Food Chemistry 132 (2012) 948–953

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Food Chemistry
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Compositional analysis of manuka honeys by high-resolution mass spectrometry:

Identification of a manuka-enriched archetypal molecule
Liam Fearnley a, David R. Greenwood a,b,c, Michael Schmitz d, Jonathan M. Stephens a,e,
Ralf C. Schlothauer a,e, Kerry M. Loomes a,b,⇑
a
School of Biological Sciences and Institute for Innovation in Biotechnology, University of Auckland, PB92019 Auckland, New Zealand
b
Maurice Wilkin’s Centre for Molecular Biodiscovery, University of Auckland, PB92019 Auckland, New Zealand
c
The New Zealand Institute for Plant & Food Research Limited, PB92169 Auckland, New Zealand
d
Department of Chemistry, University of Auckland, PB92019 Auckland, New Zealand
e
Comvita NZ Limited, Wilson South Rd., Paengaroa, PB1 Te Puke, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: Manuka honey is used medicinally as a wound-healing dressing and possesses antibacterial bioactivities.
Received 29 August 2011 It also possesses immunomodulating properties, comprising both anti-inflammatory and immune stim-
Received in revised form 4 November 2011 ulating activities. At present its active components have not been identified. Given the importance of
Accepted 15 November 2011
manuka honey as a therapeutic, we performed high-resolution Fourier-transform mass spectrometry
Available online 25 November 2011
analysis, in order to gain an insight into its complex make-up, as well as examining other honeys derived
from different floral origins and storage conditions. Our analyses show that manuka-derived honeys con-
Keywords:
tain unique compounds, particularly in the high molecular weight range, compared to other honeys from
Honey
Phenolics
other floral species. Storage conditions also directly impact on the molecular composition. An archetypal
Leptospermum scoparium (manuka) mother molecule specific to manuka honey was identified that may serve as a precursor store for free
3,4,5-trimethoxybenzoic acid and provide a means of fingerprinting manuka honeys.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction wound-healing process (Blair, Cokcetin, Harry, & Carter, 2009;

Manuka honey is derived from Leptospermum scoparium, a New
Zealand indigenous plant and has attracted much interest with
regard to its bioactive properties and medicinal use as a wound-
healing dressing (Armstrong, 2009; Lusby, Coombes, & Wilkinson,
2002). While honey exhibits antibacterial properties, due to the
presence of hydrogen peroxide formed through the catalytic activ-
ity of glucose oxidase, some region-specific manuka honeys pos-
sess further components that confer high levels of additional
bioactive properties, termed ‘unique manuka factor’ (UMF). A prin-
cipal component is the antimicrobial compound, methylglyoxal
(Adams et al., 2008; Mavric, Wittmann, Barth, & Henle, 2008),
which is reported to arise spontaneously from dihydroxyacetone
that is present in the nectar of manuka flowers and which in-
creases with ageing or controlled heating of the honey (Adams,
Manley-Harris, & Molan, 2009).
In addition to these known antimicrobial activities, manuka
honey possesses anti-inflammatory bioactivities, which aid the
⇑ Corresponding author at: School of Biological Sciences and Institute for
Innovation in Biotechnology, University of Auckland, PB92019 Auckland, New
Zealand. Tel.: +64 93737999; fax: +64 93737045.
E-mail address: k.loomes@auckland.ac.nz (K.M. Loomes).
Cooper, Jenkins, Henriques, Duggan, & Burton, 2010; Medhi et al.,
2008; Robson, Dodd, & Thomas, 2009), by arresting microbial
infections and reducing signs of inflammation (Gethin & Cowman,
2009; Henriques, Jenkins, Burton, & Cooper, 2010). Some of these
bioactivities may in part be due to the presence of phenolic and fla-
vonoid compounds in honey, which are becoming increasingly
recognised for their potential role in promoting human health
(Gomez-Caravaca, Gomez-Romero, Arraez-Roman, Segura-Carrete-
ro, & Fernandez-Gutierrez, 2006; Pyrzynska & Biesaga, 2009;
Stephens et al., 2010). Manuka and kanuka honeys were shown
recently to contain significant levels of phenolic compounds and
methoxylated derivatives including trimethoxybenzoic acid, 2-
methoxybenzoic acid, and methoxyphenyllactic acid (Stephens
et al., 2010). Potentially, phenolic compounds could participate in
free radical scavenging and modulate the immune response in
wound healing. Also, phenolic compounds can exist in a plethora
of modified forms, including methylated derivatives, which can
affect their subsequent metabolism in vivo (Wen & Walle, 2006).
These bioactive properties underpin the notion that manuka
honey possesses a rich and diverse array of small molecules. Given
the therapeutic importance of manuka honey as a medicinal
wound dressing it is clearly important to characterise fully the
bioactive components that are involved. With the advent of high-
performance tandem mass spectrometers such as Fourier-transform

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.11.074
 L. Fearnley et al. / Food Chemistry 132 (2012) 948–953
ion cyclotron resonance (FT-ICR) instruments, it has become
feasible to realistically analyse natural products for compositional
analysis (Zhang, McCombie, Guenat, & Knochenmuss, 2005). This
technology has been applied to the analysis of other complex
mixtures, such as phenolic profiling from red wine, for example,
in order to facilitate fingerprint identification (Cooper & Marshall,
2001). In principle it could therefore be applied to the analysis of
honeys to identify bioactive compounds and manuka-enriched
compounds that could be used to fingerprint geographical and
floral origin.
In this study, we applied Fourier-transform mass spectrometry
to the analysis of honey, in order to gain an appreciation into its
complex make-up and how this varies between honeys derived
from different floral origins and with storage conditions. Our anal-
yses demonstrate that honey does indeed comprise an array of
low-molecular-weight compounds and that manuka-derived
honeys in particular contain unique compounds, compared to
clover-derived honey. We also report the identification of a manu-
ka-enriched component that may serve as a precursor store for free
3,4,5-trimethoxybenzoic acid.
2. Materials and methods
2.1. Solid-phase extraction and liquid chromatography
The following representative honeys and one nectar sample
were used in the present study: L. scoparium var. incanum,
gumlands, Kaitaia, Northland; a blend of approximately 50%
L. scoparium var. incanum, native forest, Kaitaia, Northland;
L. scoparium var. linifolium, wetlands, Waikato; Kunzea ericoides,
Waikato; Trifolium spp., South Island; and nectar harvested from
L. scoparium var. incanum cultivar. These honeys were of varying
ages, floral culture, and geographical origin within New Zealand
(Table 1). Solid-phase extraction (SPE) of aqueous honey was used,
similar to the method as described (Stephens et al., 2010). To gain a
broader picture of honey composition against differing back-
grounds of floral and geographical origins as well as storage condi-
tions, only a single sample of each representative honey was used
for analysis. Each 0.1 g of honey or nectar, which was harvested
from L. scoparium (Table 1), was weighed into a glass Kimax tube,
dissolved in 4 mL of 5% aqueous methanol and applied to a precon-
ditioned 300-mg large-pore C-18 SPE cartridge (Alltech Associates/
Grace, Deerfield, IL). Each loaded cartridge was then washed with a
mixture of 4 mL of 10% aqueous methanol and 0.1% formic acid and
eluted with a mixture of 4 mL of 80% methanol and 0.1% formic
acid, to collect the non-carbohydrate honey extractives. These
were subjected to LC–MS/MS. For each honey sample there were
Table 1
Source, age and storage conditions for honey samples.
Identifier Description
Honey and nectar samples
1A Ph03 – L. scoparium var. incanum, gumlands, Kaitaia, Northland
1B Ph06 – blend, approx. 50% L. scoparium var. incanum, native forest, Kaitaia, Northland
1C Ph07 – L. scoparium var. linifolium, wetlands, Waikato
1D Ph09 – L. scoparium var. linifolium, wetlands, Waikato
1E Ph16 – Kunzea ericoides, Waikato
1F Ph20 – Trifolium spp., South Island
2A Ph01 – L. scoparium var. incanum, gumlands, Kaitaia, Northland
2B Ph04 – L. scoparium var. incanum, gumlands, Kaitaia, Northland
2C Nectar – Nectar harvested from L. scoparium var. incanum cultivar
3A PMHC1 4C – L. scoparium var. incanum, gumlands, Kaitaia, Northland
3B PMHC1 RT – L. scoparium var. incanum, gumlands, Kaitaia, Northland
Superscript 1 denotes samples prepared by Hill Laboratories (Hamilton, New Zealand), and samples labelled with superscript 2 were prepared at the University of Auckland.
949
multiple mass spectrometry runs made that were carried out un-
der slightly different separation parameters in order to optimise
compound separation and ion analysis. The results presented show
data from a single optimised LC/MS run for each honey sample.
A bulk extract was prepared from 1.0 g aged manuka honey and
subjected to the same SPE treatment as above. The resulting eluant
was concentrated 10-fold to 0.4 mL by evaporation under a gentle
nitrogen stream at room temperature and applied over 6 runs to a
150  4.6 mm, 5 lm particle size, 1.25  10 8 m pore size Aqua C-
18 column (Phenomenex, Auckland, NZ) using an aqueous acetoni-
trile gradient containing 0.1% trifluoroacetic acid (TFA) in an HP
1050 Ti series HPLC (Agilent, Waldbronn, Germany), monitoring
at 280 and 350 nm, in order to recover the major peak (containing
approximately 1.5 mg of the trimethoxy-containing parent mole-
cule), which was evaporated to dryness at room temperature under
a gentle nitrogen stream and dissolved in D3-acetonitrile contain-
ing 10% D20 to enhance solubility for the NMR studies.
2.2. Mass spectrometry
Aliquots (10 lL) of the acidified aqueous methanol SPE eluants
were injected onto a 2  150 mm Phenomenex (Torrance, CA) Luna
PFP chromatographic column containing a Security guard cartridge
of the same adsorbent housed in a column oven of a Thermo Sur-
veyor autosampler set at 45 °C. The column was developed with
a linear gradient of 5–95% acetonitrile in water containing 0.1% for-
mic acid over a 35 min period at 100 lL/min for a total run time of
60 min. The column was attached to a ThermoFinnigan LTQ-FT
(Thermo Fisher Scientific, Bremen, Germany) electrospray ion trap
– Fourier transform ion cyclotron resonance mass spectrometer in
positive ESI mode running Xcalibur 2.0 SR1 software (ThermoFish-
er Scientific). A triple top 3 tandem mass experiment was per-
formed in parallel, where a full scan in the ICR cell (accurate
mass to <2 ppm) was followed coincidently by MS2 in the ion trap
on the top three ions and then MS3 performed on the top three
daughter ions from each MS2 scan. Dynamic exclusion was exer-
cised with one repeat. The run started after a 2 min delay during
which the solvent was diverted to waste. Full scan FT data were
collected at a resolution of 100,000 at m/z 400 over the range m/z
100–1200. The capillary temperature was set at 275 °C, a nitrogen
sheath gas flow of 12 units, auxiliary gas flow of 5 units and sweep
gas flow of 1 unit. The source voltage was 3.8 kV, capillary voltage
13 V and tube lens voltage 100 V. An isolation width of 2 amu and
normalised collision energy of 35 was used for both MS2 and MS3
scans and all charge states other than 1 were rejected. MSn data
were collected from an average of 2 microscans and ion times were
150 ms for FT and 100 ms for ion trap MSn scans.
Age Date of analysis
4–5 years – room temperature March, 20091
0.75 years – room temperature March, 20091
<1 year – 4 °C March, 20091
4 years – room temperature March, 20091
3 years – room temperature March, 20091
<0.75 years – room temperature March, 20091
<0.75 years – 4 °C March, 20102
4–5 years – room temperature March, 20102
2 years – 4 °C April, 20102
2 years – room temperature April, 20102
950 L. Fearnley et al. / Food Chemistry 132 (2012) 948–953
2.3. Computerised analysis
Identification of compounds within samples was performed
using a combination of software analysis and manual techniques.
MassFrontier 5.1 (ThermoFisher Scientific) was used for qualitative
analysis and provisional identification of peaks. Tandem MS/MS
data were loaded into the software, and spectral trees consisting
of MS/MS data were assembled from the files using the Total
Extraction Component Detection (TECD) algorithm after setting a
threshold for each spectrum, in order to eliminate low-intensity
noise peaks. These data were then loaded into databases for com-
parison between samples, which was performed using library
search algorithms based on the NIST MS search algorithm (Stein,
1999), to produce a score value indicative of quality of match
and similarity. Identification of parent ions and their MSn-derived
spectral trees was performed using the HighChem MassFrontier
ESI MS–MS spectral library and verified where possible by compar-
ison with authentic standards and manual interpretation of MS/MS
data coupled with atomic composition determination from accu-
rate mass determination of parent ions.
Full-sample quantification was performed by transforming the
data into mzXML format using Mscovert (ProteoWizard), and using
a modified version of the quality threshold clustering algorithm
(Heyer, Kruglyak, & Yooseph, 1999), a near minimal set of m/z
cluster points (cluster radius ±5 ppm) containing all ions analysed
(based on the ions triggering the MS2 level threshold) was ob-
tained. In comparisons between multiple samples, the sets of ana-
lysed ions were combined into a superset and used for cluster set
generation. The cluster set was then formatted for use as input
for MetWorks 2.0 (ThermoFisher Scientific), in order to obtain
full-sample quantitative data in the form of peak areas. MetWorks
uses the MassFrontier implementation of the TECD algorithm in or-
der to detect components, and utilises identical thresholding and
filtering of the ion trees extracted. These data, and qualitative data
obtained through MassFrontier, were then processed using Excel
2007 (Microsoft) and Prism (GraphPad). Similarity of ions between
samples for profile analysis was based on monoisotopic mass and
fragmentation data, as analysed by the MassFrontier software
package.
2.4. NMR spectroscopy
NMR experiments to elucidate the structure of the mother
compound were performed at 14.1 T on a Bruker Avance-600 spec-
trometer equipped with a TCI cryoprobe. 1H-1D and 13C-1D exper-
iments were conducted with a sweep width of 14 and 240 ppm,
respectively. The 1H spectrum was centred at 4.88 ppm, the 13C
spectrum was centred at 110 ppm. 64 and 16,384 scans of 64 k
time points were acquired for the 1H and 13C spectra. Data were
apodised by exponential multiplication with 0.1 and 1 Hz line
width, respectively.
13
C-1H heteronuclear multiplicity-edited single (HSQC) and
multiple bond (HMBC) correlation experiments were performed
at 8.7  180 ppm and 8.7  240 ppm centred at 6.3 ppm for the
1
H dimension and 90 or 110 ppm respectively for the 13C dimen-
sion. 256 free induction decays (FIDs) of 1024 points were acquired
for the HSQC experiment, 300 FIDs of 2048 points were acquired
for the HMBC experiments. FIDs were apodised by application of
a 90° shifted squared sine bell window in both dimensions.
1
H homonuclear through-bond (COSY, TOCSY) and through-
space (NOESY, ROESY) correlation experiments were acquired at
8.98  9.0 ppm and centred at 4.88 ppm. 512 FIDs of 2048 points
were acquired for the COSY and TOCSY experiments, whereas
512 FIDs of 4096 points were acquired for the NOESY and ROESY
experiments. An80-ms DIPSI-2 spin lock at a power level of
10 kHz was employed for the TOCSY experiment. The NOESY
experiment used a mixing time of 600 ms, the ROESY experiment
applied a 2.1 kHz spin lock pulse during the 200-ms mixing time.
FIDs were apodised by a 90° shifted square sine bell window in
both dimensions of the TOCSY, NOESY and ROESY experiments.
3. Results and discussion
3.1. Honey contains a complex array of compounds that vary with
floral origin and storage conditions
To gain some insight into the inherent compound complexity of
manuka honey, an initial LC–FTMS comparison was made between
a set of honeys of varying ages, floral culture, and geographical ori-
gin (Fig. 1). These were run in two groups with differing concentra-
tions of sample, and assessed qualitatively using the MassFrontier
software package. As shown in Fig. 1A there was a wide variability
in the distribution of total analysed parent ions corresponding to
individual compounds across the honeys by m/z value. However,
in all cases the mass range distribution was characterised by a
greater percentage of low-molecular-weight entities in the m/z
100–400 range, with significantly fewer analysable ions at masses
greater than this.
Closer examination of the manuka (sample 1A) and clover hon-
eys (sample 1F) revealed that even with similar mass range distri-
butions there were relatively large differences in terms of
compound similarity (Fig. 1B). For example, despite a similar total
number of analysed compounds in the similarly sized m/z 100–299
mass range (99 versus 95 respectively), the aged manuka honey
shared only 36 compounds out of the detected 99 compounds with
the clover-origin honey. The analysis also revealed only 1 com-
pound in common in the m/z 500–699 mass range and no com-
pounds in common with masses greater than 700 Da.
To investigate the intrinsic variability within a honey from a
specific region, two separate manuka honey batches from the same
geographical region but with different storage conditions were
analysed (honeys 2A and 2B). The molecular masses in these sam-
ples were similarly distributed, with higher proportions of low-
molecular-weight molecules (Fig. 1C). However, in contrast with
the interfloral differences observed between manuka and clover
honeys (Fig. 1B), these two samples shared a higher and more uni-
form compound similarity across all mass ranges shown in the dis-
tribution (shown by black shaded region). In the higher (m/z 900+)
region there was a noticeable reduction in similarity. When nectar
samples collected from a subset of the manuka plants from which
honey samples (derived subsequently from apicultural activity)
were compared, the number of shared compounds were consis-
tently similar to the young sample and moreover contained more
compounds over all molecular weight classes.
These findings showed that honey contains a diverse array of
compounds whose composition varies widely, depending on floral
origin, geographical region, and storage characteristics. To look
more closely at the specific effects of storage on compound distri-
bution, two L. scoparium honey samples from the same source were
analysed (Fig. 2). These two samples differed only in the conditions
at which they were stored, with the first sample stored under
refrigeration (4 °C) and the second at room temperature for
approximately 18 months. Analyses revealed that the proportions
of parent ions by m/z value were relatively consistent across both
these samples and that the majority of the ions analysed in the
4 °C sample were similar to those found in the room temperature
sample (Fig. 2). Nevertheless, the presence of new ions in the room
temperature sample strongly suggested that storage condition di-
rectly impacts on the compositional make-up of honey.
Compared to the 4 °C sample the room temperature-stored
sample also displayed significant increases in the quantities of a
 L. Fearnley et al. / Food Chemistry 132 (2012) 948–953 951

200
A 1A
1B
400
3A (4°C)
3B (room temperature)
1C
Number of compounds

compounds in 3B similar to 3A

Number of compounds
150 1D
300
1E
1F
100 200

50 100

0 0

0-
9

9
9

0-

29

49

69

89

90
29

49

69

89

90

0-

0-

0-

0-
0-

0-

0-

0-

10

30

50

70
10

30

50

70

m/z m/z

150 1A: L. scoparium Fig. 2. Effects of differing storage conditions on molecular composition. A manuka
B 1F: Trifolium spp.
honey was divided into separate samples one of which was stored for 2 years at 4 °C
(sample 3A) and the other stored for 2 years at room temperature (sample 3B). The
Number of compounds

Compounds in 1F similar to 1A
dotted bar indicates the number of ions within the m/z window detected for honey
sample 3B that were similar to honey sample 3A.
100

ucts resulting from reactions involving higher-molecular-weight

molecules outside the analysed mass range (m/z 100–1200). Pre-
50
sumably, these reactions could involve molecules, such as various
low-molecular-weight volatiles, as well as polymers and waxes
that were not analysed by our LC–MS conditions. Overall, the dif-
0 ferences between the room temperature and 4 °C honey samples
were both subtle and complex.
0-
9

9
29

49

69

89

90
0-

0-

0-

0-
10

30

50

70

3.2. Identification of an archetypal manuka-enriched maltose

m/z trimethoxybenzoic acid parent compound
300 2A: <0.75 years at 4 °C
C 2B: 4-5 years at
Compared to other honeys, manuka honey has attracted much
room temperature
attention for its bioactive properties and in particular its use in
Number of compounds

Compounds in 2B similar to 2A wound healing. While methylglyoxal has been attributed as one
200 2C: Nectar component of this bioactivity, our data shows that manuka honey
contains an array of compounds not present in clover honey, which
essentially lacks these bioactivities. Consequently, it is possible
that other components in manuka honey exist that contribute to
100 its therapeutic bioactivities. We therefore subjected manuka honey
to preparative HPLC to separate and collect major components
(Fig. 3). Chromatographic traces revealed a significant peak eluting
0 at 15 min in the HPLC trace that appeared consistently in all
L. scoparium sourced samples. This peak was of specific interest
0-
9

9
29

49

69

89

90
0-

0-

0-

0-
10

30

50

70

Scale (arbitrary milliabsorbance units)

m/z 5000

Fig. 1. Mass range distributions of ions grouped by m/z values from representative
honeys. (1A) m/z distributions of ions analysed in 6 honeys of differing floral source, 4000
age, geographical origin, storage conditions. (1B) Comparison of honey sourced
from L. scoparium stored 4–5 years at room temperature (sample 1A), and clover
3000
honey sourced from Trifolium spp. stored <9 months at room temperature (sample
1F). The black shaded region indicates the number of ions within the m/z window
detected for Trifolium spp. that were similar to L. scoparium (1C) m/z distributions of
2000
ions analysed from nectar (sample 2C) and separate honey batches sourced from L.
scoparium from the same variety and geographic region. One of the honeys had been
stored <9 months at 4 °C (sample 2A) and the other for 4–5 years at room 1000
temperature (sample 2B). The black bar indicates the number of ions within the m/z
window detected for honey sample 2B that were similar to honey sample 2A.
0
0 5 10 15 20 25 30
large number of ions, both major (m/z 582–625 range) and minor
Time (minutes)
(m/z 300–450 range), without a significant fall in the amounts of
others (data not shown). This observation implies that some of Fig. 3. Chromatographic trace of an aged manuka honey. The major peak eluting at
the observed mass changes were attributable to breakdown prod- 15 min is denoted by an arrow. Solid line (280 nm); dashed line (340 nm).
952 L. Fearnley et al. / Food Chemistry 132 (2012) 948–953
because mass spectrometry analysis indicated that it corresponded
to only one major chemical component.
On the basis of MS/MS fragmentation assignments by manual
interpretation and by MassFrontier, together with independent
NMR analysis of the isolated HPLC peak, the species was consistent
with being a maltose (1–4-linked glucose disaccharide unit)
esterified to 3,4,5-trimethoxybenzoic acid at the anomeric carbon
with the following IUPAC name: (2R,3R,4R,5S,6R)-3,4-dihydroxy-
6-(hydroxymethyl)-5-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydro-
xymethyl)-tetrahydro-2H-pyran-2-yl)oxy)-tetrahydro-2H-pyran-
2-yl 3,4,5-trimethoxybenzoate (Fig. 4). The molecule appeared as
both a sodiated [M + Na]+ and an ammoniated [M + NH4]+ ion un-
der LC–MS conditions, with a small amount of the protonated
[M + H]+ ion detected (m/z 559.16324; C22H32O15Na+ requires
559.16334 [M + Na]+). CID fragmentation of the molecule showed
that two hexose units were present as a disaccharide and that
the esterified unit was a trimethoxybenzoic acid moiety. Key ions
produced by CID of the parent sodiated ion in the ion trap and ana-
lysed in the ion cyclotron resonance cell included m/z 397.11045
[M–hexose + Na]+ (5%; C16H22O10Na requires 397.11052) and m/z
347.09501 (100%; sodiated dihexose unit minus H2O; C12H20O10Na
requires 347.09487). When the m/z 397.1 MS2 ion was fragmented,
the resulting ions (379.0, 10%; 235.1, 75%; 185.1, 100%) indicated
the sodiated trimethoxybenzoate unit (235.1) and the sodiated
hexose unit minus water (185.1). When the MS2 ion 347.1 was
fragmented the following MS3 ions resulted: m/z 329.08446
(35%; sodiated dihexose unit minus 2H2O; C12H18O9Na requires
329.08430), m/z 259.07890 (100%; C9H16O7Na requires
259.07882), m/z 203.05267 (95%; sodiated hexose unit; C6H12O6Na
requires 203.05261).
Assignment of the 3,4,5 positions of the aglycone was achieved
by comparing the experimental MS/MS spectra obtained with
authentic trimethoxybenzoic acids with different methoxylation
patterns (2,3,4-; 3,4,5-; 2,4,6-; Stephens et al., 2010). Confirmation
that maltose was the actual disaccharide present and not isomalt-
ose or some other hexose disaccharide, was afforded by cryoprobe
analysis of a 1.5-mg sample in a series of high field 600 MHz NMR
1-D and 2-D experiments, performed to confirm the stereochemi-
cal assignments. Included were HSQC, COSY, ROESY and NOESY
experiments. Further FTMS analysis of some higher molecular
weight ions demonstrated that additional condensation products
of trimethoxybenzoate saccharides were present with one or more
hexose units detected.
The principal disaccharide-linked trimethoxybenzoate com-
pound was of potential interest, due to the known presence of
the ‘‘free’’ 3,4,5-trimethoxybenzoic acid moiety in manuka honey
which has reported antibacterial activity against Staphylococcus
aureus (Russell, Molan, Wilkins, & Hollan, 1990). Also, given its
structural similarity to the manuka compound methyl syringate,
3,4,5-trimethoxybenzoic acid has potential superoxide anion radi-
cal-scavenging activity, which may be important in the reported
Fig. 4. Structures of the maltose unit esterified with trimethoxybenzoic acid.
modulation of inflammation by manuka honey in chronic wounds
(Henriques, Jackson, Cooper, & Burton, 2006; Inoue et al., 2005).
Potentially, 3,4,5-trimethoxybenzoic acid could be liberated via
hydrolysis of its esterified form upon storage. Further quantitative
measurements of the 3,4,5-trimethoxybenzoic acid-esterified
maltose molecule and of the free 3,4,5-trimethoxybenzoic acid
ion (as confirmed by a standard from Sigma–Aldrich) showed that
their levels in manuka honey were relatively constant regardless of
storage temperature (Fig. 5). For the 3,4,5-trimethoxybenzoic
acid-esterified maltose molecule, both sodiated and ammoniated
adducts were present, although the former was the preferred form
in all but two (samples 1A and 1C) of the samples analysed.
There was a significant variation in the 3,4,5-trimethoxybenzoic
acid-esterified maltose molecule and free 3,4,5-trimethoxybenzoic
acid with floral source and geographic origin of the honey. There
were only trace levels of these compounds detected in clover
(Trifolium spp., 1F) and kanuka (K. ericoides, 1E) (data not shown)
and significantly lower levels in the manuka-blended honey
(sample 1B) (Fig. 5). Consequently, the 3,4,5-trimethoxybenzoic
acid-esterified maltose disaccharide molecule is a specific manu-
ka-enriched compound and could represent a class of compounds
that would presumably generate free 3,4,5-trimethoxybenzoic
acid. Interestingly 3,4,5-trimethoxybenzoic acid was completely
absent in the nectar sample despite the presence of a reasonable
amount of the 3,4,5-trimethoxybenzoic acid-esterified maltose
disaccharide molecule. This finding points toward a mechanism
whereby free 3,4,5-trimethoxybenzoic acid could arise during the
conversion of nectar to honey, presumably by hydrolytic reaction
by enzymes in honeybee saliva.
In summary, we report the first characterisation of honey using
Fourier transform mass spectrometry. Our profile analyses based
on high mass accuracy demonstrate that honey is a complex
mixture of small molecule compounds and that these vary signifi-
cantly depending on floral origin and storage conditions. Compared
to clover honey, manuka-derived honeys contained a unique
complement of compounds, which likely contribute to its observed
bioactive properties. In support of this possibility we identified an
archetypal molecule specific to manuka honey which may serve as
a precursor store for free 3,4,5-trimethoxybenzoic acid. This mole-
cule could well represent a broad class of very similar compounds
of hydroxylated or methoxylated benzoic acid derivatives attached
to glucose or maltose. It is therefore of interest to define their
bioactivities and whether identification of such manuka-specific
components provides a realistic strategy of fingerprinting honeys
to identify the proportion derived from manuka origin.
5.0×10 7 3,4,5-trimethoxybenzoic acid
Ammoniated mother molecule
Signal (Arbitrary units)
Sodiated mother molecule
4.0×10 7
3.0×10 7
2.0×10 7
1.0×10 7
0
1A
1D
2A
2C
1C
1F
1B
1E
2B
3A
3B
Honey origin
Fig. 5. Relative abundance of 3,4,5-trimethoxybenzoic acid and mother molecule
adducts in selected honeys.
 L. Fearnley et al. / Food Chemistry 132 (2012) 948–953
4. Disclosure statement
Jonathan Stephens and Ralf Schlothauer are employees of Com-
vita NZ Limited Paengaroa, PB1, Te Puke, New Zealand.
Acknowledgements
We would like to thank the Foundation for Research Science
and Technology. Also, Professor Joerg Kistler in the School of Biolog-
ical Sciences and Comvita located within the Institute for Innova-
tion for Biotechnology for providing additional stipend support
for Liam Fearnley.
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