Honey Authenticity Test

Honey Authenticity
Authenticity Testing of Honey

STATE-OF-THE-ART ANALYTICAL METHODOLOGY
TO DETERMINE ADULTERATION WITH SUGAR SYRUPS
13C-EA/LC-IRMS Analysis Foreign Enzyme Analysis
Dr. Lutz Elflein Dr. Kurt-Peter Raezke Dr. Vassil Valkov

APPLICA GmbH Applied Chemical Analysis
Bremen Germany
1st World Honeydew Honey Symposium, Apimondia IHC
August 1-3 2008, Tzarevo (BG)
Honey Authenticity
CONTENTS
Principle of EA/LC-IRMS
13C values of authentic honeys 1
13C values as criteria of authenticity 1
Examples of authentic and adulterated honeys 1
Interpretation of EA/LC-IRMS results 1
EA/LC-IRMS vs. EA/IRMS
Adulterated honeys: current situation
Foreign Enzyme Analysis 2
1
original paper in press (Apidologie)
patent pending
Honey Authenticity
Sugar Sources for Adulteration

Plant Origin Examples
Range of 13C values
Method
C4-Plants
Corn
Sugar Cane
-8 to -13
EA-IRMS
(AOAC 998.12)
C3-Plants
Beet
Rice
Wheat
Cichory
-22 to -30
LC-IRMS
(new)
Conclusion: combination of EA-IRMS and LC-IRMS: 13C-EA/LC-IRMS
Honey Authenticity
EA-IRMS
13C protein
13C honey
(a) autosampler
(b) combustion column
(c) reduction column
(d) water trap
(e) ion source

(f) flight tube
(g) magnetic beam deflector
(h) signal detectors
Honey Authenticity
LC-IRMS
Step 1:
Step 2:
Step 3:
Step 4:
chromatographic
separation of sugars
conversion
to CO2
separation from eluent

and transfer to IRMS
13/12C
isotope ratio
measurement
Source: Finnigan LC IsoLink Product Brochure
Honey Authenticity
LC-IRMS
Step 1:
Step 2:
Step 3:
Step 4:
chromatographic
conversion
to CO2

13/12C
isotope ratio
measurement
... by chemical oxidation
Honey Authenticity
LC-IRMS
Step 1:
Step 2:
Step 3:
Step 4:
chromatographic
conversion
to CO2

13/12C
isotope ratio
measurement
Honey Authenticity
LC-IRMS
Step 1:
Step 2:
Step 3:
Step 4:
chromatographic
conversion
to CO2

13/12C
isotope ratio
measurement
13C of fructose, glucose, di- and trisaccharides
Honey Authenticity
EA/LC-IRMS: Natural 13C Variations of Pure Honey (1)

parameter
ave.
s. d.
range
13C () protein (p)
-25.2
0.7
-22.7 to -26.7
13C () honey (h)
-25.5
0.7
-23.0 to -27.3
p-h ()
0.3
0.4
-0.9 to 1.5
C4 sugar (%) *
0.3
0.9
0 to 5.7
13C () fructose (fru)
-25.5
0.7
-23.2 to -27.5
13C () glucose (glu)
-25.5
0.7
-22.7 to -27.2
13C () disaccharides (ds)
-25.8
1.0
-22.5 to -28.2
13C () trisaccharides (ts)
-24.7
1.0
-22.6 to -27.5
fru/glu ratio
1.30
0.21
0.92 to 1.82
ds (area %)
6.8
2.4
1.2 to 14.1
ts (area %)
1.8
1.1
0.0 to 8.0
oligosaccharides (area %)
Number of samples: 451
< 0.7
* apparent C4 sugar content
13C values of individual sugars in honey are very similar!

Honey Authenticity
EA/LC-IRMS: Natural 13C Variations of Pure Honey (2)

parameter
ave.
s. d.
max. d. (abs.)
13C () fru - glu
0.0
0.3
1.0
13C () fru - ds
0.3
0.7
2.0
13C () fru - ts
-0.8
0.7
2.0
13C () fru - p
-0.3
0.4
1.5
13C () glu - ds
0.3
0.7
1.9
13C () glu - ts
-0.7
0.7
2.0
13C () glu - p
-0.3
0.4
1.6
13C () ds - ts
-0.9
0.6
2.1
13C () ds - p
-0.6
0.7
2.0
0.5
0.7
2.1
13C () ts - p
Number of samples: 451
13C values show only small variations!
Honey Authenticity
EA/LC-IRMS: 13C, Proposed Limits for Pure Honey

parameter
proposed limit
13C () fru - glu
1.0
13C () fru - ds
13C () fru - ts
13C () fru - p
13C () glu - ds
13C () glu ts
= 13C () max.
2.1
13C () glu - p
13C () ds - ts
13C () ds - p
13C () ts - p
13C () p - h
1.0
statistical certainty (confidence level): 99.7 % (3 s.d.)
Honey Authenticity
LC-IRMS Chromatogram of Pure Honey

LC-IRMS Chromatogram of Honey
20000
Fructose
18000
Glucose
Intensity (mV)
16000
m/z
14000
12000
44
10000
45
Ca2+ column
46
water eluent
Disaccharides
8000
6000
Trisaccharides
4000
2000
2464
2327
2191
2054
1917
1780
1643
1506
1369
1232
1095
958
822
685
548
411
274
137
Retention Time (s)

RT (s)
Area (Vs)
13C () vs. VPDB
trisaccharides
1167
61
-26,05
disaccharides
1393
253
-26,78
glucose
1479
2300
-25,88
fructose
1864
2687
-26,01
13C ()
vs. VPDB
protein
-25,6
honey
-26,0
13C (F-G): - 0,13

13C (max.): 1,18
Honey Authenticity
Range of 13C Values for Honey and Syrups

not all sugar syrups are outside the naturally occurring range of honey
ISS: invert sugar syrup
EA-IRMS
-11.3
-19.8
C4 sugar
C4/C3 sugar mix
EA-IRMS
LC-IRMS
-24.2
-26.1 -26.4
C3 sugars
Honey Authenticity
EA-IRMS
HONEY
TYPE
LC-IRMS: Real Life Examples
EA-IRMS (AOAC 998.12)

d13C ()
Protein
d13C ()
Honey
Acacia
pure
-24.9
Acacia
adulterated
LC-IRMS (APPLICA)
C4 Sugar
(%)
d13C ()
Fructose
d13C ()
Glucose
-25.1
-25.1
-25.3
-25.2
-24.6
3.9
-23.8
Linden
pure
-25.7
-25.6
0.6
Linden
adulterated
-25.6
-25.7
Honeydew
pure
-25.6
Honeydew
adulterated
d13C ()
Trisaccharides
d13C ()
max.
F/GRatio
-25.3
-24.4
0.9
1.57
-26.0
-25.7
-25.0
2.2
1.60
-25.5
-25.4
-25.9
-25.5
0.5
1.35
-26.0
-25.7
-23.9
-24.4
2.1
1.27
-25.8
-25.7
-25.5
-26.5
-26.1
1.0
1.33
-25.3
-25.6
-26.0
-25.7
-23.8
-23.8
2.2
1.37
Polyflora
pure
-25.6
-25.8
-25.7
-25.5
-26.5
-26.1
1.0
1.33
Polyflora
adulterated
-24.7
-24.0
4.7
-24.8
-24.4
-19.2
-20.1
5.6
1.23
Polyflora
adulterated
-25.2
-25.0
1.3
-24.4
-25.7
-24.8
-24.5
1.3
1.27
d13C ()
Disaccharides
Honey Authenticity
Adulterated Acacia Honey (Real Life Example 1)

LC-IRMS Chromatogram of Adulterated Honey
16000
Adulteration
with fructose
syrup !
Intensity (mV
14000
Fructose
12000
10000
8000
44
Glucose
Disaccharides
6000
m/z
Trisaccharides
45
Ca2+ column
46
water eluent
4000
2000
0
1
1073 2145 3217 4289 5361 6433 7505 8577 9649 10721 11793
Retention Time (s)

RT (s)
Area (Vs)
13C () vs. VPDB
trisaccharides
1219
12
-24,96
disaccharides
1334
48
-25,71
glucose
1552
331
-25,96
fructose
1953
529
-23,79
13C (F-G): 2,17
070327101
13C ()
vs. VPDB
protein
-25,2
honey
-24,6
EA-IRMS ok!
13C (max.): 2,17

Honey Authenticity
Adulterated Acacia Honey (Real Life Example 2)

40000
35000
Intensity (mV)
25000
m/z
44
Disaccharides
20000
Trisaccharides
15000
Ca2+ column
46
water eluent
5000
RT (s)
Area (Vs)
13C () vs. VPDB
oligosaccharides
1095
479
-23,47
trisaccharides
1221
303
-23,02
disaccharides
1339
573
-23,24
glucose
1538
1765
-24,98
fructose
1930
2060
-24,91
2638
2506
2374
2243
2111
1979
1847
1715
1583
1451
1319
1188
1056
924
792
660
528
396
264
133
Retention Time (s)
F/G = 1.17
45
Oligosaccharides
10000
Adulteration
with ca. 50 %
invert sugar
syrup
containing
higher
saccharides!
Fructose
Glucose
30000
070327108
13C ()
vs. VPDB
protein
-25,0
honey
-24,5
EA-IRMS ok!
Honey Authenticity
Adulterated Polyflora Honey (Real Life Example 3)

16000,0
Glucose
m/z
10000,0
44
Disaccharides
8000,0
45
Trisaccharides
6000,0
4000,0
46
Oligosaccharides
Ca2+ column
water eluent
2000,0
Retention Time (s)
13C (max.): - 3,13

RT (s)
Area (Vs)
13C () vs. VPDB
oligosaccharides
1088
73
-26,68
trisaccharides
1237
62
-24,30
disaccharides
1331
169
-26,35
glucose
1534
914
-26,96
fructose
1912
789
-27,43
2609,6
2472,3
2334,9
2197,6
2060,3
1923,0
1785,7
1648,4
1511,1
1373,8
1236,4
1099,1
961,8
824,5
687,2
549,9
412,6
275,3
137,9
0,0
13C (F-G): - 0,47
F/G = 0.86
Fructose
12000,0
0,2
Adulteration
with ca. 13 %
rice syrup
containing
higher
saccharides!
Intensity (mV
14000,0
070718018
13C ()
vs. VPDB
protein
-26,4
honey
-27,0
EA-IRMS ok!
Honey Authenticity
Polyflora Honey spiked with 20 % Rice Syrup

LC-IRMS Chromatogram of Honey spiked with 20 % Rice Syrup
14000,0
Glucose
Intensity (mV
12000,0
10000,0
Fructose
m/z
Disaccharides
8000,0
44
45
Trisaccharides
6000,0
46
Oligosaccharides
4000,0
Ca2+ column
water eluent
2000,0
Retention Time (s)
C (max.): - 2,18
13
F/G = 0.88
RT (s)
Area (Vs)
13C () vs. VPDB
oligosaccharides
1106
102
-26,16
trisaccharides
1238
49
-24,39
disaccharides
1351
270
-26,46
glucose
1553
832
-26,57
fructose
1936
735
-26,30
2609,6
2472,3
2334,9
2197,6
2060,3
1923,0
1785,7
1648,4
1511,1
1373,8
1236,4
1099,1
13C (F-G): 0,27
961,8
824,5
687,2
549,9
412,6
275,3
137,9
0,2
0,0
A20183
13C ()
vs. VPDB
protein
-26,2
honey
-26,4
EA-IRMS ok!
Honey Authenticity
Acacia Honey: Addition of Invert Sugar Syrup (ISS 1)

Parameter
0 % ISS
5 % ISS
10 % ISS
20 % ISS
50 % ISS
100 % ISS
d13C protein ()
-24.30
-24.30
-24.30
-24.30
-24.30
n.a.
d13C honey ()
-24.40
-24.30
-24.10
-23.50
-22.30
-19.80
1.4
5.5
13.7
n.a.
EA-IRMS (AOAC)
ok
ok
ok
ok
adulterated
d13C fructose ()
-24.40
-24.19
-24.26
-24.20
-23.66
-22.78
d13C glucose ()
-24.54
-24.57
-24.08
-23.02
-20.80
-10.87
d13C disaccharides ()
-24.21
-24.13
-23.52
-22.26
-19.70
-12.40
d13C trisaccharides ()
-23.64
-23.05
-22.11
-20.62
-19.12
-11.36
d13C oligosaccharides ()
n.a.
n.a.
n.a.
-12.31
-12.96
-12.05
F/G ratio
1.48
1.48
1.67
1.87
2.46
5.61
disaccharides (% DM)
8.15
9.23
9.75
9.68
8.85
4.14
trisaccharides (% DM)
2.81
2.89
3.23
3.40
2.86
2.18
oligosaccharides (% DM)
n.d.
n.d.
n.d.
0.75
1.65
3.15
ok
ok
adulterated
adulterated
adulterated
% C4 sugar (spec. < 7 %)
LC-IRMS
n.a. not analysable; n.d. not. detected < 0.7 % DM; DM dry matter; ISS 1 invert sugar syrup (80 % fructose)
Limit of detection: EA-IRMS ~ 27 %
EA/LC-IRMS ~ 8 %
misleading results causing a wrong result interpretation !

Honey Authenticity
Honey: Detection of Adulteration, EA-IRMS vs. EA/LC-IRMS

d13C EA-IRMS analysis (AOAC)
9%
d13C EA/LC-IRMS
honeys not corresponding to
purity criteria
pure
adulterated
US
24%
91%
TW
3%
IN
4% HU
4%
d13C EA/LC-IRMS analysis

29%
pure
adulterated
71%
US
DE
CA
MY
VN
9%
GR
CZ
IT
AR
GR
17%
CN
16%
CN
HK
JP
ES
VN
SK
RU
ID
HU
TR
SI
MX
IN
BG
BE
RO
TW
SA
GB
1086 samples suspected of being adulterated (08/2007 06/2008)

Honey Authenticity
Foreign Enzyme Test

foreign enzyme
metabolite
substrate
honey enzymes
honey adulterated with 10 %

invert sugar syrup
honey adulterated with 5 %
invert sugar syrup
authentic honey
authentic honey
Honey Authenticity
Thank you!

Honey Authenticity Test

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Honey Authenticity

Authenticity Testing of Honey

Dr. Lutz Elflein Dr. Kurt-Peter Raezke Dr. Vassil Valkov

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

original paper in press (Apidologie)

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

Sugar Sources for Adulteration

Range of 13C values

Conclusion: combination of EA-IRMS and LC-IRMS: 13C-EA/LC-IRMS

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

1st World Honeydew Honey Symposium, Apimondia IHC

(e) ion source

August 1-3 2008, Tzarevo (BG)

separation from eluent

Source: Finnigan LC IsoLink Product Brochure

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

separation from eluent

Source: Finnigan LC IsoLink Product Brochure

... by chemical oxidation

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

separation from eluent

Source: Finnigan LC IsoLink Product Brochure

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

separation from eluent

Source: Finnigan LC IsoLink Product Brochure

13C of fructose, glucose, di- and trisaccharides

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

EA/LC-IRMS: Natural 13C Variations of Pure Honey (1)

13C () protein (p)

13C () honey (h)

13C () fructose (fru)

13C () glucose (glu)

13C () disaccharides (ds)

13C () trisaccharides (ts)

* apparent C4 sugar content

13C values of individual sugars in honey are very similar!

August 1-3 2008, Tzarevo (BG)

EA/LC-IRMS: Natural 13C Variations of Pure Honey (2)

13C () fru - glu

13C values show only small variations!

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

EA/LC-IRMS: 13C, Proposed Limits for Pure Honey

13C () fru - glu

statistical certainty (confidence level): 99.7 % (3 s.d.)

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

LC-IRMS Chromatogram of Pure Honey

Retention Time (s)

13C () vs. VPDB

13C (F-G): - 0,13

1st World Honeydew Honey Symposium, Apimondia IHC

August 1-3 2008, Tzarevo (BG)

Range of 13C Values for Honey and Syrups

ISS: invert sugar syrup

C4/C3 sugar mix

1st World Honeydew Honey Symposium, Apimondia IHC