰)
protein (p)		 δ13C (‰)
honey (h)		 δ13C (‰) fru δ13C (‰) gru δ13C (‰) ds δ13C (‰) ts fru-gru Δδ13C (‰) Δδ13C (‰) max. (abs)
Acacia honey Hebei Province G001 − 24.18 − 24.29 − 24.31 − 24.46 − 22.01 − 23.07 0.15 2.45
G002 − 24.10 − 24.28 − 24.19 − 24.45 − 21.34 − 22.18 0.26 3.11
G003 − 24.17 − 24.48 − 24.50 − 24.85 − 21.83 − 22.92 0.35 3.02
G004 − 25.31 − 24.30 − 24.22 − 24.22 − 21.86 − 22.26 0.00 3.45
Mean ± SD − 24.44 ± 0.50 − 24.34 ± 0.08 − 24.31 ± 0.12 − 24.50 ± 0.23 − 21.76 ± 0.25 − 22.61 ± 0.39 0.19 ± 0.13 3.01 ± 0.36
Liaoning Province ‘5–1 − 23.06 − 23.21 − 23.19 − 23.51 − 19.31 − 21.15 0.32 4.20
‘5–2 − 23.93 − 24.26 − 24.10 − 24.44 − 21.37 − 22.39 0.34 3.07
‘5–3 − 23.57 − 25.35 − 25.20 − 25.77 − 22.34 − 23.68 0.57 3.43
‘5–4 − 23.65 − 24.74 − 24.68 − 25.18 − 21.96 − 22.93 0.50 3.22
‘5–5 − 23.74 − 24.08 − 24.04 − 24.27 − 21.70 − 22.33 0.23 2.57
Mean ± SD − 23.59 ± 0.29 − 24.33 ± 0.71 − 24.24 ± 0.67 − 24.63 ± 0.78 − 21.34 ± 1.06 − 22.50 ± 0.83 0.39 ± 0.12 3.30 ± 0.53
Shandong Province F001 − 23.73 − 24.68 − 24.73 − 24.84 − 22.14 − 22.71 0.11 2.70
F002 NDg − 24.73 − 24.75 − 24.97 − 22.00 − 22.86 0.22 2.97
F003 ND − 24.73 − 24.78 − 25.08 − 22.01 − 22.78 0.30 3.07
F004 ND − 24.78 − 24.90 − 25.32 − 22.67 − 23.57 0.42 2.65
F005 − 23.74 − 24.70 − 24.76 − 24.96 − 22.07 − 22.98 0.19 2.89
F006 − 23.87 − 25.04 − 24.82 − 24.94 − 21.54 − 22.94 0.12 3.50
F007 − 24.06 − 24.84 − 24.83 − 25.06 − 21.67 − 23.00 0.23 3.39
F008 − 24.01 − 24.97 − 24.90 − 25.24 − 21.56 − 22.81 0.34 3.68
F009 − 23.88 − 24.83 − 24.83 − 25.00 − 21.55 − 22.82 0.17 3.45
Mean ± SD − 23.88 ± 0.12 − 24.81 ± 0.12 − 24.81 ± 0.06 − 25.05 ± 0.14 − 21.91 ± 0.35 − 22.94 ± 0.25 0.23 ± 0.10 3.14 ± 0.35
Henan Province E001 − 23.39 − 24.07 − 23.82 − 23.43 − 24.34 − 22.98 − 0.39 1.36
E002 − 24.41 − 24.12 − 24.17 − 23.78 − 25.48 − 22.86 − 0.39 2.62
E003 ND − 24.97 − 25.21 − 24.82 − 26.59 − 23.08 − 0.39 3.51
E004 ND − 25.08 − 25.20 − 24.76 − 25.93 − 23.38 − 0.44 2.55
E005 —h – – – – – – –
E006 − 24.51 − 25.02 − 25.19 − 24.67 − 25.99 − 23.47 − 0.52 2.52
E007 − 24.35 − 25.09 − 25.07 − 24.49 − 25.98 − 23.40 − 0.58 2.58
E008 − 24.32 − 25.24 − 25.31 − 24.84 − 26.34 − 23.33 − 0.47 3.01
E009 − 24.11 − 24.37 − 24.22 − 24.00 − 24.41 − 22.84 − 0.23 1.57
E010 − 24.41 − 24.14 − 24.15 − 23.94 − 24.60 − 22.73 − 0.22 1.87
E011 − 23.16 − 24.30 − 24.33 − 24.07 − 24.07 − 23.15 − 0.26 1.18
Mean ± SD − 24.08 ± 0.48 − 24.64 ± 0.45 − 24.67 ± 0.55 − 24.28 ± 0.47 − 25.37 ± 0.88 − 23.12 ± 0.25 − 0.39 ± 0.12 2.28 ± 0.71
Rape honey Sichuan Province A003 − 27.30 − 28.93 − 29.08 − 29.25 − 31.69 ND 0.17 4.39
A004 – – – – – – – –
A005 − 27.42 − 28.94 − 28.76 − 28.96 − 28.90 ND 0.20 1.54
A006 − 27.54 − 29.49 − 29.45 − 29.37 − 31.61 ND − 0.09 4.07
A007 − 27.48 − 29.31 − 29.22 − 29.37 − 29.18 ND 0.15 1.83
A008 − 27.15 − 29.43 − 29.35 − 29.53 − 31.34 ND 0.18 4.19
A009 − 27.41 − 29.15 − 29.01 − 29.25 − 31.77 ND 0.24 4.36
A010 − 28.64 − 29.23 − 29.26 − 29.37 − 31.31 ND 0.11 2.67
Mean ± SD − 27.56 ± 0.46 − 29.21 ± 0.20 − 29.16 ± 0.21 − 29.30 ± 0.16 − 30.83 ± 1.14 ND 0.14 ± 0.10 3.29 ± 1.16
Hubei Province B001 − 26.88 − 27.53 − 27.43 − 27.45 − 29.39 ND 0.02 2.51
B002 − 27.69 − 28.02 − 27.90 − 27.91 − 28.65 ND 0.00 0.96
B003 − 27.20 − 27.85 − 27.77 − 27.76 − 29.14 ND − 0.01 1.94
B004 − 27.09 − 27.74 − 27.74 − 27.74 − 29.29 ND − 0.01 2.20
B005 − 27.14 − 28.06 − 28.03 − 28.06 − 29.13 ND 0.02 1.99
Mean ± SD − 27.20 ± 0.27 − 27.84 ± 0.19 − 27.77 ± 0.20 − 27.78 ± 0.20 − 29.12 ± 0.25 ND 0.00 ± 0.01 1.92 ± 0.52
Xinjiang Uygur Autonomous Region C001 − 26.46 − 28.40 − 28.23 − 28.51 − 24.30 ND 0.28 4.21
C002 − 25.53 − 27.30 − 27.08 − 27.41 − 26.61 ND 0.33 1.88
C003 − 26.29 − 28.33 − 28.04 − 28.10 − 26.28 ND 0.06 2.05
C004 − 27.57 − 28.09 − 28.05 − 28.22 − 26.08 ND 0.17 2.01
C005 − 27.52 − 27.99 − 27.68 − 27.80 − 26.91 ND 0.12 1.08
C006 − 25.15 − 26.84 − 26.61 − 26.80 − 26.72 ND 0.18 1.69
C007 − 25.47 − 26.87 − 26.57 − 26.68 − 26.65 ND 0.10 1.40
C008 − 26.66 − 28.31 − 28.07 − 28.13 − 27.64 ND 0.06 1.65
C009 − 26.38 − 28.14 − 27.82 − 27.94 − 27.64 ND 0.13 1.76
Mean ± SD − 26.34 ± 0.81 − 27.81 ± 0.59 − 27.57 ± 0.61 − 27.73 ± 0.60 − 26.54 ± 0.94 ND 0.16 ± 0.09 1.97 ± 0.84
Jiangsu Province D001 − 27.29 − 28.22 − 28.39 − 28.58 − 28.04 ND 0.19 1.29
D002 − 26.62 − 28.69 − 28.44 − 28.64 − 28.07 ND 0.20 2.07
D003 − 26.14 − 27.32 − 27.63 − 27.79 − 26.77 ND 0.17 1.65
D004 − 25.84 − 27.97 − 27.79 − 27.91 − 27.28 ND 0.11 2.13
D005 − 27.17 − 28.09 − 27.77 − 27.93 − 27.76 ND 0.16 0.92
D006 − 26.62 − 28.39 − 27.98 − 28.22 − 27.94 ND 0.24 1.77
D007 − 27.25 − 28.14 − 27.96 − 28.12 − 27.33 ND 0.16 0.89
D008 − 26.73 − 28.59 − 28.10 − 28.33 − 28.46 ND 0.23 1.86
D009 − 25.77 − 28.16 − 27.95 − 27.99 − 28.30 ND 0.05 2.53
D010 − 26.26 − 27.25 − 27.13 − 27.23 − 28.47 ND 0.10 2.21
D011 − 26.81 − 27.79 − 27.62 − 27.73 − 26.93 ND 0.11 0.98
Mean ± SD − 26.59 ± 0.51 − 28.06 ± 0.44 − 27.89 ± 0.35 − 28.04 ± 0.38 − 27.76 ± 0.57 ND 0.16 ± 0.06 1.66 ± 0.54
Vitex honey Hubei Province B006 − 24.11 − 25.83 − 25.78 − 26.38 − 22.22 − 23.66 0.60 4.16
B007 − 23.84 − 25.97 − 25.91 − 26.33 − 22.34 − 24.00 0.42 3.99
Mean ± SD − 23.98 ± 0.14 − 25.90 ± 0.07 − 25.85 ± 0.06 − 26.36 ± 0.03 − 22.28 ± 0.06 − 23.83 ± 0.17 0.51 ± 0.09 4.08 ± 0.09
Henan Province E015 − 24.20 − 25.86 − 25.65 − 25.64 − 24.06 − 23.13 − 0.01 2.73
E016 − 24.35 − 26.12 − 26.02 − 26.19 − 23.93 − 24.00 0.17 2.26
E017 − 23.81 − 25.73 − 25.76 − 26.17 − 23.05 − 23.80 0.41 3.12
E018 − 23.95 − 25.87 − 25.75 − 25.94 − 22.51 − 22.86 0.20 3.43
E019 − 24.11 − 26.47 − 25.99 − 26.56 − 23.08 − 24.15 0.57 3.48
E020 − 24.21 − 26.55 − 26.55 − 26.67 − 25.45 − 24.12 0.12 2.55
E021 − 24.83 − 27.34 − 27.17 − 27.54 − 26.21 − 24.43 0.37 3.11
E022 − 24.89 − 26.78 − 26.45 − 26.44 − 27.28 ND − 0.01 2.39
E023 − 25.15 − 26.43 − 26.21 − 26.18 − 26.88 ND − 0.03 1.73
Mean ± SD − 24.39 ± 0.43 − 26.35 ± 0.49 − 26.17 ± 0.46 − 26.37 ± 0.51 − 24.72 ± 1.68 − 23.78 ± 0.53 0.20 ± 0.20 2.76 ± 0.55
Cotton honey

Xinjiang

Uygur Autonomous Region

 N003 − 19.97 − 25.04 − 24.98 − 24.85 − 23.98 ND − 0.13 5.07
N004 − 22.68 − 24.67 − 24.70 − 24.66 − 24.00 ND − 0.04 2.02
N005 − 22.77 − 24.69 − 24.46 − 24.36 − 24.73 ND − 0.10 1.96
N006 − 23.07 − 24.34 − 24.30 − 24.15 − 23.82 ND − 0.15 1.27
N007 – – – – – – – –
N008 − 24.24 − 25.01 − 24.69 − 24.94 − 23.95 ND 0.25 1.06
N009 − 24.31 − 25.06 − 24.93 − 25.16 − 25.49 ND 0.24 1.18
N010 − 24.57 − 25.02 − 24.76 − 25.02 − 26.03 ND 0.26 1.46
N011 − 24.90 − 24.75 − 24.68 − 24.69 − 23.40 ND 0.02 1.50
N012 − 19.72 − 24.61 − 24.38 − 24.34 − 24.85 ND − 0.03 5.13
Mean ± SD − 22.91 ± 1.81 − 24.80 ± 0.23 − 24.65 ± 0.22 − 24.69 ± 0.32 − 24.47 ± 0.82 ND 0.04 ± 0.16 2.29 ± 1.53
Linden honey Jilin Province P001 ND − 24.78 − 24.61 − 24.87 − 22.63 ND 0.27 2.24
P002 ND − 24.71 − 24.76 − 25.12 − 22.91 ND 0.36 2.21
P003 ND − 24.56 − 24.47 − 24.79 − 22.42 ND 0.32 2.37
P004 ND − 24.86 − 25.01 − 25.34 − 23.33 ND 0.33 2.01
Mean ± SD ND − 24.73 ± 0.11 − 24.71 ± 0.20 − 25.03 ± 0.22 − 22.82 ± 0.34 ND 0.32 ± 0.03 2.21 ± 0.13
Jujube honey Shanxi Province I010 − 23.87 − 24.35 − 24.05 − 24.89 − 22.33 − 23.27 0.84 2.56
I011 − 24.03 − 24.32 − 23.96 − 24.71 − 22.65 − 23.42 0.76 2.06
I012 − 24.46 − 23.74 − 23.89 − 24.89 − 22.80 − 23.30 0.99 2.09
Mean ± SD − 24.12 ± 0.25 − 24.14 ± 0.28 − 23.97 ± 0.07 − 24.83 ± 0.08 − 22.59 ± 0.20 − 23.33 ± 0.06 0.86 ± 0.10 2.24 ± 0.23
Sunflower honey Inner Mongolia Autonomous Region J001 − 23.92 − 25.21 − 24.87 − 25.19 − 25.67 ND 0.32 1.75
J002 − 23.73 − 25.37 − 24.97 − 25.28 − 25.56 ND 0.32 1.83
Mean ± SD − 23.83 ± 0.10 − 25.29 ± 0.08 − 24.92 ± 0.05 − 25.24 ± 0.04 − 25.62 ± 0.06 ND 0.32 ± 0.00 1.79 ± 0.04

Xinjiang

Uygur Autonomous Region

 N001 − 24.32 − 25.31 − 25.22 − 25.52 − 26.73 ND 0.30 2.41
N002 − 24.55 − 25.22 − 25.13 − 25.33 − 25.85 ND 0.20 1.30
Mean ± SD − 24.44 ± 0.12 − 25.27 ± 0.04 − 25.18 ± 0.04 − 25.43 ± 0.10 − 26.29 ± 0.44 ND 0.25 ± 0.05 1.86 ± 0.56
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aFructose (fru)

bGlucose (glu)

cDisaccharides (ds)

dTrisaccharides (ts)

eDifference δ13C fru–δ13C glu

fMaximum difference (absolute) between all measured δ13C values

gNot detected

hHaven’t received this sample

Conventional δ13C-EA-IRMS Method

From the results of δ13CP and δ13CH from Qinhuangdao CIQ lab it can be summarized that different honey species have its own characteristics (Table 3). As for acacia honey, the δ13CH values were ranged from − 22.82 to − 24.90‰, the differences are subtle and have little to do with area differentiation. The δ13CP values were ranged from − 22.96 to − 24.75‰. Among those acacia honey samples, about 6.9% samples showed the Δδ13CP–H values greater than 1.0‰.

Thirty-three rape honey samples from Sichuan province, Hubei province, Xinjiang Uygur Autonomous, and Jiangsu province were analyzed. The δ13CH values were ranged from − 26.41 to − 30.40‰. The δ13CH values that greater than − 29.0‰ were found chiefly concentrated in the regions of Sichuan province. The δ13CP values were in the range of − 25.20 to − 28.42‰. Among those 33 samples, about 76% samples had the Δδ13CP–H values that greater than 1.0‰.

Vitex honey, produced from Hubei and Henan province, had δ13CH and δ13CP values in the range of − 25.38 to − 26.56‰ and − 23.73 to − 24.30‰, respectively. All these samples had Δδ13CP–H values greater than 1.0‰, and 3 of them even greater than 2.0‰.

As for 10 cotton honey samples, which originated from Xinjiang Uygur Autonomous Region, have δ13CH and δ13CP values within the range of − 24.28 to − 25.14‰ and − 19.44 to − 24.56‰, respectively. The maximum Δδ13CP–H values of N003 sample and N012 sample reached to 4.84‰ and 4.90‰, respectively, in the present work.

Apart from the above honey samples, Linden honey from Jilin Province, Jujube honey from Shanxi Province, Sunflower honey from Inner Mongolia Autonomous region and Xinjiang Uygur Autonomous region were also analyzed in this article. From the data we can see that only two Sunflower honey samples, that is, J001 and J002, had the Δδ13CP–H values that greater than 1.0‰.

From the results that were obtained by this conventional δ13C-EA-IRMS method we can conclude that the δ13C4 sugar of all honey samples were qualified, that is, fall within the theoretical ranges proposed by JW White. Affected by climatic fluctuations, some samples had δ13CH values greater than − 29.0‰. The Δδ13CP–H values of cotton honey and vitex honey were relatively larger than other honey samples.

LC-IRMS method

In this work, LC-IRMS method was carried out d by coupling a liquid chromatography to a stable isotope mass spectrometry. This method was first proposed by Intertek laboratory in Europe. As proposed by Intertek laboratory, the limits for Δδ13C values of pure honey were as follows: Δδ13C (‰) max. (maximum difference between all measured δ13C values): ≤ ± 2.1‰; and Δδ13C (‰) fru-glu (differences between fructose and glucose δ13C values): ≤ ± 1.0‰. In this work, a comprehensive survey of pure honey produced from different areas in China according to the δ13C-LC-IRMS method, and inter-laboratory comparison experiments between Jiangsu CIQ and Qinhuangdao CIQ were also involved. The statistics of Δδ13C (‰) max. values of all authentic honey samples were summarized in Table 4 according to honey botanical origin.

Table 4.

The Δδ13CP–H and Δδ13C (‰) max. (abs) results from two labs of authentic honey samples in Table 3

Honey species Δδ13C (‰) P–H Δδ13C (‰) max. (abs)
Test by Qinhuangdao CIQ Test by Jiangsu CIQ Test by Qinhuangdao CIQ Test by Jiangsu CIQ
Range Passing ratea Range Passing ratea(%) Range Passing rateb(%) Range Passing rateb (%)
Acacia honey (n = 29) − 0.78 to 1.08 100% − 1.00 to 1.78 100 1.39–4.39 17.2 1.18–4.20 14.3
Rape honey (n = 33) 0.25 to 2.50 100% 0.33 to 2.39 100 1.20–4.12 36.4 0.92–4.39 65.6
Vitex honey (n = 11) 1.44 to 2.32 100% 1.28 to 2.35 100 2.39–4.34 0 1.73–4.16 9.1
Cotton honey (n = 10) 0.36 to 4.90 100% − 0.16 to 5.07 100 0.75–5.05 70 1.06–5.13 78
Linden honey (n = 4) 0.12 to 0.48 100% to 100 2.24–2.81 0 2.01–2.37 25
Jujube honey (n = 3) − 0.56 to − 0.36 100% − 0.72 to 0.48 100 2.42–2.51 0 2.06–2.56 66.7
Sunflower honey (n = 4) 0.84 to 1.54 100% 0.67 to 1.63 100 1.21–1.98 100 1.30–2.41 75
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aThe rate of samples that fall within the range of Δδ13C (‰) P–H ≥ –1.0‰

bThe rate of samples that fall within the range of Δδ13C (‰) max. (abs) ≥ ± 2.1‰

From Tables 3 and 4 we could found that as for acacia honey, 24 among 29 pure honey samples from Qinhuangdao CIQ and 24 among 28 samples from Jiangsu CIQ were failed to pass the test, the pass rate was only 17.2% and 14.3%, respectively. Apart from this, the Δδ13C (‰) max. values from the two labs is 4.39 and 4.20, respectively. This is due to the relatively small δ13Cds (disaccharides) values and therefore have much larger difference values between δ13Cds and δ13Cmono (monosaccharide) of all acacia honey samples except those originated from Henan province. Samples from Henan province had large Δδ13C (‰) max. values between δ13Cds and δ13Ctri (trisaccharide).

As for rape honey, 21 among 33 pure honey samples from Qinhuangdao CIQ and 11 among 32 samples from Jiangsu CIQ were failed to pass the test, the qualification yield was 36.4% and 65.6%, respectively. The Δδ13C (‰) max. values from these two labs was 4.12 and 4.36, respectively. This was because of the much larger difference values between δ13Cds and δ13CP of all rape honey samples.

Among 11 vitex honey samples, 11 and 10 were unqualified from Qinhuangdao CIQ and Jiangsu CIQ, respectively. The Δδ13C (‰) max. values from the two labs was 4.34 and 4.16, respectively. About 63.6% of these samples showed large difference values between δ13Cds and δ13Cmono, while the left 36.4% showed large difference values between δ13CH and δ13CP.

Three and two honey samples from Qinhuangdao CIQ and Jiangsu CIQ, respectively, were failed to pass the test with Δδ13C (‰) max. values of 5.24 and 5.13, respectively. All the unqualified samples could be explained by the fact that these samples had large difference values between δ13CH and δ13CP.

Although only 4 linden honey samples were analyzed in this article, all of them were considered to be adulterated from Qinhuangdao CIQ and 3 of them were failed to pass the test from Jiangsu CIQ. The Δδ13C (‰) max. values from these two labs were much smaller in comparison with other honey samples, that was, 2.81 and 2.37, respectively. The difference values between δ13Cmono and δ13CP were relatively larger.

It was gratified that 100% and 75% of four sunflower honey samples did successfully pass the test from Qinhuangdao CIQ and Jiangsu CIQ, respectively. It is clear that variance of N001 sample analyzed by Jiangsu CIQ has arisen due to the smaller δ13Cds values.

In Figs. 1 and 2, it is shown the LC-IRMS chromatogram of an authentic honey and an adulterated honey sample, respectively. It can be seen from Fig. 2 that almost no disaccharides and trisaccharides presented in adulterated honey with beet syrup, and the Δδ13C max. value, 0.1‰, fall within the theoretical range of 2.1‰. However, the highest difference observed did exceed 2.1‰ in pure honey sample in Fig. 1.

Fig. 1.

Fig. 1

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LC-IRMS chromatogram of one Vitex honey sample (B007). δ13C values: fructose − 25.99‰, glucose − 26.32‰, disaccharides − 22.21‰, trisaccharides − 24.15‰. Conditions: column, Carbomix Ca-NP5 (8% of cross-linking, 300 mm × 7.8 mm, 5 μm, Sepax Technologies Company); column temperature, 55 °C; mobile phase, ultrapure water; flow rate, 300 μl/min. The CO2 reference gas pulse was introduced two times (20 s each) at the beginning and at the end of each run

Fig. 2.

Fig. 2

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LC-IRMS chromatogram of adulterated honey with beet syrup. δ13C values: fructose − 25.0‰, glucose − 24.9‰. Conditions are the same as in Fig. 1

Based on the limits proposed by Intertek laboratory for the Δδ13C values of pure honey, it could be concluded by the fact that all samples have Δδ13C (‰) fru-glu values less than or equal to ± 1.0‰. It means that this indicator is reliable and no false positive result could be obtained. While as for the Δδ13C (‰) max. indicator, a large number of natural honey samples had Δδ13C (‰) max. values larger than ± 2.1‰. According to the method of the LC-IRMS, about 70% pure honey samples in China were considered to be adulterated, although those are 100% natural honeys. The main reason is because the carbon stable isotope ratio (13C/12C) values of honey can be affected by many factors, such as climate zone, temperature, rainfall values and harvesting period, which has been proved by Karabagias et al. (2016). Karabagias et al. (2016) investigated the impact of botanical origin and harvesting period on 13C/12C values and the results showed that the botanical origin and harvesting year of Greek honey had a strong impact on 13C/12C values and other physicochemical parameters. Therefore, as presented in this work, the method that proposed by Intertek is not appropriated to be used to detect adulterated honey with sugar syrups from various C3 plant sources in China.

Conclusion

The EA/LC-IRMS method that proposed by Intertek was very important in the detection of more sophisticated adulterations with syrups produced from either C3 plant or C4 plant source to imitate the sugar profile of honey, however, based on our research, false positive results were always obtained by using this method. As presented in this work, the representativeness data are the samples had Δδ13C (‰) values falling outside the range of ± 2.1‰ for Δδ13C (‰) max. Ninety-four authentic honey samples collected in China were involved, and about 70% samples can’t pass the δ13C-LC-IRMS test, which indicates that about 70% Chinese honeys can be classified as adulterated, although these honeys were extracted from unadulterated sources, and the failure rate was a bit high. The same conclusion could be obtained by two different labs. Therefore, based on these experimental results, the LC-IRMS method, which was proposed by Intertek lab, is not appropriate to be used for the detection of adulterated honeys with C3 sugars in China. It may not be appropriate to treat those honey samples from all over the world with the same criterion, because the carbon stable isotope ratio (13C/12C) values of honey can be affected by many factors.

Acknowledgements

This research project was supported by the by the Jiangxi provincial Natural Science Foundation for Youths of China (20161BAB213096).

Compliance with ethical standards

Conflict of interest

The author declares that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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