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Analysis of chemical composition of nectars and honeys from Citrus by extractive

electrospray ionization high resolution mass spectrometry

Yuanyuan Gao, Ahui Xue, Xiang Li, Xueyong Huang, Fangjian Ning, Xiaoping Zhang,
Tao Liu, Huanwen Chen, Liping Luo

PII: S0023-6438(20)30737-4
DOI: https://doi.org/10.1016/j.lwt.2020.109748
Reference: YFSTL 109748

To appear in: LWT - Food Science and Technology

Received Date: 3 November 2019

Revised Date: 12 May 2020
Accepted Date: 11 June 2020

Please cite this article as: Gao, Y., Xue, A., Li, X., Huang, X., Ning, F., Zhang, X., Liu, T., Chen, H.,
Luo, L., Analysis of chemical composition of nectars and honeys from Citrus by extractive electrospray
ionization high resolution mass spectrometry, LWT - Food Science and Technology (2020), doi: https://
doi.org/10.1016/j.lwt.2020.109748.

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CRediT author statement:

Yuanyuan Gao: Methodology, Software, Lab. Experiment, Writing-Original Draft.

Ahui Xue: Software, Lab. Experiment, Writing-Review & Editing.

Xiang Li: Investigation, Writing-Review & Editing.

Xueyong Huang: Supervision, Writing-Review & Editing.

Fangjian Ning: Supervision, Writing-Review & Editing.

Xiaoping Zhang: Writing-Review & Editing.

Tao Liu: Writing - Review & Editing.

Huanwen Chen: Resources.

Liping Luo: Resources, Investigation, Writing-Review & Editing.

 1 Analysis of chemical composition of nectars and honeys from Citrus

2 by extractive electrospray ionization high resolution mass

3 spectrometry

5 Yuanyuan Gaoab, Ahui Xueab, Xiang Liab, Xueyong Huangab, Fangjian Ningab,

6 Xiaoping Zhangc, Tao Liuab, Huanwen Chenc, Liping Luoab*

8
a
State Key Laboratory of Food Science and Technology, Nanchang University,

9 Nanchang, 330031, China

10
b
School of Life Sciences, Nanchang University, Nanchang, 330031, China

11
c
Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China

12 University of Technology, Nanchang, 330013, China

13

14
*
Corresponding author: Tel: +86-0791-83969519; Fax: +86-0791-83969519

15 E-mail address: lluo2@126.com

16

17

18

19

20

21

22

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24 ABSTRACT

25 Analyzing the chemical composition of nectar and honey will contribute to honey

26 traceability, which has become a research hotspot in recent years. However, the rapid,

27 green, and facile analytical methods without tedious chemical pretreatment remain to

28 be developed. Herein, the extractive electrospray ionization high resolution-mass

29 spectrometry (EESI-HRMS) were used to analyze the polyphenols and amino acids

30 composition of the nectars and honeys from three Citrus species, and

31 high-performance liquid chromatography coupled to ultraviolet detector (HPLC-UV)

32 was used to validate the results. 12 polyphenols and 8 amino acids were identified by

33 EESI-HRMS, whilst 9 polyphenols were quantified by HPLC-UV. Principal

34 component analysis (PCA) revealed that the chemical composition of Citrus nectars

35 was similar, and so do honeys, but the nectar and its corresponding honey were

36 chemically differentiated. Accordingly, EESI-HRMS is a reliable analytical method

37 for the analysis of samples with limited sources and complex composition, which

38 contributes to the honey traceability.

39 Keywords: Citrus honey; Floral nectar; Chemical composition; EESI-HRMS.

40

41

42

43

44

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46

47 1. Introduction

48 Nectar is a kind of sweet liquid secreted by the nectaries of flowers and is one of the

49 most important rewards that plants provide for floral visitors (Gardener & Gillman,

50 2001). Bees collect nectar to combine with their own secretions to transform, deposit,

51 dehydrate, and store in the hive until becoming honey (Naila, Flint, Sulaiman, Ajit, &

52 Weeds, 2018). Navel orange (Citrus. sinensis Osbeck), mandarin (C. reticulata

53 Kinokuni), and tangerine (C. reticulata Blanco) are the main fruit nectariferous plants

54 and secrete a large volume of nectar per flower. Citrus honey is a high value food

55 commodity with recognized nutraceutical and medicinal properties (Svečnjak, Prđun,

56 Rogina, Bubalo, & Jerković, 2017).

57 Honey is mainly composed of sugars and other constituents such as enzymes, amino

58 acids, minerals, and polyphenols (Naila, et al., 2018). As for nectar, amino acids are

59 the second richest compounds next to sugars (Power, Stabler, Borland, Barnes, &

60 Wright, 2018). The quality and price of honey from different plants are remarkably

61 different. Therefore, it is important to know the floral and geographical origins of the

62 honey. Patrignani et al. (Patrignani, Fagúndez, Tananaki, Thrasyvoulou, & Lupano,

63 2018) evaluated the geographical origin of Argentine honeys based on their volatile

64 substances. Some specific phytochemicals, especially polyphenols, are used to

65 identify the floral origin of several honeys, e.g., hesperetin for Citrus honey (Ferreres,

66 García-Viguera, Tomás-Lorente, & Tomás-Barberán, 1993), kaempferol for rosemary

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67 honey, and acacetin for acacia honey (Truchado, Ferreres, & Tomas-Barberan, 2009).

68 Most of the secondary metabolites used as potential markers of different botanical

69 origins in honey are derived from specific chemicals in the nectar. Truchado et al.

70 (Truchado, Ferreres, Bortolotti, Sabatini, & Tomás-Barberán, 2008) demonstrated that

71 nectar flavonol rhamnosides are floral markers of acacia honey. Tu et al. (Tu, Xu,

72 Chen, & Liu, 2011) found that volatile compound benzoic

73 acid-4-hydroxy-3,5-dimethoxy-hydrazine was the floral markers of rapeseed honey.

74 Furthermore, the sugars and polyphenols composition in Robinia pseudoacacia L.

75 nectar and its monofloral honey were detected to reveal how much nectar reflected the

76 final product (Gismondi, et al., 2018). These studies have shown that investigating the

77 chemical composition and typical metabolites of nectar and analyzing their

78 distribution in honey are the keys to honey traceability research.

79 To our knowledge, the main methods of honey traceability include traditional methods

80 such as physicochemical parameters and melissopalynological analysis,

81 chromatography, and chromatography-mass spectrometry (Kaškonienė & Venskutonis,

82 2010). Traditional methods have the disadvantages of subjectivity and uncertainty. For

83 example, the determination of physicochemical parameters is broad and cannot be

84 uniformly applied to different varieties of honey, and the melissopalynological

85 analysis of some honeys like Citrus honey is not representative (Svečnjak, Prđun,

86 Rogina, Bubalo, & Jerković, 2017). Chromatography and chromatography-mass

87 spectrometry require lengthy and complicated sample pretreatments such as extraction,

88 separation, and derivatization, leading to the prolongation of analytical time, the

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 89 consumption of the sample, and the loss of information. The applications of these

90 methods to nectar, especially the hard-to-collect nectar, are also problematic. Recently,

91 direct mass spectrometry requiring no tedious pretreatment with just a small amount

92 of sample has been used widely in food, biological, environmental, and

93 pharmaceutical fields (Lara-Ortega, et al., 2018; Zhang, et al., 2018). Extractive

94 electrospray ionization-mass spectrometry (EESI-MS) is one of the main technologies

95 of direct mass spectrometry, which has the advantages of fast, high sensitivity and

96 strong matrix tolerance et al., and it has been applied to the rapid analysis of uranium

97 in natural water samples (Luo, et al., 2009), identification of isomers of

98 methoxychalcone (Zhang, et al., 2018), and the detection of pesticides in honey (Deng,

99 et al., 2017).

100 In this study, a facile and effective method for simultaneous and rapid detection of

101 polyphenols and amino acids in three species of Citrus was established by EESI-high

102 resolution mass spectrometry (EESI-HRMS) for the first time. While

103 high-performance liquid chromatography with ultraviolet detector (HPLC-UV) was

104 used to validate the results and explore the phenolic composition more

105 comprehensively. Principal component analysis (PCA) was applied to evaluate their

106 relationships. The results provide reference for identification of potential markers

107 specifically of Citrus honey and the traceability of different varieties of honey. These

108 methods could be used for the analysis of samples with limited sources and complex

109 composition rapidly as well.

110 2. Materials and methods

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111 2.1. Materials and reagents

112 Nectars were collected by microcapillary method (Power, et al., 2018) from flowers of

113 three species of Citrus, including navel orange, mandarin, and tangerine, during

114 blooming stage from April to May 2018 in Jiangxi Province, China. Monofloral

115 honeys were produced by introducing new frames with beeswax foundations into

116 hives when collecting nectar, and there were no other notable plants flowering in the

117 nectar collecting area. Nectars were kept on ice in the field, and then stored at –80°C

118 for further analysis.

119 Solid-phase extraction (SPE) cartridges (Oasis HLB, 60 mg/3 mL) were purchased

120 from Waters Corporation (Milford, MA, USA). Chlorogenic acid, gallic acid,

121 cinnamic acid, genistein, vanillic acid, and isorhamnetin were purchased from

122 Aladdin Co., Ltd. (Shanghai, China), with a purity higher than 98%. Protocatechuic

123 acid, benzoic acid, P-hydroxybenzoic acid, quercetin, rutin, kaempferol, hesperidin,

124 and hesperetin were purchased from J&K Chemicals Co., Ltd. (Beijing, China), with

125 a purity higher than 98%. HPLC grade methanol, ethanol, acetonitrile and acetone,

126 and analytical grade formic acid were purchased from Anpel Co., Ltd. (Shanghai,

127 China).

128 2.2. EESI-HRMS analysis

129 2.2.1. Sample preparation

130 Nectar (0.5 g) and honey (0.5 g) were homogenized and put into 2.5 mL and 5.0 mL

131 volumetric flasks, respectively, and the final volume was adjusted with 70% methanol.

132 After centrifuged at 6700 ×g for 10 min using TGL-20B high speed centrifuge from

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133 Anting scientific instrument Co., Ltd. (Shanghai, China), the supernatant was

134 collected through a 0.45 µm membrane filter (Aladdin, China) and stored at –20°C

135 until analysis.

136 2.2.2. Reference standards solutions

137 The reference standards stock solutions of polyphenols were prepared by dissolving

138 appropriate amounts of analytes in methanol. All reference standard stock solutions

139 were stored in amber disposable vial in the freezer at –20°C. The working mixed

140 standard solutions were diluted to 1 µg/mL for EESI-HRMS and 10 µg/mL for

141 HPLC-UV analysis with methanol respectively.

142 2.2.3. Instruments and working conditions

143 EESI-HRMS experiments were carried out on a commercial LTQ-Orbitrap-XL high

144 resolution mass spectrometer coupled with a Xcalibur data processing system

145 (Thermo Scientific, San Jose, CA, USA). And the home-made EESI source (Jia,

146 Zhang, Ding, Yang, & Chen, 2012) used for ion generation was developed by Jiangxi

147 Key Laboratory for Mass Spectrometry and Instrumentation, East China University of

148 Technology, China.

149 The EESI ion source was placed at 0.5 cm (d) in front of the MS inlet capillary, with

150 the distance of 0.1 cm and angle (α) of 60° between extractant spray and sample spray

151 respectively (Fig. 1). Mass spectrum was averaged in the range of m/z 50–800 from

152 the 30 scans obtained over the sampling period of 10 s under both the positive and

153 negative ion detection modes. The ionization voltage was 3.5 kV. The capillary

154 temperature was maintained at 250°C, and the pressure of nitrogen sheath gas was 1.2

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155 MPa. The extractant (80% methanol) and sample flow rates were 4 µL/min and 5

156 µL/min, respectively. The instrument was operated at a high resolution up to 60,000.

157 Collision induced dissociation (CID) experiments were carried out for MS2 analysis.

158 The width of the isolation window of the parent ion was set to 1.0 Da, with the

159 normalized collision energy of 20–40%, while other parameters were set to the default

160 values of the LTQ instrument.

161 2.3. HPLC analysis

162 2.3.1. Extraction of polyphenols from samples

163 The extraction method of polyphenols used was based on Sun et al. (Sun, Tan, Zhang,

164 & Zhang, 2016) with minor modifications. Nectar (2.0 g) and honey (2.0 g) samples

165 were homogenized and diluted with acidified deionized water (pH 2, 1.0 mol/L HCl

166 solution) to a final volume of 5.0 mL and 10.0 mL, respectively. The solutions were

167 centrifuged at 8940 ×g for 10 min. The supernatant was filtered through an Oasis

168 HLB SPE cartridge which was previously activated and equilibrated with methanol

169 (3.0 mL) and acidified deionized water (3.0 mL, pH 2). A volume of 2.0 mL honey

170 solution was taken into the treated cartridge and passed at 1.0 mL/min. Then, SPE

171 cartridges were washed with 3.0 mL acidified deionized water (pH 2) to remove the

172 honey matrices. Finally, the polyphenols remaining in the cartridge were eluted with

173 3.0 mL methanol and adjusted to the volume to 5.0 mL. The methanol fraction was

174 filtered through a 0.45 µm membrane and stored at –20°C until further analysis by

175 HPLC-UV.

176 2.3.2. Instruments and working conditions

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177 Analyses of the samples were carried out by HPLC Waters Alliance 2695 (Milford,

178 MA, USA) with UV detector (Waters 2489) using a Symmetry C18 column (4.6 mm

179 × 250 mm × 5 µm). The mobile phases were acetonitrile (A) and 0.5% aqueous

180 formic acid (B) (v/v) with the following linear gradient elutions (Xue, Cui, Zou, &

181 Luo, 2018): 5–14% A from 0 to 5 min, 14–21% A from 5 to 20 min, 21–27% A from

182 20 to 27 min, 27–42% A from 27 to 40 min, 42–65% A from 40 to 48 min, 65–35% A

183 from 48 to 54 min, 35–14% A from 54 to 60 min, and 14–5% A from 60 to 62 min.

184 The flow rate was maintained at 1.0 mL/min, and the column was operated at 35°C.

185 The injection volume was set to 50 µL. Chromatograms were recorded at 280 nm.

186 Fingerprinting profiles and similarity of samples and standards were constructed and

187 analyzed based on the Similarity Evaluation System for Chromatographic

188 Fingerprinting of Traditional Chinese Medicine (Version 2004A).

189 2.4 PCA analysis

190 In EESI-HRMS and HPLC-UV analysis, a total of six samples, including three groups

191 of nectar samples (navel orange, mandarin, and tangerine nectar), and three groups of

192 honey samples (navel orange, mandarin, and tangerine honey) were analyzed.

193 Min-max normalization was used to the linear transformation of raw mass spectra

194 data by Matlab R2016a software (Mathworks, Inc., Natick, USA), so that the data was

195 normalized to 0-10 scale. The transformation function was as follows:

196 y=(x-min)/(max-min). Among them, “y” is the value after normalization; “x” is the

197 raw data exported by Xcalibur data processing system; “max” is the maximum value

198 of the sample data; “min” is the minimum value of the sample data. In EESI-HRMS

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199 analysis, three pieces for each sample were collected and each sample having five

200 technical duplications, that is, 15 sets of data were collected for statistical analysis to

201 eliminate the instrument errors. In HPLC-UV, each sample was repeated three times.

202 3. Results and discussion

203 3.1. EESI-HRMS analysis

204 3.1.1. Optimization of conditions

205 To achieve the maximum sensitivity, the signal intensities of galangin (m/z 269.0444)

206 and hesperetin (m/z 301.0707) were used to optimize the extraction agent and

207 capillary temperature, because the m/z of galangin is in the middle of polyphenols

208 commonly found in honey, while hesperetin is the possible maker in Citrus honey.

209 Methanol, ethanol, acetone, acetonitrile, and water were used as extraction solvents to

210 carry out the optimization experiments. For the highest signal intensities, methanol

211 was selected as the extraction solvent (Fig. 2a) with different proportions of 50%,

212 60%, 70%, 80%, and 90%, respectively (Fig. 2b). As the proportion of methanol

213 increases, the signal intensities showed an increasing trend first and then decreasing,

214 and reached the maximum at 80% of methanol.

215 The signal intensities of the target ions increased along with the temperature and

216 reached the maximal level at 250°C (Fig. 2c), while the signal level started to

217 gradually decrease when the capillary temperature was higher than 250°C. This is

218 most likely because the capillary temperature is positively correlated with the

219 sensitivity of the instrument and determines ion desolvation efficiency, while high

220 temperature (250°C or higher) may destroy the substances such as polyphenols.

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221 3.1.2. Characterization of compounds

222 Preliminary qualitative calibration was done based on the high resolution mass

223 measurement of parent ions in full MS mode. The MS2 spectra obtained by CID

224 experiments were compared with the tandem mass spectra of references, standards, or

225 the public databases of MassBank (http://www.massbank.jp/), Human Metabolome

226 Database (http://www.hmdb.ca/), and the national institute of standards and

227 technology (https://www.nist.gov/), to identify or predict metabolites. For example,

228 the mass spectra of several polyphenols and amino acids of the mandarin nectar and

229 its corresponding monofloral honey were presented with either negative (Fig. 3) or

230 positive ion modes (Fig. 4). The MS2 spectra of other compounds identified were

231 presented in Supplementary Information.

232 In the negative ion mode, fragment ions of m/z 123, 152, owing to the loss of CO2 and

233 CH3, were yielded by the precursor ion of m/z 167 (Fig. 3a), and its fragmentation

234 patterns agreed with the deprotonated vanillic acid standard (Supplementary

235 Information Fig. S4) and those reported previously (Biesaga & Pyrzynska, 2009),

236 confirming that the m/z 167 was assigned to deprotonated vanillic acid. Precursor ion

237 m/z 169 (Fig. 3b) showed fragment ions at m/z 125, 151, and 141, due to the neutral

238 loss of CO2, H2O, and CO, respectively. These patterns were consistent with the

239 deprotonated gallic acid standard (Fig. S5) and those previously reported

240 (Oelschlaegel, et al., 2012), confirming that m/z 169 was identified as deprotonated

241 gallic acid. Studies have shown that flavonoids easily lose carboxyl and oxygen atoms

242 in the C-ring, while the retro-Diels-Alder reaction occurs at different sites to generate

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243 corresponding fragment ions (Goussé, Gandini, & Hodge, 1998). In the MS2 spectrum

244 of m/z 269 (Fig. 3c), the main fragment ion m/z 251 was formed by the loss of H2O in

245 the precursor ion, while the fragment ions of m/z 225 and 223 generated by the loss of

246 CO2 and -CH2O2 in the C-ring of precursor ion formed a stable carbon skeleton.

247 These fragment ions were consistent with the deprotonated genistein standard

248 spectrum, confirming that the m/z 269 was the deprotonated genistein (Fig. S6).

249 Precursor ion at m/z 301 (Fig. 3d) generated fragment ions at m/z 286, 257, and 283,

250 owing to the elimination of CH3, CO2, and H2O, respectively, supported by the

251 deprotonated hesperetin standard (Fig. S7).

252 By comparing with databases and previous studies (Biesaga & Pyrzynska, 2009;

253 Kıvrak & Kıvrak, 2017; J. Sun, Hou, Feng, & Shi, 2006), precursor ion at m/z 135

254 (Fig. 3e) generated fragment ions at m/z 117 and 91, due to the loss of H2O and

255 -C2H4O, respectively, suggesting that m/z 135 was revealed as deprotonated

256 phenylacetic acid. Precursor ion at m/z 179 (Fig. 3f) generated fragment ions at m/z

257 135, 161, 151, and 143, due to the elimination of CO2, H2O, CO, and H4O2,

258 respectively, consistent with the spectrum of caffeic acid as reported previously (Allen,

259 Greiner, & Wishart, 2015). Therefore, m/z 179 was recognized as deprotonated caffeic

260 acid. Characterization of the qualitative fragments of 12 polyphenols in Citrus nectars

261 and honeys detected under negative ion mode was shown in Table 1.

262 8 amino acids were detected in positive ion mode, supported by the fragments

263 reported in databases and the previous studies (Xu, et al., 2013; Piraud, et al., 2003;

264 Wang, et al., 2016). The loss of HCOOH (MW=46) from precursor ion to form

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265 [R−CH=NH2]+ was observed in seven amino acids, except for arginine. The main

266 fragment of protonated ion serine (m/z 106) (Fig. 4a), threonine (m/z 120) (Fig. 4d),

267 aspartic acid (m/z 134) (Fig. 4f), and glutamic acid (m/z 148) (Fig. 4g) were all

268 generated by the elimination of H2O to form [M+H−H2O]+. Protonated arginine (m/z

269 175) (Fig. 4h) generated fragment ions at m/z 158 and 157, due to the loss of NH3 and

270 H2O, respectively, while the fragment ions at m/z 130 and 116 were yielded by the

271 losses of [NH3+CO] and [NH=C(NH2)2] from precursor ions, respectively. Fragment

272 ions at m/z 70 suggested the loss of CO and H2O from m/z 116. Meanwhile, precursor

273 ion at m/z 116 was assigned to protonated proline (Fig. 4b). Characterization of the

274 qualitative fragments of 8 amino acids in Citrus nectars and honeys detected under

275 positive ion mode was shown in Table 2.

276 3.1.3. Various components in Citrus nectars and honeys

277 A total of 20 compounds were detected in Citrus nectars and honeys, including 12

278 polyphenols in negative ion mode (Table 1) and 8 amino acids in positive ion mode

279 (Table 2). Polyphenols detected in various Citrus nectars and honeys by EESI-HRMS

280 was illustrated in Table S1. Benzoic acid, phenylacetic acid, P-hydroxybenzoic acid,

281 gallic acid, and cinnamic acid were identified in all nectar and honey samples. As

282 reported in the previous study (Mou, Ning, & Ramp, 2017), benzoic acid in honey is

283 found in the floral nectar and is produced by phenylalanine under the action of

284 microorganisms with the cinnamic acid as an intermediate product. Polyphenols

285 contain hydroxybenzoic acid and hydroxycinnamic acid, which are derived from

286 benzoic acid and cinnamic acid, respectively. Benzoic acid and cinnamic acid

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287 detected in all samples may be substrates of other polyphenols. Vanillic acid,

288 protocatechuic acid, hydrocinnamic acid, and hesperetin were detected in all honeys

289 but not in nectar samples, probably because vanillic acid and hydrocinnamic acid are

290 mainly found in pollen (Gómez-Caravaca, Gómez-Romero, Arráez-Román,

291 Segura-Carretero, & Fernández-Gutiérrez, 2006), while the hesperetin in honey was

292 hydrolyzed from hesperidin in nectar. Hesperidin was not detected in nectar by

293 EESI-HRMS probably because the glycoside hesperidin was decomposed at a high

294 capillary temperature. Isorhamnetin was detected in all nectars, but was only found in

295 mandarin honey. Genistein was detected in tangerine nectar and mandarin nectar. As

296 an important constituent of propolis (Gargouri, Osés, Fernández-Muiño, Sancho, &

297 Kechaou, 2019), caffeic acid was detected in all honeys as well as tangerine nectar.

298 Flowers of Citrus secrete secondary metabolites to attract bees to pollinate. Plants

299 have several polyphenols derivatives with high structural diversity and complexity,

300 which are transferred from plants to honey as honeybees collect nectar.

301 In the positive ion mode, essential amino acids valine and threonine, as well as

302 non-essential amino acids proline, glutamic acid, and arginine, were detected in all

303 nectar and honey samples. Proline originates mainly from the salivary secretions of

304 honeybees during the conversion of nectar into honey (Silva, Gauche, Gonzaga, Costa,

305 & Fett, 2016), while our results demonstrated that proline also existed in nectar. All

306 samples except for mandarin and tangerine honeys contained leucine and aspartic acid,

307 while only mandarin honey did not detect serine. Previous studies (Gardener, &

308 Gillman, 2001) reported that there was no significant difference in the types of free
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309 amino acids in honey of the same genus but different botanical origins. Leucine,

310 aspartic acid, and serine were not detected in mandarin or tangerine honeys, possibly

311 because they were decomposed into other amino acids (Cometto, Faye, Caccavari,

312 Baroni, & Aldao, 2006). The compositions of amino acids and polyphenols are

313 different in each sample may due to their different botanical and geographic origin.

314 As reported previously (Tkaczewska, et al., 2020), sophisticated analytical techniques

315 such as automatic amino acid analyzer are particularly used for amino acid analysis,

316 while the precolumn derived chromatography is always used in the determination of

317 honey amino acids. These methods require complex pretreatment and high-volume of

318 samples which are the major limitations of amino acids in nectar research. However,

319 EESI-HRMS provided a rapid and sensitive method to simultaneously examine both

320 the polyphenols and amino acids composition of nectar.

321 3.1.4. PCA of Citrus nectars and honeys

322 To visualize the differences among all samples, PCA analysis was carried out. The 3D

323 PCA score plot matrix of nectars and their monofloral honeys was shown in Fig. 5a.

324 Three kinds of nectars (navel orange, mandarin, and tangerine nectar) were not

325 evidently separated from each other, indicating that there was no significant difference

326 detected by EESI-HRMS in the chemical composition of them. Similarly, the

327 biochemical changes among three kinds of honeys (navel orange, mandarin, and

328 tangerine honeys) were not significant. These results were in agreement with those

329 previously reported (Svečnjak, et al., 2017) showing that the honeys from different

330 species in the same genus exhibited similar characteristics. In contrast, nectars were
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331 clearly separated from their monofloral honeys indicating that there was notable

332 difference between the chemical composition of them. Three PCs representing about

333 71% of the total variance with the eigenvalue of 38.3% for PC1, 20.7% for PC2, and

334 12% for PC3 were selected for modeling. In the loading plots of these three principal

335 components (Fig. 5b), the mass spectral signals with the significant contribution were

336 m/z 179 (caffeic acid), m/z 269 (genistein), and m/z 301 (hesperetin) among the

337 qualitative compounds.

338 According to (Gargouri, et al., 2019), caffeic acid is an important constituent of

339 propolis, therefore, the main source of coffee acid in honey may be propolis rather

340 than nectar. The flavanone hesperetin has been suggested as a possible marker of

341 Citrus honey by (Truchado, et al., 2009). In our study, hesperetin was detected in all

342 honey, but not found in nectar. That is, the conclusion can be confirmed by

343 EESI-HRMS. Overall, the chemical composition or concentration of Citrus nectars

344 and honeys varied greatly due to the effect of hydrolytic enzymes (glucosidases) and

345 oxidative enzymes (glucose oxidase).

346 3.2. HPLC analysis

347 3.2.1 PCA

348 PCA was performed over the HPLC-UV data of nectars and honeys after normalizing

349 (Fig. 6). Citrus nectars were grouped together, indicating that they contained

350 relatively similar composition of compounds. Citrus honeys also exhibited the

351 chemical similarities. Nectars and their monofloral honeys were well-separated.

352 Therefore, we conclude that the results of EESI-HRMS can be validated by HPLC
16
353 analysis.

354 3.2.2 Method validation

355 Linearity was evaluated using working mixed standard solutions spiked at 6 level

356 concentrations by the procedures as described in 2.2.2. Linear calibration curves were

357 constructed from the peak area versus nominal analyte concentrations. All the

358 calibration curves showed good linearity (R2≥0.9941).

359 The limits of detection (LOD) and quantification (LOQ) under the chromatographic

360 conditions were separately determined at a signal-to-noise ratio (S/N) of 3 and 10.

361 And they were in the range of 0.251-5.345 ng/mL and 0.837-17.818 ng/mL for 9

362 polyphenols, respectively.

363 The intra/inter-day precision were evaluated according to the relative standard

364 deviation (RSD) of 4 consecutive sample injections of a sample in one day and

365 repeated for 3 days. Intra-day precision varied from 0.39 for protocatechuic acid to

366 2.34 for rutin. Inter-day precision varied from 0.87 for chlorogenic acid to 2.66 for

367 p-hydroxybenzoic acid.

368 Sample stability was evaluated by selecting a sample for continuous injection of 4

369 needles every 12 h within 48 h. The sample also showed good stability within 48 h,

370 with RSD of 0.51%~2.92%.

371 The parameters related to the validation process were described in detail in Table S2

372 in Supplementary Information.

373 3.2.3 Polyphenols detected in Citrus nectars and honeys by HPLC-UV

17
374 A total of 9 polyphenols were quantified by HPLC-UV (Table 3). Gallic acid,

375 protocatechuic acid, P-hydroxybenzoic acid, and hesperidin were detected in all

376 samples, while chlorogenic acid was detected in most of the samples except for navel

377 orange nectar and its monofloral honey, with higher concentration in nectars than in

378 honeys as for mandarin (0.722 µg/g in nectar and 0.158 µg/g in honey) and tangerine

379 (1.008 µg/g in nectar and 0.158 µg/g in honey).

380 For flavonoids, rutin was only detected in tangerine nectar and its monofloral honey.

381 As a potent antioxidant, the presence of a large amount of rutin in nectar (1.989 µg/g)

382 would suggest a protective role of this molecule for the plants. The existence of rutin

383 in both nectar and honey is probably due to the insufficient of bee enzymes to

384 hydrolyze rutin (Truchado, et al., 2008). Hesperidin was detected in all samples and

385 showed higher concentrations in nectars (14.999–96.822 µg/g) than in honeys (0.784–

386 2.751 µg/g). It represented the most abundant flavonoid compound in nectar as

387 reported previously (Ferreres, et al., 1993; Truchado, et al., 2009), probably due to the

388 reason that the concentration of hesperidin is high in navel oranges at their juvenile

389 fruit stage (1.4% of the fresh weight) (Barthe, Jourdan, McIntosh, & Mansell, 1988).

390 Our results showed that hesperetin, quercetin, and kaempferol were only detected in

391 honeys, possibly because there were corresponding flavanone glycosides in nectars. In

392 our work, hesperidin was detected in all three Citrus nectars. And a recent study

393 (Palmer-Young, et al., 2019) reported that among 26 species of nectar producing

394 plants, quercetin-O-glycoside and kaempferol-O-glycoside were detected in 9 and 5

395 species, respectively. To conclude, the amounts of two glycosides (rutin and

18
396 hesperidin) in honey decreased because glycosides were hydrolysed to form flavonoid

397 aglycone (quercetin and hesperetin) catalyzed by the enzymes in bee saliva. Due to

398 the same mechanism, quercetin was detected in navel orange honey while rutin was

399 not observed. Overall, the varieties of secondary metabolites were richer in Citrus

400 honeys than nectars, while flavonoids, except for rutin and hesperidin, were more

401 abundant in Citrus honeys than in nectars.

402 The chemical compositions of nectars and honeys of Citrus were thoroughly explored

403 by HPLC-UV and EESI-HRMS. The differences of substances identified by the two

404 techniques are likely due to the difference in pretreatments, i.e., samples were only

405 simply diluted in EESI-HRMS, while disposed through SPE cartridges in HPLC

406 analysis.

407 4. Conclusion

408 A rapid and facile method based on EESI-HRMS has been established to analyze the

409 polyphenols and amino acids compositions of both the nectar and honey. A total of 12

410 polyphenols and 8 amino acids were detected by EESI-HRMS as well as 9

411 polyphenols by HPLC-UV in nectars and honeys from three Citrus species. Results of

412 the PCA indicated that there was no significant difference in chemical composition

413 among Citrus nectars and among Citrus honeys, but significant chemical variation

414 was found in nectars and their corresponding honeys. The major mass spectrometry

415 signals contributing to the differentiation of nectars and honeys included m/z 179

19
416 (caffeic acid), m/z 269 (genistein), and m/z 301 (hesperetin) among the qualitative

417 compounds. Furthermore, the varieties of secondary metabolites were richer in Citrus

418 honeys than nectars, and the content of flavonoids, except for rutin and hesperidin,

419 were more abundant in honeys than nectars. Hesperidin was detected in all nectars

420 with high concentration, while hesperetin was detected in all honey samples by both

421 EESI-HRMS and HPLC-UV, suggesting that hesperetin could be a suitable marker for

422 the floral origin of Citrus honey. Hence, EESI-HRMS can be a new analytical method

423 to confirm the conclusion. The work could provide reference for identification of

424 potential marker of Citrus honey and EESI-HRMS can be a promising tool for the

425 analysis of samples with limited sources and complex composition, contributing to the

426 honey traceability. And a larger number of honey samples from other floral origins

427 should be analyzed using EESSI-HRMS in the future, in order to validate the use of

428 this promising analytical method for the determination of the floral origin of honey.

429 Acknowledgments

430 This work was supported by the National Natural Science Foundation of China (No.

431 31772067), the Department of Science and Technology of Jiangxi Province (No.

432 20165BCB18005, 20171BCB24002), Jiangxi Agriculture Research System (No.

433 JXARS-14) and Research Project of State Key Laboratory of Food Science and

20
434 Technology (SKLF-ZZB-201918).

435 Author Information

436
*
Corresponding author: Tel: +86-0791-83969519; Fax: +86-0791-83969519

437 Email address: lluo2@126.com (L.L.P.)

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26
Figure Captions

Fig. 1. Schematic flow diagram of electrospray extraction ionization high resolution

mass spectrometry.

Fig. 2. Optimization of experimental conditions for electrospray extraction ionization

high resolution mass spectrometry: (a) and (b) extraction solvent; (c) capillary

temperature.

Fig. 3. EESI-HRMS2 spectra of some polyphenols in mandarin nectar and its

monofloral honey in negative ion pattern. (a) vanillic acid; (b) gallic acid; (c)

genistein; (d) hesperetin; (e) phenylacetic acid; (f) caffeic acid.

Fig. 4. EESI-HRMS2 spectra of mandarin nectar and its monofloral honey in positive

ion pattern: (a) serine; (b) proline; (c) valine; (d) threonine; (e) leucine; (f) Aspartic

acid; (g) glutamic acid; (h)arginine.

Fig. 5. PC-three-dimensional (a) and corresponding loading plots (b) of Citrus nectars

by PCA analysis. NN, navel orange nectar; MN, mandarin nectar; TN, tangerine

nectar; NH, navel orange honey; MH, mandarin honey; TH, tangerine honey.

Fig. 6. PCA result of nectars and corresponding monofloral honeys detected by

HPLC-UV.
 Table 1 Compounds detected in Citrus nectars and honeys in negative ion mode
Error
[M-H]−, m/z -MS2[M-H]−
Compound Formula CAS (ppm)
Measured Theoretical [M-H-H2O]− [M-H-CO] − [M-H-CO2]− Others
abc
Benzoic acid C7H6O2 65-85-0 121.0280 121.0284 -3.3 93(5) 77(100)
c
Phenylacetic acid C8H8O2 103-82-2 135.0448 135.0441 5.2 117(100) 91(25) [M-H-C2H4O]−
P-hydroxybenzoic acid bc C7H5O3 99-96-7 137.0230 137.0233 -2.1 109(15) 93(100)
a
Cinnamic acid C9H8O2 140-10-3 147.0444 147.0441 2.0 129(60) 62(100) [M-H-C4H5O2]−
Hydrocinnamic acid c C9H10O2 501-52-0 149.0591 149.0597 -4.0 131(50) 121(55) 105(100)

Protocatechuic acid ac C7H6O4 99-50-3 153.0176 153.0182 -3.9 135(25) 109(100) 125(25) [M-H-C2H4]−

Vanillic acid ab C8H8O4 121-34-6 167.0332 167.0390 -4.2 123(100) 152(10) [M-H-CH3]−

Gallic acid ab C7H6O5 149-91-7 169.0133 169.0131 1.2 151(18) 141(15) 125(100)

Caffeic acid b C9H8O4 331-39-5 179.0333 179.0339 -3.4 161(70) 151(32) 135(100) 143(18) [M-H-2H2O]−

Genistein a C15H10O5 446-72-0 269.0442 269.0444 -0.7 251(100) 225(40) 223(20) [M-H-CO-H2O]−

Hesperetin a C16H14O6 520-33-2 301.0696 301.0707 -3.7 283(40) 257(40） 286(100) [M-H-CH3]−

Isorhamnetin a C16H12O7 480-19-3 315.0483 315.0499 -5.0 297(100) 271(38) 300(20) [M-H-CH3]−

Note: a, the identification was based on reference standard; b, the identification was based on literature; c, the identification was based on the databases;

the number in parentheses indicates the relative abundance (%).

 Table 2 Compounds detected in Citrus nectars and honeys in positive ion mode

Error
[M+H]+, m/z -MS2[M+H]+
Compound Formula CAS (ppm)
Measured Theoretical [M+H-H2O]+ [M+H-HCOOH]+ Others
ab
Serine C3H7NO3 56-45-1 106.0495 106.0499 -3.8 88(100) 60(30)
ab
Proline C5H9NO2 147-85-3 116.0704 116.0706 -1.9 70(100)
ab
Valine C5H11NO2 72-18-4 118.0868 118.0863 4.2 72(100) 101(15) [M+H-NH3]+
Threonine ab C H NO 72-19-5 120.0660 120.0655 4.2 102(100) 74(100)
ab
Leucine C6H13NO2 61-90-5 132.1024 132.1019 3.9 86(100)
b
Aspartic acid C4H7NO4 56-84-8 134.0441 134.0448 -5.2 116(100) 88(60)

Glutamic acid b C H NO 56-86-0 148.0599 148.0604 -3.4 130(100) 102(20) 131(20) [M+H-NH3]+

158(100) [M+H-NH3]+

ab
116(40) [M+H-NH=C(NH2)2]+
Arginine C6H14N4O2 74-79-3 175.1183 175.1190 -4.0 157(80)
130(38) [M+H-NH3-CO]+
70(5) [M+H-NH=C(NH2)2-H2O-CO]+
Note: a, the identification was based on literature; b, the identification was based on the databases; the number in parentheses indicates the relative abundance
(%).
 Table 3 Determination of polyphenols in Citrus nectars and honeys by HPLC-UV

Content (µg/g)
Compound RT (min)
Navel orange nectar Mandarin nectar Tangerine nectar Navel orange honey Mandarin honey Tangerine honey

Gallic acid 4.668 0.229±0.015 0.205±0.011 0.256±0.007 0.256±0.012 0.237±0.004 0.317±0.014

Protocatechuic acid 7.533 0.411±0.023 0.852±0.066 0.811±0.022 0.865±0.015 0.419±0.079 0.725±0.061

Chlorogenic acid 9.313 — 0.722±0.151 1.008±0.125 — 0.158±0.027 0.158±0.003

P-hydroxybenzoic acid 9.972 3.745±0.087 4.279±0.155 2.108±0.165 0.693±0.045 5.641±0.975 9.752±0.151

Rutin 17.039 — — 1.989±0.045 — — 0.847±0.021

Hesperidin 24.589 14.999±0.366 66.978±7.936 96.822±2.964 0.784±0.033 2.311±0.398 2.751±0.056

Quercetin 32.698 — — — 0.662±0.039 0.269±0.057 0.514±0.022

Kaempferol 37.886 — — — 1.291±0.028 0.937±0.155 1.045±0.007

Hesperetin 38.651 — — — 0.129±0.007 0.172±0.031 0.066±0.012

Note: All results represent the mean ± SD (X̅ ±SD µg/g) of independent measurements (n=3), “—” means not detected.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Highlights:

(1) A fast EESI-HRMS method was developed for compounds analysis in nectar and

honey.

(2) This method was firstly applied in the analysis of nectar and honey.

(3) Samples did not require tedious pretreatment.

(4) The result that hesperetin was the marker of Citrus honey can be validated by

EESI-HRMS.
Conflict of interest

The authors have no conflict of interest to declare.