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)
2
patent pending

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity

Sugar Sources for Adulteration

Plant Origin Examples Range of 13C values Method

Corn EA-IRMS
C4-Plants -8 to -13
Sugar Cane (AOAC 998.12)
Beet
Rice LC-IRMS
C3-Plants -22 to -30
Wheat (new)
Cichory

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)
Honey Authenticity

EA-IRMS

13C protein
13C honey

(a) autosampler (e) ion source

(b) combustion column (f) flight tube
(c) reduction column (g) magnetic beam deflector
(d) water trap (h) signal detectors

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity

LC-IRMS

Step 1: Step 2: Step 3: Step 4:

chromatographic conversion separation from eluent 13/12C
isotope ratio
separation of sugars to CO2 and transfer to IRMS measurement

Source: Finnigan LC IsoLink Product Brochure

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity

LC-IRMS

Step 1: Step 2: Step 3: Step 4:

chromatographic conversion separation from eluent 13/12C
isotope ratio
separation of sugars to CO2 and transfer to IRMS measurement

Source: Finnigan LC IsoLink Product Brochure

... by chemical oxidation

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity

LC-IRMS

Step 1: Step 2: Step 3: Step 4:

chromatographic conversion separation from eluent 13/12C
isotope ratio
separation of sugars to CO2 and transfer to IRMS measurement

Source: Finnigan LC IsoLink Product Brochure

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity

LC-IRMS

Step 1: Step 2: Step 3: Step 4:

chromatographic conversion separation from eluent 13/12C
isotope ratio
separation of sugars to CO2 and transfer to IRMS measurement

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)
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 %) < 0.7 - -
Number of samples: 451 * apparent C4 sugar content

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

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
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
13C () ts - p 0.5 0.7 2.1
Number of samples: 451

13C values show only small variations!

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
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.)

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity
LC-IRMS Chromatogram of Pure Honey
LC-IRMS Chromatogram of Honey

20000
18000
Fructose
m/z
16000 Glucose
Intensity (mV) 14000
12000 44
10000 45 Ca2+ column
Disaccharides
8000 46 water eluent
6000 Trisaccharides
4000
2000
0
0
137
274
411
548
685
822
958
1095
1232
1369
1506
1643
1780
1917
2054
2191
2327
2464
Retention Time (s)

RT (s) Area (Vs) 13C () vs. VPDB 13C ()

vs. VPDB
trisaccharides 1167 61 -26,05
protein -25,6
disaccharides 1393 253 -26,78
honey -26,0
glucose 1479 2300 -25,88
fructose 1864 2687 -26,01 13C (F-G): - 0,13
13C (max.): 1,18

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
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

-11.3 -19.8
EA-IRMS -24.2 -26.1 -26.4
EA-IRMS
C4 sugar C4/C3 sugar mix LC-IRMS C3 sugars

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity
EA-IRMS LC-IRMS: Real Life Examples
EA-IRMS (AOAC 998.12) LC-IRMS (APPLICA)
HONEY
TYPE d13C () d13C () C4 Sugar d13C () d13C () d13C () d13C () d13C () F/G-
Protein Honey (%) Fructose Glucose Disaccharides Trisaccharides max. Ratio

Acacia
-24.9 -25.1 0 -25.1 -25.3 -25.3 -24.4 0.9 1.57
pure
Acacia
-25.2 -24.6 3.9 -23.8 -26.0 -25.7 -25.0 2.2 1.60
adulterated
Linden
-25.7 -25.6 0.6 -25.5 -25.4 -25.9 -25.5 0.5 1.35
pure
Linden
-25.6 -25.7 0 -26.0 -25.7 -23.9 -24.4 2.1 1.27
adulterated
Honeydew
-25.6 -25.8 0 -25.7 -25.5 -26.5 -26.1 1.0 1.33
pure
Honeydew
-25.3 -25.6 0 -26.0 -25.7 -23.8 -23.8 2.2 1.37
adulterated
Polyflora
-25.6 -25.8 0 -25.7 -25.5 -26.5 -26.1 1.0 1.33
pure
Polyflora
-24.7 -24.0 4.7 -24.8 -24.4 -19.2 -20.1 5.6 1.23
adulterated
Polyflora
-25.2 -25.0 1.3 -24.4 -25.7 -24.8 -24.5 1.3 1.27
adulterated

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
 Honey Authenticity
Adulterated Acacia Honey (Real Life Example 1)
LC-IRMS Chromatogram of Adulterated Honey

16000

14000
12000 Fructose m/z
Intensity (mV

10000 44
Adulteration Glucose
8000 Disaccharides 45 Ca2+ column
with fructose
syrup ! 6000 46 water eluent
Trisaccharides
4000
2000

0
1 1073 2145 3217 4289 5361 6433 7505 8577 9649 10721 11793

Retention Time (s)

070327101

RT (s) Area (Vs) 13C () vs. VPDB 13C ()

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

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
 Honey Authenticity
Adulterated Acacia Honey (Real Life Example 2)
LC-IRMS Chromatogram of Adulterated Honey

40000

35000
Fructose
Intensity (mV) 30000 Glucose m/z
25000
Adulteration Disaccharides 44
20000 45 Ca2+ column
with ca. 50 % Trisaccharides
invert sugar 15000 46 water eluent
syrup 10000 Oligosaccharides
containing 5000
higher
0
saccharides!
0
133
264
396
528
660
792
924
1056
1188
1319
1451
1583
1715
1847
1979
2111
2243
2374
2506
2638
Retention Time (s) 070327108

RT (s) Area (Vs) 13C () vs. VPDB 13C ()

vs. VPDB
oligosaccharides 1095 479 -23,47
protein -25,0
trisaccharides 1221 303 -23,02
honey -24,5
disaccharides 1339 573 -23,24
glucose 1538 1765 -24,98
F/G = 1.17 EA-IRMS ok!
fructose 1930 2060 -24,91

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
 Honey Authenticity
Adulterated Polyflora Honey (Real Life Example 3)
LC-IRMS Chromatogram of Adulterated Honey

16000,0
14000,0 Glucose Fructose
Intensity (mV 12000,0 m/z
10000,0 44
Disaccharides
Adulteration 8000,0 45
Trisaccharides Ca2+ column
with ca. 13 % 6000,0 46
rice syrup water eluent
4000,0 Oligosaccharides
containing
2000,0
higher
0,0
saccharides!
0,2
137,9
275,3
412,6
549,9
687,2
824,5
961,8
1099,1
1236,4
1373,8
1511,1
1648,4
1785,7
1923,0
2060,3
2197,6
2334,9
2472,3
2609,6
13C (F-G): - 0,47
Retention Time (s)
13C (max.): - 3,13 070718018

RT (s) Area (Vs) 13C () vs. VPDB 13C ()

vs. VPDB
oligosaccharides 1088 73 -26,68
protein -26,4
trisaccharides 1237 62 -24,30
honey -27,0
disaccharides 1331 169 -26,35
glucose 1534 914 -26,96
F/G = 0.86 EA-IRMS ok!
fructose 1912 789 -27,43

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
 Honey Authenticity
Polyflora Honey spiked with 20 % Rice Syrup
LC-IRMS Chromatogram of Honey spiked with 20 % Rice Syrup

14000,0

12000,0 Glucose Fructose

Intensity (mV 10000,0 m/z
Disaccharides 44
8000,0
45
6000,0 Trisaccharides Ca2+ column
46
4000,0 Oligosaccharides water eluent
2000,0

0,0
0,2
137,9
275,3
412,6
549,9
687,2
824,5
961,8
1099,1
1236,4
1373,8
1511,1
1648,4
1785,7
1923,0
2060,3
2197,6
2334,9
2472,3
2609,6
13C (F-G): 0,27 Retention Time (s)
A20183
C (max.): - 2,18
13

RT (s) Area (Vs) 13C () vs. VPDB 13C ()

vs. VPDB
oligosaccharides 1106 102 -26,16
protein -26,2
trisaccharides 1238 49 -24,39
honey -26,4
disaccharides 1351 270 -26,46
glucose 1553 832 -26,57
F/G = 0.88 EA-IRMS ok!
fructose 1936 735 -26,30

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
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
% C4 sugar (spec. < 7 %) 0 0 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
LC-IRMS ok ok adulterated adulterated adulterated
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 !
1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity

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

d13C EA-IRMS analysis (AOAC) d13C EA/LC-IRMS

9% honeys not corresponding to
purity criteria
pure
adulterated
US
24%
91%

TW
d13C EA/LC-IRMS analysis 3%
IN
GR
29% 4% HU
4% VN CN 17%
9% 16%
pure
adulterated US GR CN VN HU IN TW
71% DE CZ HK SK TR BG SA
CA IT JP RU SI BE GB
MY AR ES ID MX RO

1086 samples suspected of being adulterated (08/2007 06/2008)

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
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

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)
Honey Authenticity

Thank you!

1st World Honeydew Honey Symposium, Apimondia IHC August 1-3 2008, Tzarevo (BG)