Plausible Authentication of Manuka Honey and Related Products by

Jun 18, 2014 - 23, South Island, –, Japan, NZ, 592, 345. 24, “high-grade manuka honey, 92.5%”, –, Japan, NZ, 720, 587. 25, with 0.25% propolis...
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Plausible Authentication of Manuka Honey and Related Products by Measuring Leptosperin with Methyl Syringate Yoji Kato,*,†,‡ Rie Fujinaka,† Akari Ishisaka,†,‡ Yoko Nitta,§ Noritoshi Kitamoto,†,‡ and Yosuke Takimoto⊥ †

School of Human Science and Environment, and ‡Research Institute for Food and Nutritional Sciences, University of Hyogo, Hyogo, Japan § Department of Nutritional Science, Okayama Prefectural University, Okayama, Japan ⊥ Healthcare Systems, Inc., Chikusa-ku, Nagoya, Aichi, Japan ABSTRACT: Manuka honey, obtained from Leptospermum scoparium flowers in New Zealand, has strong antibacterial properties. In this study, plausible authentication of the manuka honey was inspected by measuring leptosperin, methyl syringate 4-O-β-D-gentiobiose, along with methyl syringate. Despite a gradual decrease in methyl syringate content over 30 days at 50 °C, even at moderate 37 °C, leptosperin remained stable. A considerable correlation between nonperoxide antibacterial activity and leptosperin content was observed in 20 certified manuka honey samples. Leptosperin and methyl syringate in manuka honey and related products were analyzed using HPLC connected with mass spectrometry. One noncertified brand displayed significant variations in the leptosperin and methyl syringate contents between two samples obtained from different regions. Therefore, certification is clearly required to protect consumers from disguised and/or low-quality honey. Because leptosperin is stable during storage and specific to manuka honey, its measurement may be applicable for manuka honey authentication. KEYWORDS: manuka honey, chemical marker, authentication, leptosperin, methyl syringate

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Sardinia and is widely distributed in plants,10 indicating that methyl syringate is not suitable as a chemical marker to certify manuka honey, at least as a single agent. In previous studies, we identified leptosperin (leptosin) as a novel glycoside of methyl syringate, which was found almost exclusively in manuka honey.9 [Leptosin has been renamed “leptosperin” in order to avoid confusion of other leptosin(s), which isolated from a marine fungus Lestoshaeria sp. (Takahashi C. et al. Leptosins, antitumor metabolites of a fungus isolated from a marine alga. J. Chem. Soc., Perkin Trans. 1994, 1, 1859−1864.)] Because the leptosperin content tends to correlate with the UMF value presented on the jar labels, this novel compound may be a good candidate chemical marker for manuka honey authentication. A positive correlation between methylglyoxal and leptosperin contents was recently reported.11 In this study, we investigated the possibility of leptosperin along with its aglycone, methyl syringate, as chemical marker(s) for manuka honey authentication.

INTRODUCTION Manuka honey, produced in New Zealand, shows high antibacterial activity. Honey samples are classified on the basis of their antibacterial activity using the unique manuka factor (UMF), which is standardized as the equivalent of the phenol concentration showing antibacterial activity. That is, UMF 5+ manuka honey displays the same nonperoxide antibacterial activity (NPA) as a >5% phenol solution. The term “Active” is also used for honey authentication. Active signifies antibacterial activity, including peroxide-dependent bactericidal activity, which is also expressed as a phenol equivalent. Methylglyoxal is a major known antibacterial constituent of manuka honey.1 Antibacterial activity and methylglyoxal content are considerably correlated.2 The MGO rating system is also used to certify premium honey, whereby MGO 100+ indicates a methylglyoxal content of >100 mg/kg honey. High values of UMF, Active, or MGO directly influence the market price of honey. However, methylglyoxal contents are not consistent during the storage period because dehydroxyacetone in honey gradually converts to methylglyoxal.2,3 Because methylglyoxal is probably generated from the dihydroxyacetone by the amino-carbonyl reaction (Maillard reaction), it can be artificially increased through moderate heating or prolonged storage of honey. These facts suggest that the actual antibacterial activity, often expressed as UMF, Active, and MGO, is not constant. Manuka honey contains a unique phenolic compound, methyl syringate, displaying the scavenging activity of superoxides4 and inhibitory effects on aflatoxin production.5 Methyl syringate, identified in extracts of the first leaves of Kalopanax pictus Nakai (Araliaceae), is a selective agonist of transient receptor potential channel, ankryn 1 (TRPA1).6,7 It has also been identified in asphodel honey8 or some honeys9 from © 2014 American Chemical Society

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MATERIALS AND METHODS

Materials. Methyl syringate (methyl 3,5-dimethoxy-4-hydroxybenzoate) was purchased from Alfa Aesar, Johnson Matthey Co. (Ward Hill, MA). p-Nitrophenyl-β-D-glucopyranoside (PNPG) was obtained from Nacarai Tesque Inc. (Kyoto, Japan). Trimethoxybenzoic acid (TMBZ) was obtained from Sigma-Aldrich Japan (Tokyo, Japan). Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) was purchased from TCI Co., Ltd. (Tokyo, Japan). Isolation of Leptosperin from Manuka Honey. Leptosperin was purified from manuka honey according to a documented method9 with some modifications. First, 500 g of honey was dissolved in 500 Received: March 27, 2014 Accepted: June 18, 2014 Published: June 18, 2014 6400

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Figure 1. Time-dependent changes in the amounts of leptosperin or methyl syringate in manuka honey during incubation under warm conditions [37 °C (A) or 50 °C (B)]. mL of water, stirred at room temperature (r.t.) for 30 min, and filtered to remove impurities. The filtrate and 400 mL of (wet) Diaion Sepabeads HP20 resins (Mitsubishi Chemical Co., Tokyo, Japan), pretreated with methanol and then water, were mixed in a bottle and incubated with gentle shaking for 3 h at r.t. and subsequently filtered. HP20 resins were washed with water (500 mL) and 20% methanol (500 mL). Subsequently, the materials were eluted with methanol (2 L). Following the evaporation of methanol, materials were dissolved in 100 mL of water and transferred to a separating funnel. An equal volume of ethyl acetate was added, and the mixture washed three times. The water fraction was collected and concentrated. The concentrate was applied to an open column filled with HP20 resin and preequilibrated with water. The column was washed with 200 mL of water and 20% methanol, and elution performed with 50% methanol (500 mL). The eluate was fractionated, and absorbance at 262 nm was measured. The fractions containing leptosperin were pooled in a flask and evaporated. The concentrate was purified using reversed-phase HPLC with Develosil Combi-RP (20 × 100 mm, Nomura Chemical Co., Ltd., Aichi, Japan) containing 0.1% acetic acid/CH3CN = 85/15 at a flow rate of 5 mL/min with monitoring at 262 nm. The peak was collected and repurified under the same conditions. Honey and Related Samples. Twenty certified samples of manuka honey were obtained from the Unique Manuka Factor Honey Association. Information on NPA (phenol equivalent) and methylglyoxal contents of the 20 samples was provided by Hill Laboratories. Other kinds of honey and samples were obtained from retail stores between 2011 and 2014 in Japan, China, Brazil, and New Zealand. The samples were dissolved in water at a concentration of 0.1 g/mL and centrifuged. The supernatant was collected and directly used for HPLC or diluted 100-fold with water before application of liquid chromatography tandem mass spectrometry (LC/MS/MS), as described below. In addition, in the case of sample no. 26 (Table 2), the original honey solution (0.1 g/mL) was directly analyzed using LC/MS/MS. Samples of candy, cosmetics, or toothpaste were dissolved in 0.1% formic acid/CH3CN (9/1) at a concentration of 0.1 g/mL. In some cases, in particular for candy, samples were crushed into small pieces and dissolved at 60 °C for 30 min. After centrifugation, the supernatant was collected and diluted 2−100 fold, which was estimated by preliminary analysis of each sample. Vodka was eightfold diluted with water and then used. Tea samples were extracted with 500 mL of boiled water for 5 min using two bags and directly used for mass analyses.

HPLC Measurement. The honey solution (0.1 g honey/mL) was analyzed using a HPLC-photodiode-array detector (PDA), as described previously,9 with some modifications. In brief, a 5 μL sample was injected into the HPLC connected to a Develosil ODSHG-5 column (4.6 × 150 mm, Nomura Chemical Co., Ltd.). The separation was performed with gradient elution using a two-solvent system (solvent A, 0.1% formic acid; solvent B, CH3CN) at a flow rate of 0.8 mL/min with the following gradient program: 0 min (A90%), 10 min (A60%), 11 min (A90%), 25 min (A90%). Leptosperin and methyl syringate were quantified at 262 and 275 nm, respectively. LC/MS/MS Measurement. Analyses were performed using the API3000 tandem mass spectrometer (AB Sciex Instruments, Foster City, CA) connected with a HPLC system using a Develosil ODS-HG3 column (2 × 50 mm, Nomura Chemical Co., Ltd.). The flow rate was 0.2 mL/min, and 0.1% formic acid (A) and CH3CN (B) were used as solvents. The time program was as follows: 0 min (A90%), 6 min (A60%), 6.4 min (A90%), 15 min (A90%). The leptosperin content in honeys and samples was measured using electrospray ionization (ESI) with multiple reaction monitoring (MRM) negative mode (ESI-MS/MS). Simultaneous measurement of leptosperin and methyl syringate was accomplished using atmospheric pressure chemical ionization (APCI) with MRM negative mode (APCI-MS/ MS). For APCI-MS/MS, the duration time was divided into two periods (6 and 9 min) for effective ionization settings of the respective compounds. Prior to MS analyses, internal standards (100 nM PNPG and 250 nM TMBZ) were added to both samples and standard cocktails. The MRM transitions by collision-induced fragmentation were as follows: leptosperin, 581.0/323.2 (APCI), 581.0/210.9 (ESI); methyl syringate, 211.1/181.1; PNPG, 346.2/138.1; TMBZ, 211.1/ 152.1. In addition, [M−HCOO−]− (581.10) was selected for Q1 transition of leptosperin (M.W. 536). Marker Stability. Honey samples (raw) were directly aliquoted into screw-capped microtubes, which were sealed tightly and incubated at 37 or 50 °C. At various intervals, tubes were individually removed and stored at −80 °C until analysis. The samples were dissolved in water at a concentration of 0.1 g/mL, and the changes in the leptosperin and methyl syringate contents were analyzed using HPLC as described above. Statistical Analysis. The relationship between the two markers was calculated using SPSS Statistics ver. 17.0 with Spearman’s correlation. 6401

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Table 1. Quantitation of Leptosperin and Methyl Syringate in 20 Certified Manuka Honey Samples leptosperin (nmol/g honey)

methyl syringate (nmol/g honey)

sample no.

methylglyoxal (MGO) (mg/kg honey)

NPA

HPLC

ESI-MS

APCI-MS

HPLC

APCI-MS

A B C D E F G H I J K L M N O P Q R S T

261 361 631 830 131 289 196 162 611 761 686 135 416 562 739 1207 109 535 315 999

9.5 11.7 17.2 20.7 6.4 10.1 8.0 7.2 16.8 19.5 18.2 6.5 12.9 15.8 19.2 26.3 5.9 15.3 10.7 23.4

380 1069 997 753 343 568 631 592 599 863 729 314 877 626 1268 782 339 761 472 928

347 999 1090 671 245 491 502 591 490 760 576 265 935 474 851 723 244 584 465 851

131 217 293 205 132 191 219 181 232 268 297 130 194 210 423 296 129 262 175 321

511 325 636 293 122 360 183 409 479 503 365 283 327 420 525 381 146 403 681 314

301 156 395 154 60 240 111 219 332 295 252 157 144 300 324 273 64 257 520 197

Figure 2. Correlation of the amount of leptosperin (A) or methyl syringate (B) with nonperoxide antibacterial activity (NPA). Methyl syringate and leptosperin contents were measured using HPLC.

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RESULTS

using HPLC. Leptosperin and methyl syringate amounts varied from 314 to 1268 and 122 to 681 μmol/g of honey, respectively (Table 1). NPA of honey samples were independently determined by an authentic organization, as described in Materials and Methods. NPA has a high correlation with leptosperin content (r = 0.732, p < 0.001) but not significant with that of methyl syringate (Figure 2A, B). Our results are similar to those reported in a previous study.9 Establishment of LC/MS/MS Analyses for Leptosperin and/or Methyl Syringate. Leptosperin can be measured using the negative mode of ESI with MRM using 581/211 or 581/323 transitions. MRM chromatograms of a standard and typical honey sample using ESI (MRM 581.10/210.9) are shown in Figure 3A. The limit of detection (LOD) for

Heat Stability of Leptosperin and Its Aglycone, Methyl Syringate. Stabilities of leptosperin and methyl syringate in manuka honey were examined. As shown in Figure 1, methyl syringate was significantly decreased with increasing incubation times. We observed a loss of approximately 30% of methyl syringate over a period of 30 days at 37 °C. At a higher temperature of 50 °C, approximately 50% of methyl syringate was lost over the 30 day period. In contrast, leptosperin was relatively stable at the same temperatures. Correlation of Leptosperin and Methyl Syringate with Antibacterial Activity. Leptosperin and methyl syringate contents in 20 certified manuka honey samples were analyzed 6402

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Figure 3. Electrospray ionization (ESI) with multiple reaction monitoring (MRM) negative mode (ESI-MS/MS) or atmospheric pressure chemical ionization (APCI) with MRM negative mode (APCI-MS/MS) analyses of standards and honey sample. (A) Chromatograms of ESI-MS/MS analysis of leptosperin with internal standard (I.S.), p-nitrophenyl-β-D-glucopyranoside (PNPG). Upper chart, manuka honey; lower chart, standard leptosperin. (B) Chromatograms of APCI-MS/MS analysis of leptosperin or methyl syringate with internal standards, PNPG or 3,4,5trimethoxybenzoic acid (TMBZ). Upper chart, manuka honey; lower chart, standard of leptosperin and methyl syringate. (C) Quadratic standard curve of leptosperin using APCI-MS/MS.

leptosperin was 3 nM (1.5 fmol/injection), and the peak area linearly increased up to 2000 nM. PNPG was used as the internal standard for leptosperin measurement. However, methyl syringate was barely detected using ESI-MS/MS at a concentration lower than approximately 1 μM. Leptosperin contents in the 20 manuka samples were analyzed using ESI (Table 1). High linearity of leptosperin data was observed between HPLC and ESI-MS/MS (r = 0.947, p < 0.001). In addition, APCI-MS/MS negative mode was developed to analyze methyl syringate with leptosperin (Figure 3B). In this ionization mode, methyl syringate could be analyzed at concentrations of 10−1000 nM, but LOD of leptosperin (30 nM) was lower than that of ESI ionization (3 nM). TMBZ was selected as the internal standard for methyl syringate (Figure 3B). The standard curve of leptosperin was not linear and was then adapted using a quadratic curve fitting (Figure 3C). Leptosperin and methyl syringate contents in the 20 samples

were also analyzed using APCI-MS/MS. Each correlation of leptosperin and methyl syringate between APCI-MSMS and HPLC was significantly high (r = 0.812 and 0.949, respectively). However, the amount of leptosperin detected with LC/MS/MS methods, in particular APCI-MS/MS, was lower than that observed with HPLC-PDA, indicating that the reliability of APCI-MS/MS for determining leptosperin content is relatively lower compared with that of the other two methods. Similar to the HPLC analyses, the correlation between leptosperin and NPA observed using both LC/MS/ MS methods was significant. On the other hand, the methyl syringate content was not correlated with antibacterial activity. Plausible Authentication of Manuka Honey by Measuring Leptosperin. We analyzed the leptosperin and methyl syringate contents in commercial manuka honey samples, along with some nonmanuka honey samples (Table 2). Manuka honey authenticated using UMF, MGO, or Active 6403

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Table 2. Quantitation of Leptosperin and Methyl Syringate in Honeya sample no., information

purchased

origin

leptosperina (nmol/g honey)

UMF (Manuka Honey) UMF 15+ China NZ UMF 15+ China NZ UMF 15+ China NZ UMF 15+ Japan NZ UMF 15+ Japan NZ UMF 15+ Japan NZ UMF 12+ Japan NZ UMF 5+ NZ NZ Active + (Manuka Honey or Jelly Bush Honey) Active 15+ China NZ certified chloramphenicol-free Active 15+ China NZ stick type Active 13+ Japan NZ Active 12+ NZ NZ Active 10+ NZ NZ 100% raw unpasteurized Active 5+ NZ NZ Jelly bush honey Active 5+ Japan Australia MGO (Manuka Honey) MGO 250+ Japan NZ MGO 250+ China NZ Noncertified (Or Self-Certified) Manuka Honey (or Jelly Bush Honey) − Japan NZ “self-rigid inspection passed” − Japan NZ bottled in Japan, liquid type − Japan NZ − Japan NZ with kiwi fruit (2%) − Japan NZ South Island − Japan NZ “high-grade manuka honey, 92.5%” − Japan NZ with 0.25% propolis, 5+ certificated by a laboratory − Japan NZ same jar and label with no. 27 − Japan NZ same jar and label with no. 26 − NZ NZ squeeze type, Jelly bush honey − Japan Australia Other Honeys (Nonmanuka Honey) Scottish heather honey − Japan U.K. − Brazil Brazil buckwheat − Japan Japan multifloral honey, Awaji island − Japan Japan multifloral honey − Japan Japan kiawe tree − Japan U.S.

methyl syringatea (nmol/g honey)

01 02 03 04, similar label with no. 26, 27 05 06 07 08, beech trees blended, Southern Alps

860 530 758 835 414 579 487 852

432 243 369 382 335 335 316 485

09 10, 11, 12 13 14, 15,

396 262 483 294 147 1044 58

185 152 603 90 303 343 N.D.

690 386

329 260

113 372 438 767 74 592 720 258 22b 164 35

346 1048 433 572 224 345 587 307 27 85 27

N.D. N.D. N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D. N.D. N.D.

16 17 18 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30 31, 32, 33, 34, a

certified

N.D., not detected. bQuantified by ESI-MS/MS.

“O” and 7.5 fold richer than that of no.26. Certified UMF15+ honey (no. 03, Table 2) showed strong signals derived from leptosperin and methyl syringate (Figure 4, trace line A). Plausible Authentication of Manuka Honey Products by Measuring Leptosperin with Methyl Syringate. Manuka honey is added to commercial foods, cosmetics, and toothpaste for expected medicinal purposes, which may not be expressed on the label or attached brochure. We then investigated the detection of leptosperin using ESI-MS/MS and methyl syringate using APCI-MS/MS from some market products. As shown in Tables 3 and 4, leptosperin was detected in the examined samples, including manuka honey, but not in the toothpaste containing manuka honey. In addition, no leptosperin signals from nonmanuka candies were observed. The methyl syringate pattern was similar to that of leptosperin, although one nonmanuka candy contained trace amounts of methyl syringate. These results collectively validate the

contained high concentrations of leptosperin (664, 383, or 538 nmol leptosperin/g honey on average, respectively), whereas the leptosperin contents in noncertified manuka honey were lower (on average, 323 nmol leptosperin/g honey). However, some noncertified honey samples (no. 21 and 24) were also rich in leptosperin. As shown in Figure 4 and Table 2, one nonauthenticated manuka honey sample (no. 26) obtained at an online store by a Japanese agent contained only trace amounts of leptosperin (22 nmol/g of honey), which could be quantified using ESI-MS/MS without 100-fold dilution but not using HPLC (Figure 4, trace line B′). The leptosperin level was only 1.7%−2.5% compared with the relatively high content in manuka honey “O” (Table 1, ESI or HPLC). Manuka honey (no. 27), which had the same label as sample no. 26, except for the expiration date and was purchased at a retail store in New Zealand, contained 164 nmol leptosperin per g of honey [Figure 4, trace line B, and Table 2 (HPLC)]. The leptosperin content in no. 27 was calculated as 13% of the manuka honey 6404

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Table 4. Detection of Leptosperin and Methyl Syringate in Liquid-Type Honey-Related Products samples (origin) manuka honey vodka (NZ) chamomile with lemon balm and manuka honey tea (U.K.)

Table 3. Detection of Leptosperin and Methyl Syringate in Solid-Type Honey-Related Productsa samples (origin) manuka honey lozenges with blackcurrant, UMF10+ (NZ) manuka lozenges, NPA 12+, 100% manuka honey (NZ) manuka honey nuglets (NZ) manuka honey candy with propolis, MGO 400+ (NZ) manuka’s cosmet-cleansing manuka’s cosmet-soap, 15% manuka honey manuka’s cosmet-drop lotion manuka’s cosmet-gel cream manuka and propolis toothpaste with tea tree oil (NZ) tablet type of manuka honey, for portable, 100% (NZ) manuka honey candy with green tea powder, UMF15+ (JP) candy with honey and lemon with vitamin C (JP)b candy with honey and apple (JP)b mint candy with milk and xylitol (JP)b fruit candy with Chinese quince and orange oil (JP)b a

methyl syringate (nmol/g sample)a (APCI-MS)

43

19

589

417

44 52

22 28

3 32

3 41

22 19 N.D.

13 15 8

135

923

36

31.5

N.D.

0.03

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

methyl syringate (pmol/mL sample) (APCI-MS)

3792 240

1900 9

using UMF, Active, or MGO. The honey is applied for medicinal purposes, such as treatment of wounded skin. Honey intake/treatment is also expected to promote health; therefore, not only manuka honey but also products containing manuka honey such as candies and cosmetics have been available in the market. Methylglyoxal is the key compound underlying the antibacterial activity of honey. Twenty certified honey samples displayed a high correlation between NPA and methylglyoxal contents (Table 1). Methylglyoxal is probably generated from the predicted precursor molecule, dihydroxyacetone, in the honey.3 This is consistent with the finding that the UMF rating is enhanced by storage, which is known to the beekeeper/ honey industry.12 Moreover, because both methylglyoxal and dihydroxyacetone are available as reagents, artificial addition of these compounds into manuka or nonmanuka honey to increase antibacterial activity is possible. Supplementation with chemicals is often used for preparation of manuka honey mimics.13,14 That is, it is possible to disguise other kinds of honey, including multifloral honeys, as manuka honey. The association of UMF is setting up traceability of UMF-certified manuka honey from the marketplace to ensure that the products are from the stated origin to protect the consumer from counterfeiting. The main purpose of this study was to evaluate the application of leptosperin9 as a potential chemical marker for authentication of manuka honey. Stability and reliability are important characteristics of an effective chemical marker. The leptosperin level remained constant during incubation at both 37 °C and higher temperatures of 50 °C (Figure 1A, B). This is probably because leptosperin has less reactive moieties than phenolics, such as methyl syringate, which has phenolic hydroxyl. Methylation of phenolic components in honey has been suggested.12 As discussed above, the amount of the other major key component, methylglyoxal, is known to be nonconstant because methyglyoxal is generated from dihydroxyacetone and could be lost during further incubation/storage.3 These findings suggest that either methylglyoxal or dihydroxyacetone is not suitable as chemical markers for manuka honey. As shown in Figure 2A, B, NPA is highly correlated with leptosperin (r = 0.732, p < 0.001) but not with methyl syringate. Oelschlaegel et al. also reported that the leptosperin content in manuka honey has good correlation with that of methylglyoxal (r = 0.848, p = 0.000), which is a well-known strong germinant.11 On the other hand, leptosperin only displays antibacterial activity at a high concentration range.9 The underlying reason for the strong association of leptosperin content with antibacterial activity remains to be established. A number of plausible chemical candidates have been identified for manuka honey authentication. A recent study showed that 2-formyl-5-(2-methoxyphenyl)pyrrole, a unique compound in manuka honey, is correlated with NPA (r2 = 0.364).15 Another study also reported that 2-methoxybenzoic acid in fresh honey (n = 10) is highly correlated with

Figure 4. Different contents of leptosperin and methyl syringate in the same manuka honey product. Product A was UMF 15+ honey as a positive control. Honey samples B (no. 27) and B′ (no. 26) were filled into the same type of bottle with the same label and sealed. Honey sample B was purchased in New Zealand (NZ) and B′ in Japan. Leptosperin appeared at 10 min and methyl syringate at 15.5 minutes.

leptosperin (nmol/g sample)a (ESI-MS)

leptosperin (pmol/mL sample) (ESI-MS)

N.D., not detected. bManuka honey was not included.

application of leptosperin as a preferable chemical marker for manuka honey authentication.

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DISCUSSION Manuka honey is one of the most premium kinds of honey worldwide. On the basis of its unique antibacterial activity, the quality of manuka honey is certified and ranked for consumers 6405

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methylglyoxal (r2 = 0.7968).12 Fearney et al. identified a novel glycoside of TMBZ in honey, but its correlation with NPA (UMF) or methylglyoxal (MGO) is yet to be established.16 In this study, we developed a plausible authentication method on the basis of leptosperin measurement, which is a specific compound of manuka honey.9 Apart from the reports on manuka honey, few studies focused on authentication of different kinds of honey. For example, over 100 chemicals in lavandin honey, a monofloral product of recent proliferation obtained from a hybrid of Lavandula angustifolia and L. latifolia species, were assessed as possible markers for authentication, with the aim of distinguishing it from the more common Lavender honey (L. latifolia).16 Authentication of monofloral Yemeni Sidr honey using ultraviolet spectroscopy and chemometric analysis has also been proposed.17 As shown in Figure 4, one sample of manuka honey (no. 26) obtained from an online shop in Japan contained an extremely low level of leptosperin (22 nmol/g honey), which could be only analyzed using ESI-MS/MS. However, the honey sample (no. 27) with the same label, obtained from a retail store in New Zealand, displayed relatively low amount of leptosperin. We suspected that sample no. 26 was produced and sold improperly. In a masked study, five of six volunteers felt that the taste of “manuka honey” in no. 27 was more prominent compared with no. 26 (unpublished observation). In addition, UMF 15+ honey (no. 04), made by the same maker as no. 26 and no. 27 products, contained significant amounts of leptosperin and methyl syringate (Table 2). Therefore, the quantification of leptosperin with methyl syringate as a supplement may serve as an effective authenticator of manuka honey. Few cosmetics or foods in the market indicate additional inclusions of manuka honey as a component. However, there is no simple way to chemically validate supplementation of honey by consumers and even producers at the present time. In our study, leptosperin (or methyl syringate) was detected in vodka, soap, or candy using mass spectrometry. Some of the samples contained trace amounts of leptosperin and methyl syringate, which could be detected using mass spectrometry. Gaps in data were observed between HPLC and mass spectrometry analyses (Table 1), but mass spectrometry was suitable for detection of trace leptosperin and methyl syringate. In this study, we have also selected PNPG as the internal standard for leptosperin analyses using the MS/MS, but the PNPG is the substrate for β-glycosidase. Therefore, if active βglycosidase is contaminated in the sample, it may interfere with the quantitation of leptosperin. On the other hand, TMBZ, the internal standard for quantitation of methyl syringate, is a possible component of manuka honey.12 In the future, stable isotopic leptosperin and methyl syringate should be synthesized as the preferable internal standards.18 In summary, we investigated the possible application of leptosperin with methyl syringate for authentication of premium manuka honey using HPLC, ESI-MSMS, and APCIMS/MS. The detection and quantitation of leptosperin with methyl syringate as an adjunct would be applicable for manuka honey authentication.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Mr. John Rawcliffe, UMF Honey Association, for providing honey samples and Hill laboratories in New Zealand for providing analytical data.

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ABBREVIATIONS USED UMF, unique manuka factor; TRPA1, transient receptor potential channel ankryn 1; PNPG, p-nitrophenyl-β-D-glucopyranoside; TMBZ, 3,4,5-trimethoxybenzoic acid; NPA, nonperoxide antibacterial activity; LC/MS/MS, liquid chromatography tandem mass spectrometry; ESI, electrospray ionization; MRM, multiple reaction monitoring; APCI, atmospheric pressure chemical ionization; I.S, internal standard

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REFERENCES

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(14) Rogers, K.; Grainger, M.; Manley-Harris, M. The unique manuka effect: Why New Zealand manuka honey fails the AOAC 998.12 C-4 sugar method. Part 2. J. Agric. Food Chem. 2014, DOI: 10.1021/jf404767b. (15) Chan, C. W.; Deadman, B. J.; Manley-Harris, M.; Wilkins, A. L.; Alber, D. G.; Harry, E. Analysis of the flavonoid component of bioactive New Zealand manuka (Leptospermum scoparium) honey and the isolation, characterisation and synthesis of an unusual pyrrole. Food Chem. 2013, 141, 1772−81. (16) Castro-Vázquez, L.; Leon-Ruiz, V.; Alañon, M. E.; Pérez-Coello, M. S.; González-Porto, A. V. Floral origin markers for authenticating Lavandin honey (Lavandula angustifolia x latifolia). Discrimination from Lavender honey (Lavandula latifolia). Food Control 2014, 37, 362−370. (17) Roshan, A.-R. A.; Gad, H. A.; El-Ahmady, S. H.; Khanbash, M. S.; Abou-Shoer, M. I.; Al-Azizi, M. M. Authentication of monofloral Yemeni Sidr honey using ultraviolet spectroscopy and chemometric analysis. J. Agric. Food Chem. 2013, 61, 7722−7729. (18) Aitken, H. M. R.; Johannes, M.; Loomes, K. M.; Brimble, M. A. Synthesis of leptosin, a glycoside isolated from manuka honey. ̅ Tetrahedron Lett. 2013, 54, 6916−6919.

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dx.doi.org/10.1021/jf501475h | J. Agric. Food Chem. 2014, 62, 6400−6407