Article

pubs.acs.org/JAFC

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

■ 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
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 authenti-
cation.

influence the market price of honey. However, methylglyoxal
contents are not consistent during the storage period because
dehydroxyacetone in honey gradually converts to methylglyox-
al.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 super-
oxides4 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
■
MATERIALS AND METHODS
Materials. Methyl syringate (methyl 3,5-dimethoxy-4-hydroxyben-
zoate) 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

© 2014 American Chemical Society 6400 dx.doi.org/10.1021/jf501475h | J. Agric. Food Chem. 2014, 62, 6400−6407
Journal of Agricultural and Food Chemistry
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 methyl-
glyoxal 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.
6401
Article
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 ODS-
HG-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-HG-
3 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.
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Journal of Agricultural and Food Chemistry Article

Table 1. Quantitation of Leptosperin and Methyl Syringate in 20 Certified Manuka Honey Samples
leptosperin methyl syringate
(nmol/g honey) (nmol/g honey)
sample no. methylglyoxal (MGO) (mg/kg honey) NPA HPLC ESI-MS APCI-MS HPLC APCI-MS
A 261 9.5 380 347 131 511 301
B 361 11.7 1069 999 217 325 156
C 631 17.2 997 1090 293 636 395
D 830 20.7 753 671 205 293 154
E 131 6.4 343 245 132 122 60
F 289 10.1 568 491 191 360 240
G 196 8.0 631 502 219 183 111
H 162 7.2 592 591 181 409 219
I 611 16.8 599 490 232 479 332
J 761 19.5 863 760 268 503 295
K 686 18.2 729 576 297 365 252
L 135 6.5 314 265 130 283 157
M 416 12.9 877 935 194 327 144
N 562 15.8 626 474 210 420 300
O 739 19.2 1268 851 423 525 324
P 1207 26.3 782 723 296 381 273
Q 109 5.9 339 244 129 146 64
R 535 15.3 761 584 262 403 257
S 315 10.7 472 465 175 681 520
T 999 23.4 928 851 321 314 197

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.

■ RESULTS
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
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
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Journal of Agricultural and Food Chemistry Article

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 3,4,5-
trimethoxybenzoic 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
6403
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
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Journal of Agricultural and Food Chemistry Article

Table 2. Quantitation of Leptosperin and Methyl Syringate in Honeya

leptosperina methyl syringatea

sample no., information certified purchased origin (nmol/g honey) (nmol/g honey)
UMF (Manuka Honey)
01 UMF 15+ China NZ 860 432
02 UMF 15+ China NZ 530 243
03 UMF 15+ China NZ 758 369
04, similar label with no. 26, 27 UMF 15+ Japan NZ 835 382
05 UMF 15+ Japan NZ 414 335
06 UMF 15+ Japan NZ 579 335
07 UMF 12+ Japan NZ 487 316
08, beech trees blended, Southern Alps UMF 5+ NZ NZ 852 485
Active + (Manuka Honey or Jelly Bush Honey)
09 Active 15+ China NZ 396 185
10, certified chloramphenicol-free Active 15+ China NZ 262 152
11, stick type Active 13+ Japan NZ 483 603
12 Active 12+ NZ NZ 294 90
13 Active 10+ NZ NZ 147 303
14, 100% raw unpasteurized Active 5+ NZ NZ 1044 343
15, Jelly bush honey Active 5+ Japan Australia 58 N.D.
MGO (Manuka Honey)
16 MGO 250+ Japan NZ 690 329
17 MGO 250+ China NZ 386 260
Noncertified (Or Self-Certified) Manuka Honey (or Jelly Bush Honey)
18 − Japan NZ 113 346
19, “self-rigid inspection passed” − Japan NZ 372 1048
20, bottled in Japan, liquid type − Japan NZ 438 433
21 − Japan NZ 767 572
22, with kiwi fruit (2%) − Japan NZ 74 224
23, South Island − Japan NZ 592 345
24, “high-grade manuka honey, 92.5%” − Japan NZ 720 587
25, with 0.25% propolis, 5+ certificated by a laboratory − Japan NZ 258 307
26, same jar and label with no. 27 − Japan NZ 22b 27
27, same jar and label with no. 26 − NZ NZ 164 85
28, squeeze type, Jelly bush honey − Japan Australia 35 27
Other Honeys (Nonmanuka Honey)
29, Scottish heather honey − Japan U.K. N.D. N.D.
30 − Brazil Brazil N.D. N.D.
31, buckwheat − Japan Japan N.D. N.D.
32, multifloral honey, Awaji island − Japan Japan N.D. N.D.
33, multifloral honey − Japan Japan N.D. N.D.
34, kiawe tree − Japan U.S. N.D. N.D.
a
N.D., not detected. bQuantified by ESI-MS/MS.

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
“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
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Table 4. Detection of Leptosperin and Methyl Syringate in

Liquid-Type Honey-Related Products
leptosperin methyl syringate
(pmol/mL sample) (pmol/mL sample)
samples (origin) (ESI-MS) (APCI-MS)
manuka honey vodka (NZ) 3792 1900
chamomile with lemon balm and 240 9
manuka honey tea (U.K.)

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
Figure 4. Different contents of leptosperin and methyl syringate in the antibacterial activity of honey. Twenty certified honey samples
same manuka honey product. Product A was UMF 15+ honey as a displayed a high correlation between NPA and methylglyoxal
positive control. Honey samples B (no. 27) and B′ (no. 26) were filled contents (Table 1). Methylglyoxal is probably generated from
into the same type of bottle with the same label and sealed. Honey the predicted precursor molecule, dihydroxyacetone, in the
sample B was purchased in New Zealand (NZ) and B′ in Japan. honey.3 This is consistent with the finding that the UMF rating
Leptosperin appeared at 10 min and methyl syringate at 15.5 minutes. is enhanced by storage, which is known to the beekeeper/
honey industry.12 Moreover, because both methylglyoxal and
Table 3. Detection of Leptosperin and Methyl Syringate in dihydroxyacetone are available as reagents, artificial addition of
Solid-Type Honey-Related Productsa these compounds into manuka or nonmanuka honey to
leptosperin methyl syringate increase antibacterial activity is possible. Supplementation
(nmol/g sample)a (nmol/g sample)a with chemicals is often used for preparation of manuka
samples (origin) (ESI-MS) (APCI-MS) honey mimics.13,14 That is, it is possible to disguise other kinds
manuka honey lozenges with 43 19 of honey, including multifloral honeys, as manuka honey. The
blackcurrant, UMF10+
(NZ) association of UMF is setting up traceability of UMF-certified
manuka lozenges, NPA 12+, 589 417 manuka honey from the marketplace to ensure that the
100% manuka honey (NZ) products are from the stated origin to protect the consumer
manuka honey nuglets (NZ) 44 22 from counterfeiting.
manuka honey candy with 52 28 The main purpose of this study was to evaluate the
propolis, MGO 400+ (NZ) application of leptosperin9 as a potential chemical marker for
manuka’s cosmet-cleansing 3 3 authentication of manuka honey. Stability and reliability are
manuka’s cosmet-soap, 15% 32 41 important characteristics of an effective chemical marker. The
manuka honey
leptosperin level remained constant during incubation at both
manuka’s cosmet-drop lotion 22 13
manuka’s cosmet-gel cream 19 15
37 °C and higher temperatures of 50 °C (Figure 1A, B). This is
manuka and propolis N.D. 8
probably because leptosperin has less reactive moieties than
toothpaste with tea tree oil phenolics, such as methyl syringate, which has phenolic
(NZ) hydroxyl. Methylation of phenolic components in honey has
tablet type of manuka honey, 135 923 been suggested.12 As discussed above, the amount of the other
for portable, 100% (NZ)
major key component, methylglyoxal, is known to be
manuka honey candy with 36 31.5
green tea powder, UMF15+ nonconstant because methyglyoxal is generated from dihydrox-
(JP) yacetone and could be lost during further incubation/storage.3
candy with honey and lemon N.D. 0.03 These findings suggest that either methylglyoxal or dihydrox-
with vitamin C (JP)b yacetone is not suitable as chemical markers for manuka honey.
candy with honey and apple N.D. N.D. As shown in Figure 2A, B, NPA is highly correlated with
(JP)b
leptosperin (r = 0.732, p < 0.001) but not with methyl
mint candy with milk and N.D. N.D.
xylitol (JP)b syringate. Oelschlaegel et al. also reported that the leptosperin
fruit candy with Chinese N.D. N.D. content in manuka honey has good correlation with that of
quince and orange oil (JP)b methylglyoxal (r = 0.848, p = 0.000), which is a well-known
a
N.D., not detected. bManuka honey was not included. 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
application of leptosperin as a preferable chemical marker for content with antibacterial activity remains to be established.
manuka honey authentication.

■
A number of plausible chemical candidates have been
identified for manuka honey authentication. A recent study
DISCUSSION showed that 2-formyl-5-(2-methoxyphenyl)pyrrole, a unique
Manuka honey is one of the most premium kinds of honey compound in manuka honey, is correlated with NPA (r2 =
worldwide. On the basis of its unique antibacterial activity, the 0.364).15 Another study also reported that 2-methoxybenzoic
quality of manuka honey is certified and ranked for consumers acid in fresh honey (n = 10) is highly correlated with
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Journal of Agricultural and Food Chemistry
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 distinguish-
ing 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 β-
glycosidase 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 APCI-
MS/MS. The detection and quantitation of leptosperin with
methyl syringate as an adjunct would be applicable for manuka
honey authentication.
■
AUTHOR INFORMATION
Corresponding Author
*Tel.: +81-79-292-9413. Fax: +81-79-293-5710. E-mail:
yojikato@shse.u-hyogo.ac.jp.
6406
Article
Notes
The authors declare no competing financial interest.
■ 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.
■
ABBREVIATIONS USED
UMF, unique manuka factor; TRPA1, transient receptor
potential channel ankryn 1; PNPG, p-nitrophenyl-β-D-gluco-
pyranoside; TMBZ, 3,4,5-trimethoxybenzoic acid; NPA, non-
peroxide antibacterial activity; LC/MS/MS, liquid chromatog-
raphy tandem mass spectrometry; ESI, electrospray ionization;
MRM, multiple reaction monitoring; APCI, atmospheric
pressure chemical ionization; I.S, internal standard
■
REFERENCES
(1) Mavric, E.; Wittmann, S.; Barth, G.; Henle, T. Identification and
quantification of methylglyoxal as the dominant antibacterial
constituent of manuka (Leptospermum scoparium) honeys from New
Zealand. Mol. Nutr. Food Res. 2008, 52, 483−9.
(2) Atrott, J.; Henle, T. Methylglyoxal in manuka honey
Correlation with antibacterial properties. Czech J. Food Sci. 2009, 27,
S163−S165.
(3) Adams, C. J.; Manley-Harris, M.; Molan, P. C. The origin of
methylglyoxal in New Zealand manuka (Leptospermum scoparium)
honey. Carbohydr. Res. 2009, 344, 1050−3.
(4) Inoue, K.; Murayama, S.; Seshimo, F.; Takeba, K.; Yoshimura, Y.;
Nakazawa, H. Identification of phenolic compound in manuka honey
as specific superoxide anion radical scavenger using electron spin
resonance (ESR) and liquid chromatography with coulometric array
detection. J. Sci. Food Agric. 2005, 85, 872−878.
(5) Jermnak, U.; Yoshinari, T.; Sugiyama, Y.; Tsuyuki, R.; Nagasawa,
H.; Sakuda, S. Isolation of methyl syringate as a specific aflatoxin
production inhibitor from the essential oil of Betula alba and aflatoxin
production inhibitory activities of its related compounds. Int. J. Food
Microbiol. 2012, 153, 339−44.
(6) Kim, M. J.; Son, H. J.; Song, S. H.; Jung, M.; Kim, Y.; Rhyu, M. R.
The TRPA1 agonist, methyl syringate suppresses food intake and
gastric emptying. PLoS One 2013, 8, e71603.
(7) Son, H. J.; Kim, M. J.; Park, J. H.; Ishii, S.; Misaka, T.; Rhyu, M.
R. Methyl syringate, a low-molecular-weight phenolic ester, as an
activator of the chemosensory ion channel TRPA1. Arch. Pharmacal
Res. 2012, 35, 2211−8.
(8) Tuberoso, C. I.; Bifulco, E.; Jerkovic, I.; Caboni, P.; Cabras, P.;
Floris, I. Methyl syringate: A chemical marker of asphodel (Asphodelus
microcarpus Salzm. et Viv.) monofloral honey. J. Agric. Food Chem.
2009, 57, 3895−900.
(9) Kato, Y.; Umeda, N.; Maeda, A.; Matsumoto, D.; Kitamoto, N.;
Kikuzaki, H. Identification of a novel glycoside, leptosin, as a chemical
marker of manuka honey. J. Agric. Food Chem. 2012, 60, 3418−23.
(10) Fujimatu, E.; Ishikawa, T.; Kitajima, J. Aromatic compound
glucosides, alkyl glucoside, and glucide from the fruit of anise.
Phytochemistry 2003, 63, 609−16.
(11) Oelschlaegel, S.; Gruner, M.; Wang, P. N.; Boettcher, A.;
Koelling-Speer, I.; Speer, K. Classification and characterization of
manuka honeys based on phenolic compounds and methylglyoxal. J.
Agric. Food Chem. 2012, 60, 7229−37.
(12) Stephens, J. M.; Schlothauer, R. C.; Morris, B. D.; Yang, D.;
Fearnley, L.; Greenwood, D. R.; Loomes, K. M. Phenolic compounds
and methylglyoxal in some New Zealand manuka and kanuka honeys.
Food Chem. 2010, 120, 78−86.
(13) Jervis-Bardy, J.; Foreman, A.; Bray, S.; Tan, L.; Wormald, P. J.
Methylglyoxal-infused honey mimics the anti-Staphylococcus aureus
biofilm activity of manuka honey: Potential implication in chronic
rhinosinusitis. Laryngoscope 2011, 121, 1104−7.
dx.doi.org/10.1021/jf501475h | J. Agric. Food Chem. 2014, 62, 6400−6407
Journal of Agricultural and Food Chemistry Article

(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.

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