Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/264296006

PhytochemicalFingerprintsofLimeHoneyCollectedinSerbia

ArticleinJournalofAOACInternational·July2014

DOI:10.5740/jaoacint.SGEGasic

CITATIONS

3

READS

201

7authors,including:

UrošM.GašićUniversityofBelgrade

19PUBLICATIONS110CITATIONS

SEEPROFILE

BrankoSikoparija

UniversityofNoviSad

50PUBLICATIONS441CITATIONS

SEEPROFILE

TomislavTosti

UniversityofBelgrade

15PUBLICATIONS51CITATIONS

SEEPROFILE

ZivoslavTesic

UniversityofBelgrade

164PUBLICATIONS885CITATIONS

SEEPROFILE

Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate,

lettingyouaccessandreadthemimmediately.

Availablefrom:UrošM.GašićRetrievedon:10October2016

Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014 1

Phytochemical Fingerprints of Lime Honey Collected in SerbiaUroš GašićUniversity of Belgrade, Faculty of Chemistry, PO Box 51, 11158 Belgrade, SerbiaBranko ŠikoparijaUniversity of Novi Sad, Faculty of Sciences, Laboratory for Palynology, Trg Dositeja Obradovića 2, 21000 Novi Sad, SerbiaTomislav TosTi, Jelena Trifković, DUšanka miloJković-opsenica, maJa naTić, and Živoslav Tešić1

University of Belgrade, Faculty of Chemistry, PO Box 51, 11158 Belgrade, Serbia

Guest edited as a special report on “Chromatographic and Biological Fingerprinting of Herbal Samples” by Łukasz Cieśla and Monika Waksmundzka-Hajnos.

1 Corresponding author’s e-mail: ztesic@chem.bg.ac.rsDOI: 10.5740/jaoacint.SGEGasic

SPECIAL GUEST EDITOR SECTION

Composition of phenolic compounds and the sugar content were determined as the basis for characterization of lime honey from Serbia. Particular attention was given to differences in phytochemical profiles of ripe and unripe lime honey and lime tree nectar. Melissopalynological analysis confirmed domination of Tilia nectar in all analyzed samples. Phenolic acids, abscisic acid, flavonoids, and flavonoid glycosides were determined by means of ultra-HPLC coupled with a hybrid mass spectrometer (UHPLC–OrbiTrap). Sugar content was determined using high-performance anion-exchange chromatography with amperometric detection. Similar phenolic compounds characterized unripe and ripe honeys, while the lime tree nectar profile showed notable differences. Compared to lime tree nectar, a high amount of chrysin, pinocembrin, and galangin were detected in both ripe and unripe lime honey. Fructose and glucose were the major constituents of all investigated samples, and amounts were within the limits established by European Union legislation. Sucrose content in the nectar sample was up to two-fold higher when compared to all honey samples. Isomaltose and gentiobiose with turanose content were different in analyzed production stages of lime honey.

Lime trees (Tilia) are considered among the major honey sources in moderate climate areas of the Northern Hemisphere. However, unifloral lime honey

is considered rare (1). This blend is commonly found on the honey market in Germany, The Netherlands, and Poland, and it is rare in Italy, Spain, France, Switzerland, Belgium, Finland, Norway, and Sweden (2, 3). Such distribution is related to the distribution of Tilia species and size of their populations (4).

Phytochemicals are found in plant-based foods and are known to have protective or disease preventive properties, acting as antioxidants, nutrient protectors, and anticarcinogens. A comprehensive database on food phytochemicals and their health-promoting effects was published recently by Scalbert et al. (5).

Several major groups of phytochemicals exist. Phenolic

phytochemicals, i.e., flavonoids and phenolic acids, comprise an important and large group of compounds. These compounds are also identified as microcomponents among the wide range of phytochemicals present in honey. A number of publications regarding the health benefits of honey, especially its antioxidant properties and its role against cancer, cardiovascular diseases, inflammatory disorders, neurological degeneration, infectious diseases, and aging can be found in literature (6).

In finding characteristic phytochemical patterns various qualitative and quantitative methods are helpful, especially hyphenated techniques that combine chromatographic and spectral methods. Such techniques provide phytochemical fingerprints valuable for identifying the source plant. In our previous investigations, 43 phenolic compounds were identified in Serbian unifloral honeys (7) and 36 in Serbian polyfloral honey (8).

In order to trace phytochemicals that characterize lime honey produced in Serbia, we focused on the phenolic content of lime mature honeys. For that purpose, ultra-HPLC (UHLPC)/LTQ OrbiTrap MS, already proven to be reliable for the unambiguous detection of phenolic acids and their derivatives and flavonoid aglycones and glycosides, was used. Individual sugars were determined and quantified using high performance anion-exchange chromatography with a pulsed amperometric detector (HPAEC-PAD). A total of 27 mature honey samples were collected from two areas of Serbia (Fruška Gora Mountain and Eastern Serbia). The aim of this work was to examine whether there is any notable difference in the content of phytochemicals, especially flavonoid glycosides, among lime tree nectar, unripe lime honey, and mature lime honeys. Therefore, analysis included one sample of unripe lime honey and lime tree nectar collected from Fruška Gora Mountain.

Experimental

Sample Collection

A total of 27 mature honey samples declared as unifloral lime honey (Table 1) were provided by The Association of the Beekeeping Organizations of Serbia (SPOS; www.spos.info) and the Beekeeping Society (Dr Jovan Zivanovic; http://www.pcelarins.org.rs). The honeys were produced in Eastern Serbia (seven samples) and on Fruška Gora Mountain (20 samples), which are considered as the most important lime tree forage sources in Serbia (9). Analyzed samples contained honey produced in different seasons from 2001 to 2011. Seasonal variability (2001–2003 and 2006–2011) was assessed for nine samples produced by the same beekeeper at the same locality at Fruška Gora Mountain. All samples were received and

2 Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014

transferred to the laboratory in their original packages and kept at 20–22°C before the analysis. One sample of mature honey from Fruška Gora Mountain (No. 27, Table 1) was obtained from bees that, in addition to nectar foraging, were also fed by sugar syrup (each day 2 kg of sugar beet sugar in 2 L H2O).

In order to provide samples for nectar analysis, flowers were picked from Tilia trees at Fruška Gora Mountain (Serbia) in the middle of the flowering season in June, 2013 and brought to the laboratory. During the same day, nectar was aspirated from the flowers using a glass capillary and then collected in Eppendorf test tubes and stored at –20°C until analysis. About 100 µL of nectar was collected. At the same time, unripe lime honey was taken from the bee hive situated at the same locality on Fruška Gora Mountain.

Reagents and Standards

Acetonitrile and formic acid (both MS grade), methanol (HPLC grade), and hydrochloric acid were purchased from Merck (Darmstadt, Germany). The SPE cartridges used for

extraction and concentration of samples were Strata C18–E (500 mg/3 mL) obtained from Phenomenex (Torrance, CA). An ultrapure water TKA MicroPure water purification system (0.055 µS/cm) was used to prepare standard solutions and blanks (Thermo Fisher Scientific, Bremen, Germany). Flavonoid standards (rutin, naringin, luteolin, quercetin, apigenin, naringenin, kaempferol, chrysin, pinocembrin, and galangin) were purchased from Fluka AG (Buchs, Switzerland), while catechin, gallic, chlorogenic, caffeic, p-coumaric, and cis, trans-abscisic acids were supplied by Sigma-Aldrich (Steinheim, Germany). Syringe filters (13 mm, PTFE membrane 0.45 µm) were purchased from Supelco (Bellefonte, PA). Filter paper (Whatman No. 1) was supplied by Merck. Sugar standards (trehalose, glucose, fructose, isomaltose, sucrose, melezitose, gentiobiose, turanose, isomaltotriose, maltose, panose, and maltotriose) were purchased from Tokyo Chemical Industry (TCI Europe, Zwijndrecht, Belgium).

Tablets of Lycopodium tracer spores were purchased from the Department of Geology, Lund University, Sweden. Microscopic slide mounting medium, a glycerol gelatin mixture that contains gelatin, glycerin, phenol, distilled water, and basic fuchsine, was prepared in the Laboratory for Palynology, University of Novi Sad.

Melissopalynological Analysis

Qualitative melissopalynological analysis was performed to identify major nectar sources in the analyzed Serbian honey samples. Microscopic slides for qualitative analysis of samples were prepared and analyzed according to the Harmonized Methods of Melissopalynology (10). Slides were analyzed until a minimum of 1000 pollen grains were counted. Pollen was identified using referent slides and pollen identification atlases (11, 12) and counted along with honeydew elements, i.e., algae, fungal spores and hyphae, anemophilous pollen, and pollen of nectarless plants (1, 13). Relative frequency of identified pollen was calculated (14, 15). Honeydew contribution was evaluated as the ratio between the number of honeydew elements (HDE) and pollen of nectariferous plants (PN) according to scale given by Louveaux et al. (16).

Because pollen content in nectar of particular nectariferous species depends on flower morphology and pollen size (17), relative pollen frequency does not necessarily indicate the exact contribution of nectar in honey samples. Therefore, nectar contribution was determined using the concept applied in paleopalynology (18). Pollen coefficients experimentally determined by Demianowicz (19) were used for transformation of pollen relative frequency into nectar relative frequency. Missing pollen coefficients were derived from known values determined by alternative methodology (20) or deduced taking into consideration pollen and flower morphology (13).

Instruments

(a) UHPLC-LTQ OrbiTrap XL.—Separations and quantification of compounds of interest were performed using a Thermo Fisher Scientific system that consisted of a quaternary Accela 600 pump and Accela autosampler connected to a linear ion trap-orbitrap hybrid mass spectrometer (LTQ OrbiTrap XL) with heated electrospray ionization probe (HESI-II).

(b) HPAEC-PAD.—Sugar content was determined by means

Table 1. Geographical area, locality, and year of honey extraction

Sample No. Geographical area Locality

Year of honey

extraction

1 Eastern Serbia Crni vrh, Knjaževac 2008

2 Eastern Serbia Zvezdan, Zaječar 2009

3 Eastern Serbia Blagojev kamen 2009

4 Eastern Serbia Blagojev kamen 2009

5 Eastern Serbia Ujevac, Kučevo 2009

6 Eastern Serbia Ceremošnja 2009

7 Eastern Serbia Bor 2009

8 Fruška Gora Mountain Erdevik 2008

9 Fruška Gora Mountain Bešenovo, Nova Pazova 2008

10 Fruška Gora Mountain Stara Bingula 2008

11 Fruška Gora Mountain Beočin, Crveni Čot 2010

12 Fruška Gora Mountain Beočin, Crveni Čot 2007

13 Fruška Gora Mountain Beočin, Crveni Čot 2006

14 Fruška Gora Mountain Beočin, Crveni Čot 2006

15 Fruška Gora Mountain Beočin, Crveni Čot 2003

16 Fruška Gora Mountain Beočin, Crveni Čot 2002

17 Fruška Gora Mountain Ledinci 2001

18 Fruška Gora Mountain Paragovo 2001

19 Fruška Gora Mountain Sviloš 2001

20 Fruška Gora Mountain Vrdnička kula 2001

21 Fruška Gora Mountain Beočin, Crveni Čot 2001

22 Fruška Gora Mountain Banstol, Veliki vrhovi 2001

23 Fruška Gora Mountain Rakovac 2001

24 Fruška Gora Mountain Beočin, Crveni Čot 2011

25 Fruška Gora Mountain Beočin, Crveni Čot 2011

26 Fruška Gora Mountain Beočin, Crveni Čot 2008

27 Fruška Gora Mountain Beočin, Crveni Čot 2009

Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014 3

of ion chromatography with amperometric detection. Honeys were analyzed on an ICS 3000 DP LC system equipped with a quaternary gradient pump (Dionex, Sunnyvale, CA).

Preparation of Standard Solutions

(a) Preparation of phenolic standards.—A 1000 mg/L stock solution mixture of all phenolic standards was prepared in methanol. Dilution of the stock solution with methanol yielded the working solution at concentrations of 0.025, 0.050, 0.100, 0.250, 0.500, 0.750, and 1.000 mg/L. All stock and working solutions were stored in the dark at 4°C and were stable for at least 3 months. Calibration curves were obtained by plotting the peak areas of the compounds identified against the concentrations of the standard solutions. Calibration curves revealed good linearity, with R2 values exceeding 0.99.

(b) Preparation of sugar standards.—The calibration was performed with standard solutions of sugars. Each individual sugar standard was dissolved in ultrapure water, and stock solutions with concentration of about 1000 mg/L were prepared. Dilution of the stock solutions with ultrapure water yielded the working solutions at concentration ranges that corresponded to the content of each sugar in honey. For glucose and fructose the the concentration range was from 10.0 to 100.0 mg/L, for sucrose from 1.0 to 10.0 mg/L, for isomaltose from 0.5 to 5.0 mg/L, and for the other sugar standards from 0.1 to 1.0 mg/L. Evaluation of the sugar content of honey samples was obtained from calibration curves of each sugar contained in standard solutions. Under these chromatographic conditions, the last sugar was detected after approximately 25 min, and the analysis was ended at 30 min.

Sample Preparation

The method used for extraction and isolation of phenolics from lime honey samples was a modification of a method described in the literature (21). Honey samples (5 g) were mixed with 5 mL ultrapure water adjusted to pH 2 with 0.1% HCl and homogenized in an ultrasonic bath (Bandelin Electronic, Berlin, Germany) for 30 min at room temperature. The samples were then filtered through filter paper to remove solid particles. The SPE cartridge was conditioned by washing with 3 mL acetonitrile and 9 mL ultrapure water. The filtrate was passed through the cartridge, which was then washed with 6 mL acidified water to remove all sugars and other polar constituents of honey. The adsorbed compounds were eluted with acetonitrile (1.5 mL). The extracts were filtered through a 0.45 µm PTFE membrane filter and analyzed by UHPLC/HESI-MS/MS. The water fraction was used for determination of sugars by HPAEC-PAD. The described procedure was applied for the extraction and analysis of the lime tree nectar.

UHPLC/MS/MS Conditions

The analytical column used for separation was Hypersil gold C18 (100 × 2.1 mm id, 1.9 µm particle size) from Thermo Fisher Scientific. The mobile phase consisted of (eluent A) water + 0.1% formic acid and (eluent B) acetonitrile + 0.1% formic acid. A linear gradient program at a flow rate of 0.300 mL/min was used: 0.0–1.0 min 5% B, 1.0–9.9 min from 5 to 95 % (B),

9.9–10 min from 95 to 5 % (B), and 5% (B) for 3 min. The injection volume was 5 μL.

The mass spectrometer was operated in negative mode. HESI source parameters were as follows: source voltage, 3 kV; capillary voltage, –20 V; tube lens voltage, –150 V; capillary temperature, 275°C; and sheath and auxiliary gas flow (N2) 30 and 8 (arbitrary units), respectively. Mass spectra were acquired by full range acquisition covering 100–1000 m/z. For the fragmentation study, a data dependent scan was performed by deploying collision-induced dissociation (CID). The normalized collision energy of CID cell was set at 35 eV.

Phenolics were identified and quantified according to the corresponding spectral characteristics: mass spectra, accurate mass, characteristic fragmentation, and characteristic retention time. Quantification of all compounds was performed according to the method described previously by Gašić et al. (8). Xcalibur software (version 2.1) was used for instrument control, data acquisition, and data analysis. The fragmentation mechanism and characteristic fragments were confirmed using Mass Frontier software (version 6.0; Thermo Fisher Scientific). The internet database of accurate mass spectrometry data, ChemSpider (www.chemspider.com), was used as a reference library to calculate mass of the compounds of interest.

HPAEC-PAD Conditions

Carbohydrates were separated on a Carbo Pac® PA10 pellicular anion-exchange column (250 × 4 mm id; Dionex) at 30°C. Each sample (25 µL) was injected with an ICS AS-DV 50 autosampler (Dionex). Carbohydrates were eluted with flow rate set to 0.7 mL/min using a gradient prepared from 600 mM sodium hydroxide (eluent A), 500 mM sodium acetate (eluent B), and ultrapure water (eluent C). The gradient program was as follows: 0.0–20.0 min, 15% A; 20.1–30.0 min, 20% A; 0.0–5.0 min, 0% B; 5.1–12.0 min, 2% B; 12.1–20.0 min, 4% B; and 20.1–30.0 min, 20% B; and 0.0–5.0 min, 85% C; 5.1–12.0 min, 83% C; 12.1–20.0 min, 81% C; and 20.1–30.0 min, 60% C.

Calculations

LOD and LOQ values were calculated using SD of the response and the slope (S) of the calibration curve of each analyte according to the formulas: LOD = 3(SD/S) and LOQ = 10(SD/S). The values of SD and S were obtained from the calibration curves created in Microsoft Excel.

Results and Discussion

Melissopalynological Analysis of Lime Honey

According to melissopalynological analysis, none or very little honeydew contributed to analyzed Serbian lime tree honey (maximum HDE/PN = 0.94). Tilia pollen was recorded in all analyzed samples with an average contribution of 77%, which is notably higher than the maximum recorded for European lime honeys (22, 23). Apart from lime tree nectar that was found at levels of 27.81–99.83% in analyzed samples, important constituent of Serbian lime tree honey was nectar from Robinia pseudoacacia, Brassica napus, or Amorpha fruticosa.

Unripe lime tree honey had exceptionally high Tilia pollen contribution (>90%), indicating high lime tree nectar content

4 Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014

(>99%). In seven ripe honey samples (No. 6, 7, 8, 10, 11, and 15) Tilia pollen content exceeded 90%, but such an exceptionally high lime tree nectar content was found in only two samples (No. 6 and 10). Similar characteristics of honey produced only by nectar feeding and honey produced by simultaneous nectar and sugar feeding (sample No. 27) imply that bees from both hives foraged nectar from the same plant sources.

Phenolic Profile of Lime Honey and Lime Nectar

SPE combined with UHPLC/LTQ Orbitrap MS was used to determine the content of flavonoids, phenolic acids, and abscisic acid in unripe and ripe lime honeys and lime tree nectar. A total of 16 compounds were quantified by comparing retention times and mass spectra with available standards. Chromatograms of the investigated samples showed similar profiles. A selected

base peak chromatogram of ripe honey extract (sample No. 11) from Fruška Gora Mountain is shown in Figure 1A.

Phenolics were identified and quantified using the available standards. The identification was confirmed by a direct comparison of the retention times and mass spectra. LOD, LOQ, and recovery at three concentration levels were determined according to an earlier described method (8). These results are given in Table 2.

Unripe and ripe lime tree honey profiles were characterized by similar phenolic compounds, while the lime tree nectar profile showed notable differences (Table 3). High amounts of chrysin, pinocembrin, and galangin in both ripe and unripe honey were found. Chrysin and pinocembrin were present in all samples. Abscisic acid, a plant hormone, was also found in all samples, ranging from 75 to 360 µg/100 g. The absence of chrysin, pinocembrin, and galangin, which are known to be propolis-derived flavonoids (24), was noticeable in the lime tree nectar sample. A similar phenolic profile was reported for Slovenian lime honey (25).

There were some differences in phenolic profiles of honeys collected in Eastern Serbia and on Fruška Gora Mountain, mostly in the presence of catechin, naringin, and naringenin. A more detailed analysis is needed to show whether it is possible to differentiate samples according to geographical region on the basis of such findings, and it will certainly be the subject of extensive research in the future. Also, the phenolic profile of honey extracted from the hive additionally fed by sugar syrup was similar to that obtained from bees that foraged nectar (Table 3).

In the absence of standards, identification of flavonoid glycosides was based on the search for the [M–H]– deprotonated molecule and its fragmentation using UHPLC/LTQ OrbiTrap MS. The exact mass search and the study of the fragmentation pathways described in the literature enabled us to obtain as much

Table 2. Regression equations, R2, LOD, LOQ, and recovery of the method for three different concentrations

Regression equation (Y = A + BX) Recovery, %

Compound (A ± SE) × 105 (B ± SE) × 105 R2 LOD, mg/L LOQ, mg/L Level 1a Level 2a Level 3a

Gallic acid –2.10 ± 0.58 334.68 ± 1.12 0.9903 0.09 0.30 109 107 93

Chlorogenic acid –2.32 ± 1.13 286.00 ± 2.08 0.9997 0.02 0.07 115 93 98

Catechin –1.72 ± 0.70 107.71 ± 1.35 0.9943 0.07 0.23 95 114 108

Caffeic acid 0.20 ± 1.60 417.02 ± 3.24 0.9997 0.02 0.07 112 105 105

Rutin –1.22 ± 1.31 206.49 ± 2.31 0.9994 0.03 0.11 119 113 81

p-Coumaric acid 1.43 ± 0.50 4.17 ± 0.12 0.9977 0.07 0.22 87 110 106

Naringin –0.64 ± 0.48 112.09 ± 0.91 0.9939 0.08 0.27 89 105 96

Quercetin –11.52 ± 2.86 319.38 ± 4.92 0.9988 0.05 0.16 85 104 86

cis, trans-Abscisic acid –0.85 ± 0.26 21.04 ± 0.41 0.9985 0.05 0.18 99 112 108

Luteolin –4.34 ± 2.99 423.84 ± 5.75 0.9991 0.04 0.13 102 104 97

Naringenin –1.13 ± 0.37 49.01 ± 0.72 0.9991 0.04 0.13 104 95 107

Apigenin –4.31 ± 0.76 192.40 ± 1.46 0.9997 0.02 0.07 83 98 86

Kaempferol –5.08 ± 1.43 197.97 ± 2.58 0.9991 0.04 0.12 101 108 99

Chrysin –2.29 ± 0.80 76.88 ± 1.59 0.9979 0.06 0.19 106 90 97

Pinocembrin –1.48 ± 0.41 103.15 ± 0.79 0.9997 0.02 0.07 87 96 93

Galangin –29.45 ± 3.17 994.97 ± 6.35 0.9998 0.01 0.05 106 121 99a Level 1 (0.25 mg/L); Level 2 (0.50 mg/L); and Level 3 (0.75 mg/L).

Figure 1. Flavonoid glycoside fingerprint of (A) selected ripe lime honey from Fruška Gora Mountain - sample No. 11 and (B) lime nectar.

Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014 5

Table 3. The content of some polyphenolics in ripe and unripe lime honey and lime tree nectar (µg/100 g)

Sample No. GaAa ChAb Catc CaAd Rute CoAf Narg Queh AbAi Lutj Nank Apil Kaem Chrn Pino Galp

1 0.31 NDq ND 22.22 ND 16.91 ND 1.60 85.91 5.01 ND 7.70 1.66 230.34 43.17 191.93

2 0.31 ND ND 6.69 0.11 3.07 ND 0.33 77.61 1.14 ND 2.41 0.76 168.21 35.18 85.85

3 0.69 ND ND 21.14 0.17 18.57 ND 0.92 83.97 2.89 ND 6.89 2.26 205.02 56.22 255.89

4 ND ND ND 12.26 0.10 12.94 ND 0.66 77.19 1.91 ND 5.72 2.21 204.97 50.87 190.64

5 ND 1.33 ND 9.10 0.05 16.75 ND 1.11 53.23 3.05 ND 14.99 3.20 212.63 60.68 177.89

6 ND ND ND 18.94 ND 20.43 ND 0.16 75.32 1.56 ND 3.02 0.47 188.28 20.74 62.86

7 1.53 ND ND 22.41 0.70 ND ND 1.39 74.97 3.09 ND 4.92 1.98 192.43 44.22 143.93

8 2.29 ND ND 25.41 0.46 5.70 ND 4.03 109.04 2.72 ND 2.98 1.68 230.48 29.28 228.79

9 11.39 3.88 ND 41.83 0.88 9.74 ND 1.47 100.04 9.20 ND 8.31 5.77 220.61 40.56 264.28

10 1.08 ND ND 87.68 0.07 9.48 ND 0.26 94.63 2.10 ND 5.04 0.42 251.66 11.86 81.09

11 6.32 ND 29.59 0.50 ND 5.81 ND ND 140.18 ND ND 2.40 3.74 192.35 211.65 5.60

12 72.72 ND 2.46 80.67 ND 71.51 2.26 5.47 279.35 0.55 ND 13.96 5.47 278.20 198.08 88.20

13 47.93 ND 2.24 77.51 ND 81.58 4.58 3.29 275.02 1.05 1.10 11.40 4.95 238.63 182.30 86.17

14 12.16 ND 2.65 4.16 ND 16.02 2.09 ND 226.51 ND ND 4.48 0.34 227.50 203.48 18.22

15 30.59 0.68 16.10 21.76 ND 47.63 6.63 ND 237.13 ND 1.61 2.76 ND 172.99 48.17 10.81

16 20.10 ND 18.36 1.67 ND 10.01 ND ND 176.77 ND ND 1.26 ND 128.05 36.81 1.36

17 6.08 ND 10.39 39.29 ND 28.00 2.85 ND 175.71 ND 0.40 1.52 ND 120.24 10.51 1.28

18 10.05 ND 5.42 0.61 ND 10.00 1.96 ND 153.34 ND 0.43 0.79 ND 87.88 24.57 0.54

19 6.18 2.59 9.38 10.84 ND 31.68 2.10 ND 148.34 ND 0.27 3.73 ND 152.45 63.27 13.21

20 0.86 2.74 17.71 ND ND 3.26 ND ND 125.93 ND 0.50 ND ND 28.62 8.59 ND

21 6.62 ND 17.97 0.25 ND 10.14 2.23 ND 229.69 ND 0.47 1.69 ND 201.70 85.94 2.22

22 31.07 1.50 4.19 54.12 ND 44.15 1.73 0.52 256.44 0.65 0.14 6.75 0.71 247.13 83.92 37.94

23 3.73 0.63 1.06 15.15 ND 34.31 3.17 6.28 243.74 0.50 ND 16.18 11.16 250.76 283.83 101.22

24 80.07 0.83 6.95 207.92 1.24 166.36 ND 7.54 285.28 2.45 ND 36.07 31.41 278.89 336.90 195.68

25 172.04 ND ND 96.84 0.54 130.57 ND 3.25 215.09 3.74 ND 55.65 37.46 348.75 477.58 206.35

26 49.08 1.54 4.12 95.52 ND 79.57 ND 3.05 358.03 0.31 ND 7.02 4.00 220.19 177.72 76.02

27r 1.88 ND ND 48.94 ND 38.51 1.15 5.69 130.40 0.70 ND 23.54 11.46 337.94 444.31 123.58

28s ND ND ND 58.94 12.11 125.96 ND ND 230.64 3.06 0.66 66.84 60.21 275.48 101.96 150.94

29t ND 0.02 2.38 ND 1.31 ND ND ND 0.33 0.93 ND ND 0.10 1.01 0.34 0.37a GaA = Gallic acid.b ChA = Chlorogenic acid.c Cat = Catechin. d CaA = Caffeic acid. e Rut = Rutin.f CoA = p-Coumaric acid. g Nar = Naringin.h Que = Quercetin.i AbA = cis, trans-Abscisic acid.j Lut = Luteolin. k Nan = Naringenin. l Api = Apigenin.m Kae = Kaempferol.n Chr = Chrysin.o Pin = Pinocembrin.p Gal = Galangin.q ND = Not detected.r Honey produced by simultaneous nectar foraging and sugar feeding. s Unripe honey.t Lime tree nectar.

6 Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014

structural information as possible. In this way, it was possible to identify 15 glycosides (Table 4). A base peak chromatogram of lime tree nectar from Fruška Gora Mountain is shown in Figure 1B. The qualitative picture obtained in the case of the nectar was notably different when compared to ripe honey samples, which is expected due to hydrolytic enzymatic activity. Since the flavonoid glucosides are readily hydrolyzed, their aglycones were detected and characterized in ripe honey. On contrary, flavonoid rhamnosides and rutinosides cannot be hydrolyzed by the glucosidase activity of bee saliva (26); therefore, they were found in both unripe and mature honeys.

Chemical composition of honey is very dependent on nectar-providing plant species, climatic conditions (such as sunlight and moisture), and soil characteristics (27). When seasonal variability within ripe honey samples was analyzed, differences in the amount of gallic acid, cafeic acid, cis, trans–abscisic acid, apigenin, chrysin, pinocembrin, and galangin could be observed. Also, the content of quantified phenolic compounds in honey samples collected in 2011 was notably different from all other analyzed samples. This suggests the importance of climatic conditions (considered seasonally as the most important variable) on phenolic profiles of honey.

Determination of Sugars

Sugars were determined using HPAEC-PAD. The R2, LOD, LOQ, and recovery values at two concentration levels

are given in Table 5. A chromatogram of a selected ripe lime honey sample from Fruška Gora Mountain (No. 11) is depicted in Figure 2, and the quantitative sugar content is given in Table 6. Quantification was performed with 12 available sugar standards. Reducing sugars, fructose and glucose, were found to be the major constituents of all investigated samples, and their amounts were within the limits established by European Union (EU) legislation (Council Directive 2001/110/EC). Six disaccharides and four trisaccharides were recovered from all analyzed samples. Among the disaccharides, sucrose and isomaltose were the major components in ripe honey samples, and sucrose and maltose in unripe honey and lime tree nectar.

Contents of glucose and fructose in mature honey samples varied from 23.06 to 30.78 and from 36.42 to 44.44 g/100 g, respectively. Unripe honey and lime tree nectar had almost the same content of fructose and glucose, with a lower content of fructose compared to mature honey samples. Several samples showed sucrose content higher than 5 g/100 g. Although lime tree honey is not included among those for which a 10 g/100 g limit is allowed by the European Directive (Council Directive 2001/110/EC), higher sucrose concentration was observed in European unifloral lime honey (22). Sucrose content in the unripe honey sample was within the limit of the European Directive. On the other hand, the sucrose content in the nectar sample was 19.06 g/100 g, which is more than two-fold higher than the content in honey samples. Lower content of sucrose in honey might result from activities of enzymes introduced by

Table 4. Presence of flavonoid glycosides in selected ripe lime honey from Fruška Gora Mountain—sample No. 11, unripe lime honey, and lime tree nectar

Peak assignmenta Compound name tR, minb

Exact mass, [M–H]–, m/z

Accurate mass,

[M–H]–, m/z ppmcMS/MS fragmentation,

m/zLime

honeydUnripe honey Nectar

A Kaempferol-3-O-glucosyl-rhamnosyl-glucoside

5.09 755.20269 755.20203 0.87 593.15, 447.09, 285.04 + + +

B Quercetin-3-O-rhamnosyl-galactoside 5.12 609.14524 609.14392 2.17 447.09, 301.03 – – +

C Quercetin-3-O-sophoroside 5.14 625.14060 625.13910 2.40 463.09, 301.03 – – +

D Isorhamnetin-3-O-sophoroside 5.31 639.15622 639.15443 2.81 459.09, 315.05 + + +

E Kaempferol-3,7-O-diglucoside 5.37 609.14522 609.14429 1.53 447.09, 285.04 – + +

F Isorhamnetin-3-O-rutinosyl-7- O-glucoside

5.41 785.21371 785.21271 1.27 623.16, 461.11, 315.05 + + +

G Quercetin 3-O-rhamnosyl-rhamnosyl-glucoside

5.51 755.20401 755.20294 1.42 593.15, 447.09, 301.03 + + +

H Isorhamnetin-3-O-rhamnosyl-galactoside 5.52 623.16086 623.15973 1.81 461.11, 315.05 + + +

I Quercetin-3-O-rutinoside (Rutin) 5.53 609.14611 609.14539 1.18 463.09, 301.03 + + +

J Quercetin-3-O-glucoside 5.75 463.08777 463.08633 3.11 301.03 – – +

K Quercetin-3-O-(6”-malonyl-glucoside) 5.90 549.08861 549.08844 0.31 301.03 – – +

L Isorhamnetin-3-O-galactoside 5.98 477.10340 477.10269 1.49 315.05 + + +

M Kaempferol-3-O-glucoside 6.05 447.09284 447.09210 1.66 285.04 – + +

N Kaempferol-3-O-rhamnoside 6.51 431.09792 431.09708 1.95 285.04 + + +

O Isorhamnetin-3-O-glucoside 6.52 477.10340 477.10272 1.43 315.05 + + +

a Peak assignments correspond to Figure 1.b tR = Retention time.c Mean mass accuracy.d + = Detected, – = not detected.

Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014 7

bees. The amounts of sucrose in mature honeys ranged from 3.07 to 9.89 g/100 g. The amount of isomaltose in the tested ripe honey samples was higher than that of maltose and varied from 1.01 to 3.66 g/100 g. The content of isomaltose in unripe honey and lime tree nectar was very low or not detected. The content of gentiobiose with turanose was much higher in mature honey samples compared to unripe honey and lime tree nectar. Sugar content of honey extracted from the hive additionally fed by sugar syrup was within the limits for honeys obtained from bees that foraged nectar (Table 6).

When analyzing the sugar content in ripe honey samples collected in different harvesting seasons, a small variability only in sucrose and isomaltotriose could be noticed. Similarly, as in case of quantified phenolics, the content of quantified sugar compounds in honey samples obtained in 2011 was quite different from all the others.

Conclusions

Phytochemical profiles of 27 ripe lime honeys, one unripe lime honey, and one lime tree nectar were determined in order to characterize lime honey collected in Serbia and examine its chemical composition in different stages of its production. Phenolic acids, their derivatives, and flavonoid aglycones and glycosides were detected by using UHPLC/LTQ OrbiTrap MS. The sugar content, as the major honey ingredient, was determined using HPAEC-PAD.

Melissopalynological analysis confirmed predominance of Tilia nectar in all analyzed samples. Unripe and ripe honeys were characterized by similar phenolic compounds, while the lime tree nectar profile was notably different. Compared to lime tree nectar, high amounts of chrysin, pinocembrin, and galangin were determined. Fructose and glucose were the major sugar constituents of all investigated samples and within the limits established by EU legislation. The sucrose content in the nectar sample was more than two-fold higher than the sucrose content in all honey samples, which could be attributed to the enzyme activity on nectar sucrose. Sugars, such as isomaltose and

gentiobiose with turanose, indicated differences in production stages of honey.

Finally, it is important to note that honey originating from Serbia was not included in the study that defined characteristics of the unifloral lime honey in Europe (28). Therefore, the results presented in this paper are essential for positioning Serbian honey samples in the European honey market.

Acknowledgments

This work was performed within the framework of the research project No. 172017 supported by the Ministry of Education, Science and Technological Development, Republic of Serbia. The authors acknowledge the support of the FP7 RegPot project FCUB ERA GA No. 256716. The EC does not share responsibility for the content of the article. This work was partly funded by the Ministry of Education, Science and Technological Development, Republic of Serbia (project Nos. III43002 and OI173002) and Provincial Secretariat for Science

Table 5. Equation for regression line, R2, LOD, LOQ, and recovery at two concentration levels for sugar quantification

Regression equation (Y = A + BX) Recovery, %

Sugar A ± SE B ± SE R2 LOD, g/L LOQ, g/L Level 1a Level 2a

Trehalose –0.28 ± 0.37 1.44 ± 0.29 0.9965 0.09 0.30 111 107

Glucose 4.50 ± 7.18 2.22 ± 0.11 0.9967 4.07 13.57 115 114

Fructose 9.78 ± 7.97 1.82 ± 0.12 0.9974 3.73 12.43 93 91

Isomaltose –0.09 ± 0.29 2.16 ± 0.21 0.9999 0.02 0.07 108 108

Sucrose –1.13 ± 1.63 1.41 ± 0.25 0.9974 0.38 1.27 88 103

Melezitose –0.08 ± 0.18 1.43 ± 0.13 0.9994 0.04 0.13 99 109

Gent+Tur 0.27 ± 0.48 0.78 ± 0.07 0.9993 0.22 0.73 109 104

Isomaltotriose 0.01 ± 0.21 1.49 ± 0.15 0.9988 0.06 0.20 114 89

Maltose –1.23 ± 1.01 1.45 ± 0.16 0.9992 0.20 0.67 104 85

Panose –0.05 ± 0.15 1.37 ± 0.10 0.9995 0.04 0.13 88 115

Maltotriose –0.13 ± 0.09 0.37 ± 0.06 0.9999 0.01 0.03 96 112

a For glucose and fructose Level 1 (25.00 g/L), Level 2 (50.00 g/L), respectively, for sucrose Level 1 (2.50 g/L), Level 2 (5.00 g/L); for isomaltose Level 1 (1.25 g/L), Level 2 (2.50 g/L); for the other sugars Level 1 (0.25 g/L), Level (0.50 g/L).

Figure 2. HPAEC-PAD chromatogram of selected ripe lime honey from Fruška Gora Mountain—sample No. 11; I - trehalose; II - glucose; III - fructose; IV - unknown; V - isomaltose; VI - sucrose; VII - melezitose; VII - gentiobiose + turanose; IX - isomaltotriose; X - maltose; XI - unknown; XII - unknown; XIII - panose; XIV - maltotriose; and XV - unknown.

8 Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014

Table 6. Sugars in ripe and unripe lime honey and lime tree nectar (g/100 g)

Sample No. Treha Glucb Frucc iMaltd Suce Melf Gent+Turg iMaltotrih Malti Panj Maltotrik

1 0.09 23.06 42.53 2.08 4.89 0.66 2.81 0.16 1.02 0.24 0.03

2 0.08 28.18 42.13 1.86 4.74 0.65 2.86 0.11 0.85 0.21 0.08

3 0.13 24.83 42.22 2.02 4.92 0.73 2.84 0.12 1.17 0.25 0.04

4 0.14 25.54 42.46 1.73 4.57 0.64 2.82 0.10 0.84 0.22 0.05

5 0.14 29.84 40.28 1.85 4.61 0.50 2.85 0.05 0.68 0.16 0.07

6 0.30 28.21 39.33 2.41 5.10 1.05 2.54 0.21 0.84 0.25 0.03

7 0.09 26.24 40.11 1.74 4.16 0.97 2.32 0.10 0.64 0.30 0.04

8 0.05 26.30 41.40 1.49 3.58 1.05 2.47 0.10 1.80 0.30 0.15

9 0.17 24.30 40.75 1.70 4.48 0.64 2.99 0.07 0.54 0.14 0.12

10 0.13 23.31 39.13 3.32 6.70 1.22 3.13 0.33 0.70 0.27 0.06

11 0.16 26.28 43.69 1.86 4.45 0.31 1.32 0.06 0.48 0.28 0.04

12 0.28 27.78 40.57 2.95 7.45 0.35 2.00 0.33 0.51 0.52 0.01

13 0.24 24.71 41.33 2.68 6.15 0.39 2.44 0.17 0.52 0.64 0.00

14 0.30 27.24 44.44 2.75 6.67 0.38 2.67 0.13 0.55 0.09 0.01

15 0.17 25.07 38.95 3.66 9.78 0.32 2.45 0.69 0.53 0.65 0.01

16 0.15 27.63 38.56 3.11 8.22 0.38 2.17 0.41 0.47 0.51 0.01

17 0.51 25.78 41.91 3.59 9.89 0.31 2.39 0.57 0.52 0.58 0.01

18 0.18 25.04 36.62 3.63 9.29 0.30 2.14 0.99 0.55 0.82 0.01

19 0.32 29.34 36.82 2.92 6.49 0.28 1.66 0.53 0.49 0.10 0.01

20 0.31 29.09 36.42 2.89 6.52 0.28 1.67 0.54 0.49 0.10 0.01

21 0.53 24.37 42.90 3.02 8.84 0.50 2.07 0.44 0.57 0.03 0.00

22 0.18 26.92 38.98 2.77 7.54 0.45 1.75 0.41 0.43 0.03 0.02

23 0.22 27.46 40.14 3.07 7.47 0.48 1.50 0.59 0.48 0.79 0.01

24 0.61 30.78 41.11 1.57 3.72 0.54 1.54 0.07 0.29 0.06 0.00

25 0.40 30.00 42.40 1.01 3.07 0.59 1.16 0.04 0.22 0.06 0.01

26 0.17 24.78 40.10 2.51 5.50 0.29 1.63 0.23 0.53 0.13 0.02

27l 0.30 27.02 40.31 2.75 6.94 0.39 1.91 0.39 0.48 0.34 0.01

28m 0.21 33.76 33.48 0.04 4.69 0.05 0.30 0.18 0.17 0.06 0.09

29n 0.22 27.80 27.35 0.00 19.06 0.05 0.23 0.05 0.19 0.03 0.01

a Treh = Trehalose.b Gluc = Glucose.c Fruc = Fructose.d iMalt = Isomaltose.e Suc = Sucrose.f Mel = Melezitose.g Gent + Tur = Gentiobiose + Turanose.h iMaltotri = Isomaltotriose.i Malt = Maltose.j Pan = Panose.k Maltotri = Maltotriose.l Honey produced by simultaneous nectar foraging and sugar feeding.m Unripe honey.n Lime tree nectar.

Gašić et al.: Journal of aoaC international Vol. 97, no. 5, 2014 9

and Technological Development APV, Republic of Serbia (project No. APV- 114-451-3846/2013).

References

(1) Riccardelli D‘Albore, G. (1997) Textbook of Melissopalynology, Apimondia Publishing House, Bucharest, Romania, p. 308

(2) Persano Oddo, L., Piana, L., Bogdanov, S., Bentabol, A., Gotsiou, P., Kerkvliet, J., Martin, P., Morlot, M., Ortiz Valbuena, A., Ruoff, K., & von der Ohe, K. (2004) Apidologie 35, S82–S93. http://dx.doi.org/10.1051/apido:2004045

(3) Lušić, D., Koprivnjak, O., Ćurić, D., Sabatini, A.G., & Conte, L.S. (2007) Food Technol. Biotechnol. 45, 156–165

(4) Browicz, K. (1968) in Flora Europaea, Vol. 2, T.G. Tutin, V.H. Heywood, N.A. Burges, D.M. Moore, D.H. Valentine, S.M. Walters, D.A. Webb, P.W. Ball, A.O. Chater, & I.K. Ferguson (Eds), Cambridge University Press, Cambridge, UK, 2, pp 247–248

(5) Scalbert, A., Andres-Lacueva, C., Arita, M., Kroon, P., Manach, C., Urpi-Sarda, M., & Wishart, D. (2011) J. Agric. Food Chem. 59, 4331–4348. http://dx.doi.org/10.1021/jf200591d

(6) Viuda-Martos, M., Ruiz-Navajas, Y., Fernandez-Lopez, J., & Perez-Alvarez, J.A. (2008) J. Food Sci. 73, R117–R124. http://dx.doi.org/10.1111/j.1750-3841.2008.00966.x

(7) Kečkeš, S., Gašić, U., Ćirković Veličković, T., Milojković-Opsenica, D., Natić, M., & Tešić, Ž. (2014) Food Chem. 138, 32–40. http://dx.doi.org/10.1016/j.foodchem.2012.10.025

(8) Gašić, U., Kečkeš, S., Dabić, D., Trifković, J., Milojković-Opsenica, D., Natić, M., & Tešić, Ž. (2014) Food Chem. 145, 599–607. http://dx.doi.org/10.1016/j.foodchem.2013.08.088

(9) Momirovski, J., & Šimić, F. (1953) Pčelinja paša, Poljoprivredni nakladni zavod Zagreb, p. 108

(10) von der Ohe, W., Persano Odo, L., Piana, M.L., Morlot, M., & Martin, P. (2004) Apidologie 35, S18–S25. http://dx.doi.org/10.1051/apido:2004050

(11) Moore, P.D., & Webb, J.A. (1978) An Illustrated Guide to Pollen Analysis, Hodder & Stoughton, London, UK, p. 133

(12) Bucher, E., Kofler, V., Vorwohl, G., & Zieger, E. (2004) Das Pollenbild der SudtirolerHonige, Biologisches Labor der Landesagentur fur Umwelt und Arbeitsschutz, Bolzano, Italy, pp 17–37

(13) Riccardelli D’Albore, G. (1998) Mediterranean Melissopalynology, Università degli Studi di Perugia, Italy, p. 466

(14) Vergeron, P. (1964) Apidologie 7, 349–364. http://dx.doi.org/10.1051/apido:19640407

(15) Behm, F., von der Ohe, K., &, Henrich, W. (1996) Deut. Lebensm-Rundsch. 92, 183–187

(16) Louveaux, J., Maurizio, A., & Vorwohl, G. (1978) Bee World 59, 139–157

(17) Bryant, V.M., Jr, & Jones, G.D. (2001) Palynology 25, 11–28. http://dx.doi.org/10.1080/01916122.2001.9989554

(18) Davis, M.B. (1963) Am. J. Sci. 261, 897–912. http://dx.doi.org/10.2475/ajs.261.10.897

(19) Demianowicz, Z. (1964) Apidologie 7, 273–288. http://dx.doi.org/10.1051/apido:19640401

(20) Sawyer, R. (1988) Pollen Identification for Beekeepers, University College Cardif Press, London, UK, p. 115

(21) Michalkiewicz, A., Biesaga, M., & Pyrzynska, K. (2008) J. Chromatogr. A 1187, 18–24. http://dx.doi.org/10.1016/j.chroma.2008.02.001

(22) Persano Oddo, L., & Piro, R. (2004) Apidologie 35, S38–S81. http://dx.doi.org/10.1051/apido:2004049

(23) Pelimon, C. (1960) Apidologie 3, 339–347. http://dx.doi.org/10.1051/apido:19600404

(24) Tomás-Barberán, F.A., Martos, I., Ferreres, F., Radovic, B.S., & Anklam, E. (2001) J. Sci. Food Agric. 81, 485–496. http://dx.doi.org/10.1002/jsfa.836

(25) Bertoncelj, J., Polak, T., Kropf, U., Korošec, M., & Golob, T. (2011) Food Chem. 127, 296–302. http://dx.doi.org/10.1016/j.foodchem.2011.01.003

(26) Truchado, P., Ferreres, F., & Tomas-Barberan, F.A. (2009) J. Chromatogr. A 1216, 7241–7248. http://dx.doi.org/10.1016/j.chroma.2009.07.057

(27) Kaškonienė, V., & Venskutonis, P.R. (2010) Compr. Rev. Food Sci. F 9, 620–634. http://dx.doi.org/10.1111/j.1541-4337.2010.00130.x

(28) Piazza, M.G., & Persano Oddo, L. (2004) Apidologie 35, S94–S111. http://dx.doi.org/10.1051/apido:2004046