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Fluorescent Pteridine Derivatives as New Markers for the Characterization of Monofloral Genuine New Zealand Manuka (Leptospermum scoparium) Honey Nicole Beitlich, Tilo Lübken, Martin Kaiser, Lilit Ispiryan, and Karl Speer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03984 • Publication Date (Web): 03 Nov 2016 Downloaded from http://pubs.acs.org on November 7, 2016

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Journal of Agricultural and Food Chemistry

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Fluorescent Pteridine Derivatives as New Markers for the Characterization of

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Monofloral Genuine New Zealand Manuka (Leptospermum scoparium) Honey

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Nicole Beitlich,† Tilo Lübken,‡ Martin Kaiser,§ Lilit Ispiryan,† Karl Speer*†

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Food Chemistry Department,† Organic Chemistry Department,‡ and Inorganic

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Chemistry,§ Technische Universität Dresden, Bergstrasse 66, 01069 Dresden,

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Germany

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* Corresponding author (Tel: +49 351 463 33132; Fax: +49 351 464 33132; Email: [email protected])

ACS Paragon Plus Environment


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ABSTRACT

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New Zealand manuka honey is well-known for its unique antibacterial activity. Due to

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its high price and limited availability, this honey is often subject to honey fraud. Two

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pteridine derivatives, 3,6,7-trimethyl-2,4(1H,3H)-pteridinedione and 6,7-dimethyl-

16

2,4(1H,3H)-pteridinedione, have now been identified in New Zealand manuka honey.

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Their structures were elucidated by LC-QTOF-HRMS, NMR, and single-crystal X-ray

18

diffraction after isolation via semi-preparative HPLC. Their marker potential for

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authentic manuka honey was proved as both substances were neither detectable in

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the pollen-identical kanuka honey nor in the nine other kinds of monofloral New

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Zealand honey analyzed (clover, forest, kamahi, pohutukawa, rata, rewarewa, tawari,

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thyme, and vipers bugloss). The fluorescence property of the pteridine derivatives

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can be used as an easy and fast TLC screening method for the authentication of

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genuine manuka honey. 6,7-dimethyl-2,4(1H,3H)-pteridinedione has been described

25

for the first time.

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KEYWORDS: genuine manuka honey, Leptospermum scoparium, single-crystal X-

27

ray

diffraction,

NMR,


marker,

FLD

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INTRODUCTION

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New Zealand manuka (Leptospermum scoparium J.R. Forst. & G. Forst) honey has

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become more and more important for its medicinal application in treating wounds,

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even more so than for its nutritional value.1 Its antibacterial activity is derived from the

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low pH value, high sugar concentration, individual proteins as well as peptides, and

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secondary plant metabolites.2 For the latter, especially methylglyoxal, a strong

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cytotoxic effective compound, which hitherto had been found in high amounts only in

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manuka honey, is of particular importance.3-7 In contrast, hydrogen peroxide, another

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antibacterially effective compound responsible for great success in regard to wound

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healing, e.g. cornflower honey, is present in manuka honey only in traces.8,9

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Due to its special value, manuka honey is often adulterated. Even though only 1,700

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tons of manuka honey are actually produced in New Zealand each year, the so-

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called manuka honey sold worldwide is estimated at 10,000 tons. Therefore, the

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UMFHA (Official Unique Manuka Factor Honey Association)10 and the New Zealand

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Government11 require clear and robust chemical and physical methods for defining

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genuine manuka honey.

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Pollen analysis, the most common method for honey differentiation, is not suitable for

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manuka honey authentication as the simultaneously flowering kanuka bush in New

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Zealand produces pollen indistinguishable from manuka.12 Other analytical

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parameters for honey authentication such as sensory properties (color, aroma, smell,

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and viscosity) and physico-chemical characteristics (electrical conductivity, sugar

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composition) have proved to be unpromising as well.13 This also applies to the

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analysis of methylglyoxal and its precursor dihydroxyacetone which is tentatively

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unique to manuka honey and not naturally present in kanuka honey. Both substances

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are commercially available so that upgrading the scarcely antibacterially active ACS Paragon Plus Environment


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kanuka honey by adding methylglyoxal or dihydroxyacetone is easily feasible as has

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been reported recently.14

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During the last decade, secondary plant metabolites seemed to be most helpful for

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manuka honey authentication. Next to volatile compounds, non-volatile compounds

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such as phenolic acids, norisoprenoids, and flavonoids are analyzed by means of

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HPLC/UHPLC-PDA-MS/MS.15-21 By comparing the PDA-profiles of manuka honeys

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with various other New Zealand monofloral honeys, in particular kanuka honey,

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distinguishing peaks can be selected as markers. A marker is defined as a unique

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compound for a monofloral honey or as a compound present in significantly higher

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amounts. The markers for manuka honey are leptosperin (former: leptosin), 1, acetyl-

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2-hydroxy-4-(2-methoxyphenyl)-4-oxobutanoate,

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methoxyphenyl)-penta-1,4-dione, 3, whereas 4-methoxyphenyllactic acid, 4 and

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lumichrome, 5, are kanuka markers (Figure 1).15,16,18,20

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Recently, two additional peaks with marker potential could be detected in the PDA-

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profiles of manuka honey by our working group (Figure 1).22 During our studies for

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their elucidation a new marker compound for manuka honey named lepteridine was

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proposed, which turned out to be one of these two peaks.17 Therefore, the aim of this

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study concentrated on the identification of the still unknown compound in addition to

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the recently reported, using semi-preparative HPLC for separation and concentration

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for the subsequent elucidation by NMR and single-crystal X-ray diffraction.

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Subsequently, the marker potential of the isolated substances needs to be tested.

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The more marker compounds exist for a certain kind of honey, the more difficult it

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becomes to adulterate the honey.

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MATERIALS AND METHODS

2,


and

3-hydroxy-1-(2-

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Honey Samples. 64 genuine monofloral manuka honeys and 18 genuine monofloral

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kanuka (Kunzea ericoides) honeys harvested in regions with monocultures were

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analyzed by UHPLC-PDA-MS/MS. Other relevant New Zealand monofloral honeys,

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nine in total, were considered in the study: 18 clover (Trifolium species), 7 forest, 10

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kamahi (Weimannia racemosa), 7 pohutukawa (Metrosideros excelsa), 6 rata

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(Metrosideros umbellata), 13 rewarewa (Knightia excelsa), 6 tawari (Ixerba

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brexioides), 15 thyme (Thymus vulgaris), and 5 vipers bugloss (Echium vulgare). All

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the samples were provided by the UMFHA and were stored in darkness at 8 °C until

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analyzed.

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Chemicals. Methanol (HPLC grade; LC-MS grade) and acetonitrile (HPLC grade)

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were acquired from Fisher Scientific (Schwerte, Germany). A daidzein standard was

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purchased from Alfa Aesar (Karlsruhe, Germany). 4-methoxy-13C,d3-benzoic-2,3,5,6-

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d4 acid as internal standard, acetic acid, 100% glacial, as well as chlorogenic acid

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were supplied by Sigma-Aldrich (Steinheim, Germany). Sodium chloride, sodium

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sulfate, acetonitrile (HPLC grade), and methanol (HPLC grade) were purchased from

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VWR International (Darmstadt, Germany). Ethyl acetate and caffeine were from

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Sigma Aldrich (Steinheim, Germany). 6,7-dimethyl-2,4(1H,3H)-pteridinedione was

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acquired from AKos (Steinen, Germany). Deuterated solvents dimethyl sulfoxide-d6

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(99.8%) and methanol-d4 (99.8%) were purchased from Deutero GmbH (Kastellaun,

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Germany). All the chemicals were of analytical grade. Bi-distilled water was

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generated by a Bi-Distillation Apparatus Bi 18E (QSC GmbH, Maintal, Germany).

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SPE-UHPLC-PDA-MS/MS

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metabolites was performed as reported by Oelschlaegel et al.8 Peak data for

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statistical analysis were secured by using internal standards. The statistical

Analysis.

The

analysis


of

the

secondary

plant


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6

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interpretation of the analytical results was expressed as boxplots and significance

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tests (Kruskal-Wallis, P < 0.01) with SPSS 22.0 (IBM, Ehningen, Germany).

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Liquid Extraction and semi-preparative HPLC for the Fractionation of Unknown

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Compounds. 30 g honey were dissolved in an aqueous sodium chloride solution

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(2%, m/v) and then extracted twice with 60 mL ethyl acetate. The combined organic

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extracts were carefully evaporated to dryness in nitrogen flow, and the residue was

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dissolved in 5 mL methanol/water (1:1, v/v). A Elite LaChrom semi-preparative HPLC

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system (VWR, Darmstadt, Germany) was combined with a fraction collector.8 In sum,

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more than 2 kg of manuka honey were liquid-liquid extracted.23 The concentrated

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extract was chromatographed employing a 250 mm x 10 mm i. d., 10 µm, Nucleodur

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C18 Pyramid column (Macherey&Nagel, Düren, Germany;). Due to co-eluting

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substances a second separation step using a 250 mm x 10 mm i. d., 10 µm, Synergi

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Polar-RP 80 Å column (Phenomenex, Aschaffenburg, Germany) was necessary. The

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oven temperature was set at 40 °C, and the mobile phase consisted of 0.1% formic

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acid and methanol with a flow rate of 2.5 mL/min. The collected fractions were

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evaporated to dryness and analyzed by UHPLC-QTOF-HRMS, single-crystal X-ray

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diffraction, and, after dissolving, in deuterated solvent by NMR.

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UHPLC-QTOF-HRMS Analysis. For determining the exact mass, the isolated

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compounds were analyzed by a 1290 Infinity UHPLC system coupled with a 6540

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QTOF (Agilent, Waldbronn, Germany). The same UHPLC parameters, as described

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in the SPE-UHPLC-PDA-MS/MS section, were used. Mass spectrometric data were

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acquired ranging from m/z 40 – 1000 in the positive mode with an acquisition rate of

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6 spectra/s. The following source parameters were set: drying gas temperature, 200

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°C; drying gas flow rate, 8 L/min ; nebulizer pressure, 35 psi; sheath gas

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temperature, 350 °C, sheath gas flow, 11 L/min; capillary voltage, 4000 V; and nozzle


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voltage 300 V. Unknown 1: m/z 207.0868 [M+H]+ (calculated for C9H11N4O2, m/z

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207.0882, error 0.0014 Da); Unknown 2: m/z 193.0721 [M+H]+ (calculated for

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C8H9N4O2, m/z 193.0725, error 0.0004 Da).

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NMR Analysis. The one-dimensional 1H (600 MHz),

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MHz) NMR spectra of the unknown compounds were acquired on an Avance AV-III

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600 spectrometer (Bruker Bio Spin GmbH, Rheinstetten, Germany) using

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tetramethylsilane (TMS) as internal standard. Chemical shifts were given on a δ

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(ppm) scale relative to TMS (1H,

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NMR spectra included HSQC, HMBC, and NOESY. Unknown 1: 1H NMR (600 MHz,

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DMSO-d6): δ 2.654 (3H, s, C(10)H3), 2.670 (3H, s, C(11)H3), 3.466 (3H, s, C(9)H3).

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13

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147.2 (C-8a), 150.7 (C-6), 152.0 (C-4), 160.8 (C-7), 163.2 (C-2).

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MHz, DMSO-d6): 57.9 (N-5), 85.7 (N-8); Unknown 2: 1H NMR (600 MHz, DMSO-d6):

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2.485 (3H, s, C(9)H3), 2.506 (3H, s, C(9)H3).

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(C-9), 22.3 (C-10), 123.9 (C-4a), 147.3 (C-6), 148.6 (C-2), 150.8 (C-8a), 157.6 (C-7),

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161.5 (C-4).

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Single-crystal X-Ray Diffraction. The unknown compound 1 was crystallized from

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methanol (mp = 256 - 257 °C), and an appropriate well-shaped single crystal

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fragment was selected for the experiment. The crystal was glued to a glass fiber.

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Single-crystal X-ray diffraction was measured on a four-circle Kappa APEX II CCD

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diffractometer (Bruker Karlsruhe, Germany) with a graphite(002)-monochromator,

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and a CCD-detector at T = 200(2) K. MoKα radiation (λ = 71.073 pm) was used. A

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multi-scan absorption was applied.24 The structure was solved with direct methods

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and refined against Fo2.25,26 Further details on the crystal structure investigation can

13

13

C (151 MHz), and

15

N (60.8

C) and MeNO2 (15N), respectively. Two-dimension

C NMR (151 MHz, DMSO-d6): 21.4 (C-10), 22.8 (C-11), 28.2 (C-9), 124.0 (C-4a), 15

N NMR (60.8

13

C NMR (151 MHz, DMSO-d6): 21.2



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8

150

be

obtained

from

the

Cambridge

Crystallographic

151

(http://www.ccdc.cam.ac.uk/) by quoting depository number CSD-1509900.

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Fluorescence Analysis. Fluorescence properties were checked using a 4500 FL

153

spectrophotometer (Hitachi High-Technologies Europe GmbH, Krefeld, Germany) in

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the 3-D scan type. The excitation wavelength ranged from λ 220 - 620 nm (sampling

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interval 10 nm) while the emission wavelength ranged between λ 220 - 620 nm

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(sampling interval 10 nm). The scan speed was set at 2400 nm/min, the PMT voltage

157

was 700 V, and the response was 0.004 s. Furthermore, the shutter control was on.

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For instrument control as well as for data acquisition and analysis, FL Solutions

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software was used.

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Results and Discussion

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UHPLC-PDA-MS/MS Analysis.

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As stated in the introduction, apart from the known markers for manuka, two

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unknown compounds were discovered in the PDA-profiles (Figure 1, named unknown

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1, 6, and unknown 2, 7). However, they were not detected in the scarcely

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antibacterially effective kanuka honey profiles (B), the most prevalent fraudulent

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substitute for manuka honey (A), or in the other nine monofloral NZ honeys analyzed.

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Identification of unknown 1 and unknown 2

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To elucidate the chemical structure of the unknown compounds it was necessary to

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isolate the substances from the honey matrix, using semi-preparative HPLC. In

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regard to the smaller peaks, in our case it was necessary to extract more than 2 kg of

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honey to obtain adequate amounts of the two substances for subsequent UHPLC-

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MS/MS, NMR, and single-crystal X-ray diffraction measurements.


Data

Centre

(CCDC)

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9

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Compound unknown 1

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Unknown 1 was gained as a silvery-white crystalline compound (about 8 mg) with a

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UV maximum of 326 nm, an exact molecular weight of 207.0868 Da [M+H]+. The

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different NMR analyses (1H;

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1

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methyl groups: two methyl groups were located at C-6 and C-7 whereas the third

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methyl group, despite sufficient sample material of high purity, could not be

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unambiguously assigned to N-1 or N-3 (Figure 1). The crystalline form of unknown 1

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allowed for the subsequent application of the single-crystal X-ray diffraction. The

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structural refinement of unknown 1 revealed two crystallographic independent

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molecules with the same chemical structure in the unit cell (Figure 2). Unknown 1

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was clearly identified as a pteridine derivative named 3,6,7-trimethyl-2,4(1H,3H)-

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pteridinedione, 6, quite recently reported as lepteridine (Figure 1).17

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Compound unknown 2

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Unknown 2 showed a UV spectrum with a maximum at 326 nm, similar to unknown 1.

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With a molecular weight of 192 Da (a mass difference of 14 Da from unknown 1) and

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a similar fragmentation pattern as unknown 1, a structural relationship could be

190

assumed. About 2 mg of unknown 2 were isolated as a white powder. The 1H;

191

13

C{1H}; DEPT 135;

1

H/13C-HSQC;

1

H/13C-HMBC;

H/15N-HSQC; 1H/15N-HMBC; NOESY) indicated a pteridinedione structure with three

13

C{1H}; DEPT 135; 1H/13C-HSQC; 1H/13C-HMBC analyses resulted in the following

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structure: 6,7-dimethyl-2,4(1H,3H)-pteridinedione, 7 (Figure 1). Finally, the structure

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identification could be confirmed with a commercially available standard (AKos,

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Steinen, Germany). The occurrence of the demethylated lepteridine in honey has

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thus been described for the first time.

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Possible biosynthesis



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As the two newly identified compounds were very similar to riboflavin, 8, and its light

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induced acidic degradation product lumichrome, 5, it may be possible that they are a

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part of the riboflavin biosynthesis (Figure 3). 6,7-Dimethyl-8-ribityllumazine, 9, has

200

already been described as a direct precursor of riboflavin (Figure 3).27 As shown in

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Figure 3, the reaction of two molecules of 9 resulted in riboflavin and 5-amino-6-

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ribitylamino-2,4(1H,3H)-pyrimidinedione,

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degradation of riboflavin to lumichrome, it can be hypothesized that under the acidic

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honey condition 6,7-dimethyl-8-ribityllumazine converts to 6,7-dimethyl-2,4(1H,3H)-

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pteridinedione,

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pteridinedione, 6.

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Marker potential of the identified pteridines in manuka honey in comparison to

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other monofloral New Zealand honeys

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Daniels et al.17 identified 3,6,7-trimethyl-2,4(1H,3H)-pteridinedione in manuka honey

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authenticated

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dihydroxyacetone, methylglyoxal, and phenolic compounds. These authors also

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analyzed one or two samples from a number of other monofloral New Zealand

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honeys. However, no data were presented concerning color, conductivity, and pollen

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analysis. Thus, the authenticity of these honey samples has not been defined clearly.

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In our study, in addition to authentic kanuka honeys, nine other monofloral New

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Zealand honeys (clover, forest, kamahi, pohutukawa, rata, rewarewa, tawari, thyme,

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and vipers bugloss) with at least five authentic monofloral samples of each were

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analyzed. As shown in the boxplots and employing the significance test (Kruskal-

219

Wallis, P < 0.01), both substances are not only markers for distinguishing manuka

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from kanuka honey but, moreover, also for significantly differentiating manuka honey

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from the nine New Zealand honeys named above (Figure 4). The variability of the

7.

by

A

methylation

floral-source

field

10.

may

site

In

accordance

create

analysis


with

the

acidic

3,6,7-trimethyl-2,4(1H,3H)-

and

determining

the

Page 11 of 22


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pteridine derivatives within the manuka honey was caused by the different

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geographical origins.

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Fluorescent properties

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identification of manuka honey; for example, leptosperin has recently been described

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as a fluorophore.28 As riboflavin and lumichrome are also well-known for their

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fluorescence activity, fluorescence was also checked for the pteridine derivatives due

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to their structural similarity. The excitation wavelength was determined at 329 nm

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while the corresponding emission wavelength was 470 nm. The blue fluorescence of

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the newly identified pteridine derivatives was used for a TLC screening, where no

231

other kind of honey tested had a fluorescent spot at the same Rf values as the

232

manuka honey in accordance with the boxplots results (Figure 5). Therefore, TLC is

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an easy and fast screening method for unknown honey samples or honey samples

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declared as manuka honey in order to detect genuine manuka honey.

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For final manuka honey characterization and authentication, our HAHSUS method

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(Honey Authentication by HS-SPME-GC/MS and UHPLC-PDA-MS/MS combined

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with Statistics) is still necessary.29 In addition to the determination of the floral origin it

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is possible to estimate the percentage of manuka honey in manuka-kanuka mixed

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honeys. With the new fluorescent marker compounds, a valuable contribution for

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identification and characterization of genuine monofloral manuka honey was

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achieved.

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Supporting Information

243

1

244

dimethyl-2,4(1H,3H)-pteridinedione, 7; X-ray crystallographic data of 3,6,7-trimethyl-

245

2,4(1H,3H)-pteridinedione,6; TLC of isolated fractions unknown 1 and unknown 2

H and

13

are becoming

increasingly

important

for the

quick

C NMR spectra of 3,6,7-trimethyl-2,4(1H,3H)-pteridinedione, 6, and 6,7-



Page 12 of 22

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including manuka honey and 6,7-dimethyl-2,4(1H,3H)-pteridinedione standard

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substance

248

This material is available free of charge via the Internet at http://pubs.acs.org

249

Author Information

250

* Nicole Beitlich Tel: +49 351 463 37868; Fax: +49 351 464 33132; E-

251

mail: [email protected]

252

Acknowledgement

253

We sincerely thank the Landesuntersuchungsanstalt Sachsen, Dr. Thomas Frenzel

254

(LUA Sachsen, Dresden, Germany), for the QTOF-HRMS measurements.

255

Conflict of interest

256

The authors declare no competing financial interests.


Page 13 of 22


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Figure Captions Figure 1. UHPLC-PDA-MS/MS profiles: comparison of monofloral manuka honey (A) and monofloral kanuka honey (B), λ 254 nm (I), λ 326 nm leptosin, 1, acetyl-2hydroxy-4-(2-methoxyphenyl)-4-oxobutanoate,

2,

3-hydroxy-1-(2-methoxyphenyl)-

penta-1,4-dione, 3, 4-methoxyphenyllactic acid, 4, lumichrome, 5, unknown 1, 6, unknown 2, 7, internal standard, IS Figure 2. ORTEP for crystal structure of 3,6,7-trimethyl-2,4(1H,3H)-pteridinedione, 6. Figure 3. Possible relationship of the identified compounds 3,6,7-trimethyl2,4(1H,3H)-pteridinedione, 6, and 6,7-dimethyl-2,4(1H,3H)-pteridinedione, 7, in riboflavin, 8, biosynthesis. Lumichrome, 5; 6,7-Dimethyl-8-ribityllumazine, 9; 5-amino6-ribitylamino-2,4(1H,3H)-pyrimidinedione, 10. Figure 4. Boxplots of 3,6,7-trimethyl-2,4(1H,3H)-pteridinedione and 6,7-dimethyl2,4(1H,3H)-pteridinedione in different New Zealand monofloral honeys. Figure 5. TLCs of manuka honey, kanuka honey and other New Zealand honeys taken with TLC Visualizer (WinCATS, λ 366 nm); 3,6,7-trimethyl-2,4(1H,3H)pteridinedione, 6, and 6,7-dimethyl-2,4(1H,3H)-pteridinedione, 7.


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Table of Contents Graphic


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