A P P L I C AT I O N N O T E

Liquid Chromatography

Authors:
Chi Man Ng
Wilhad M. Reuter
PerkinElmer, Inc.
USA

Analysis of Sugars
in Honey Using the Introduction
Honey consumption has grown
PerkinElmer Altus HPLC significantly during the last few
decades due to its high nutritional
System with RI Detection value and unique flavor. The price of
natural bee honey is much higher than
other sweeteners making it susceptible
to adulteration with cheaper sweeteners, primarily sucrose. Besides lower levels of non-
sugar ingredients, natural honey primarily consists of glucose and fructose and may
contain low levels of sucrose and/or maltose.1, 2 However, according to the international
regulations, any commercially available “pure”-labeled honey products that are found to
have in excess of 5% by weight of sucrose or maltose are considered to be adulterated.3
With the focus on possible honey adulteration, this application highlights the LC separation
of various sugars found in honey and the analysis of these components in four store-
bought honey samples. Method conditions and performance data, including linearity and
repeatability, are presented.
 Experimental Solvents, Standards and Samples
Hardware/Software All solvents and diluents used were HPLC grade and filtered via
For all chromatographic separations, a PerkinElmer Altus™ 0.45-µm filters.
HPLC system was used, including the Altus A-10 Solvent and The sugar standards were obtained from Supelco® (Irvine, CA)
Sample Module, Column Module, integrated vacuum degasser/ and consisted of fructose, glucose, maltose and sucrose. Stock
column oven and an Altus A-10 RI Detector. All instrument sugar standards were made using 65:35 acetonitrile/water as
control, analysis and data processing was performed using the diluent. For the 1333 µg/mL (ppm) stock solution, the standards
Waters® Empower® 3 CDS platform. were first dissolved in 17.5 mL of water before adding 32.5 mL
Method Parameters of acetonitrile. The lower level standards were then prepared
The HPLC method parameters are shown in Table 1 from this stock solution.
All commercially available honey products were purchased at
Table 1. HPLC Method Parameters.
local stores. They were labeled Honey W, Honey X, Honey Y
HPLC Conditions and Honey Z. Each honey was prepared by dissolving 2.5 g into
PerkinElmer Brownlee™ Analytical Amino 3 µm, 50 mL of 65:35 acetonitrile/water, followed by another 1:1
Column:
4.6 x 150 mm (Part# N9303505)
dilution using the same solvent.
Solvent A: 65:35 acetonitrile/water
Solvent program: Prior to injection, all calibrants and samples were filtered through
Mobile Phase: Time Flow Rate
%A %B %C %D Curve
0.45-µm filters to remove small particles.
(min) (mL/min)
Initial 1.000 100.0 0.0 0.0 0.0 Initial
ChromatogramOnly Report
Results and Discussion
Analysis Time 6 min. Figure 1 shows the chromatographic separation of the 1333-µg/mL
Flow Rate: 1.0 mL/min. (2300 psi) (ppm) sugar standard containing the four target sugars using
Oven Temp.: 25 ºC the optimized conditions described above. The analysis time was
Detection: Altus A-10 RI; cell temp.: 35 °C under six minutes.
Injection Volume: 5 µL
Sampling (Data) Rate: 10 pts./sec

22.00
Fructose

20.00

18.00

16.00

14.00

12.00
µRIU

Sucrose

10.00

8.00
Glucose

6.00
Maltose

4.00

2.00

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00

Minutes

Figure 1. Chromatogram of the 1333 µg/mL sugar standard.

2
0.88 12 Sugar
14 No Std No
cal inj 10uL 3538 Maltose Level 5 1333.000 24914.904 1322.233 -0.81 No -0.09 5 Sugar
13 No Std No
cal inj 2uL 3520 Maltose Level 2 267.000 2844.989 259.151 -2.94 No
-0.23 13 No
15 Sugar Std No
cal inj 10uL 3540 Maltose Level 5 1333.000 25349.509 1340.124 0.53 No -0.81 6 Sugar
14 No Std No
cal inj 2uL 3522 Maltose Level 2 267.000 2724.392 252.166 -5.56 No
-0.62 14 No No 7 Sugar Std cal inj 5uL 3524 Maltose Level 3 667.000 10845.862 687.319 3.05 No
P eak : Maltos e
Reported
8 Sugarby User:
Std cal injSystem
5uL 3526 Maltose Level 3 667.000 Project Name: 640.276
9908.221 Sugars in Honey
-4.01 with RI
No
% %
Manual
Reported Ignore
by User: System Manual Ignore Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed:
Deviation Deviation 9 Sugar Std cal
Report Method: LC Calibration Report Date Printed: Report Method ID:inj 5uL
2927 3528 Maltose Level 3 667.000 10421.724 666.129 -0.13 2/4/2015No
1 No MethodNo
-5.11 Report 0.53 15 No
ID: 2927 No 2/4/2015 10 Sugar Std cal inj 7uL 3530 Maltose Level 4 933.000 16482.005 12:38:27 PM
956.178 2.48US/Eastern
No
Figure
7.88 2 No 2 shows
No the overlay of 12 replicate 667-µg/mL sugar standard injections,
11 Sugar Std caldemonstrating
inj 7uL exceptional
3532 Maltose reproducibility.
Level 4 933.000 15595.366 Retention
12:38:27 PM US/Eastern
915.334 -1.89 No
12.18 3 No No 12 Sugar Std cal inj 7uL
time %
-0.37 4 No
RSDs
No
were also quite exceptional, exemplified by 0.026% RSD for fructose. 3534 Maltose Level 4 933.000 16195.141 943.018 1.07 No
13 Sugar Std cal inj 10uL 3536 Maltose Level 5 1333.000 25146.237 1331.767 -0.09 No
-2.94 5 No No 14 Sugar Std cal inj 10uL 3538 Maltose Level 5 1333.000 24914.904 1322.233 -0.81 No
-5.56 6 No No P eak : Maltos e 15 Sugar Std cal inj 10uL
80000.0 3540 Maltose Level 5 1333.000 25349.509 1340.124 0.53 No
3.05 7 No No Calc. %
Sample Name Result Id Peak Name Level X Value Response Manual P eak : Maltos e
-4.01 8 No 8.00
No
Value Deviation 70000.0

% %
1 Sugar Std cal inj 1uL 3507 Maltose Level 1 133.000 604.361 126.202 -5.11 No Manual Ignore Manual Ignore
-0.13 9 No No Deviation60000.0 Deviation
2 Sugar Std cal inj 1uL 3514 Maltose Level 1 133.000 889.048 143.482 7.88 No
2.48 10 No No -5.11 150000.0
No No 0.53 15 No No
3 7.00
Sugar Std cal inj 1uL 3516 Maltose Level 1 133.000 983.771 149.205 12.18 No

A r ea
-1.89 11 No No 7.88 2 No No
4 Sugar Std cal inj 2uL 3518 Maltose Level 2 267.000 2963.948 266.023 -0.37 No 40000.0
1.07 12 No No 12.18 3 No No
5 Sugar Std cal inj 2uL 3520 Maltose Level 2 267.000 2844.989 259.151 -2.94 No 30000.0
-0.09 13 No No -0.37 4 No No
-0.81
6
14 No 6.00
Sugar Std cal inj 2uL
No
3522 Maltose Level 2 267.000 2724.392 252.166 -5.56 No
-2.94 520000.0
No No
7 Sugar Std cal inj 5uL 3524 Maltose Level 3 667.000 10845.862 687.319 3.05 No
-5.56 6 No No
10000.0
8 Sugarby
Reported Std cal inj
User: 5uL
System 3526 Maltose Level 3 667.000 9908.221
Project Name: 640.276 -4.01 with RINo
Sugars in Honey
3.05 7 No No
Report Method:
9 Sugar Std calLC
injCalibration
5uL Report
3528 Maltose Level 3 667.000 10421.724 666.129 -0.13 Date Printed:
No
5.00
Report Method ID: 2927 2/4/2015 -4.01 8
0.0
No No
10 Sugar Std cal inj 7uL 3530 Maltose Level 4 933.000 16482.005 956.178 2.48
12:38:27 PM No
US/Eastern -0.13 9 No 0.00 No 200.00 400.00 600.00 800.00 1000.00 1200.00
11 Sugar Std cal inj 7uL 3532 Maltose Level 4 933.000 15595.366 915.334 -1.89 No Concentration
2.48 10 NoPeak Name:
No Fructose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954;
12 Sugar Std cal inj 7uL 3534 Maltose Level 4 933.000 16195.141 943.018 1.07 No
4.00 -1.89 11 NoR^2: 0.999908;
No Weighting: None; Equation: Y = 8.88e-003 X^2 + 4.57e+001 X - 4.23e+003;
13 Sugar Std cal inj 10uL 3536 Maltose Level 5 1333.000 25146.237 1331.767 -0.09 No Normalized Intercept/Slope: ; RSD(E): 1.220112
µRIU

1.07 12 No No
14 Sugar Std cal inj 10uL 3538 Maltose Level 5 1333.000 24914.904 1322.233 -0.81 No
-0.09 13 No No
15 Sugar Std cal inj 10uL 3540 Maltose Level 5 1333.000 25349.509 1340.124 0.53 No 50000.0
80000.0
3.00 -0.81 14 No No

70000.0
P eak : Maltos e
40000.0
% % Reported by User: System Project Name: Sugars in Honey with RI
Manual Ignore Manual Ignore
Deviation60000.0 Deviation Report Method: LC Calibration Report Date Printed:
-5.11 1 No 2.00
No 0.53 15 No No
Report Method ID: 2927
30000.0
2/4/2015
50000.0 12:38:27 PM US/Eastern

A r ea
7.88 2 No No
A r ea

40000.0
12.18 3 No No 20000.0
-0.37 430000.0
No 1.00
No
-2.94 520000.0
No No 10000.0
-5.56 6 No No
10000.0 80000.0
3.05 7 No 0.00
No
0.0
0.0 70000.0
-4.01 8 No No
-0.13 9 No 0.00 No 200.00 400.00 600.00 800.00 1000.00 1200.00 60000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
Concentration Concentration
2.48 10 No -1.00
No
Peak Name: Fructose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954; 50000.0Peak
Name: Glucose; RT: 3.772; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3504; R: 0.999959;
-1.89 11 NoR^2: 0.999908;
No R^2: 0.999918; Weighting: None; Equation: Y = 5.30e-003 X^2 + 2.79e+001 X - 2.54e+003;
0.00
Weighting: None;0.50
Equation: Y = 1.00 1.50 X - 4.23e+003;
8.88e-003 X^2 + 4.57e+001 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
A r ea

40000.0Normalized Intercept/Slope: ; RSD(E): 1.148411

1.07 12 NoNormalized
No Intercept/Slope: ; RSD(E): 1.220112
Minutes
-0.09 13 No No 30000.0
50000.0
-0.81 14 No No
Figure 2. Overlay of 12 replicates of the 667 µg/mL sugar standard. 20000.0
Reported by User: System Project Name: Sugars in Honey with RI
Reported
40000.0 by User: System Project Name: Sugars in Honey with RI 10000.0 Method: LC Calibration Report
Report Date Printed:
Report Method: LC Calibration Report Date Printed: Report Method ID: 2927 2/4/2015
Figure 3 shows the calibration results for all four sugars over a concentration
Report Method ID: 2927 0.0 range of 133 to 1333 µg/mL. All four sugars followed a 2/4/2015 12:38:27 PM US/Eastern
30000.0 12:38:27 PM US/Eastern
quadratic (2nd order) fit and had R2 coefficients > 0.999 (n = 3 at each level). 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
A r ea

Concentration

Peak Name: Fructose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954;
20000.0
R^2: 0.999908; Weighting: None; Equation: Y = 8.88e-003 X^2 + 4.57e+001 X - 4.23e+003;
Normalized Intercept/Slope: ; RSD(E): 1.220112
50000.0
10000.0
50000.0
80000.0
Fructose 40000.0
Glucose
0.0
70000.0
Reported by User: System 40000.0 Project Name: Sugars in Honey with RI
60000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
Report Method: Overlay Report
Concentration 30000.0
Date Printed:
50000.0Peak Name: Glucose; RT: 3.772; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3504; R: 0.999959; 30000.0
e ar e a

Report
R^2: 0.999918; Method
Weighting: ID:2232
None; Equation: Y 2232
= 5.30e-003 X^2 + 2.79e+001 X - 2.54e+003; 1/30/2015
A r ea

Ar A

40000.0Normalized Intercept/Slope: ; RSD(E): 1.148411 20000.0

30000.0
Page: 1 of 1 20000.0
3:05:55 PM US/Eastern
10000.0
20000.0 10000.0
Reported by User: System Project Name: Sugars in Honey with RI
10000.0
Report Method: LC Calibration Report Date Printed: 0.0
Report Method ID: 2927 R2 = 0.999954 2/4/2015 0.0 R2 = 0.999959
0.0
12:38:27 PM US/Eastern 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0.00 200.00 400.00 Concentration
600.00 800.00 1000.00 1200.00
Concentration Concentration
Peak Name: Sucrose; RT: 4.464; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3505; R: 0.999952;
Peak Name: Fructose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954; Peak Name: Glucose;
R^2: 0.999903; RT: 3.772;
Weighting: None; Fit Type: Quadratic
Equation: (2nd Order);
Y = 5.14e-003 Cal CurveXId:
X^2 + 2.66e+001 3504; R: 0.999959;
- 2.48e+003;
R^2: 0.999908; Weighting: None; Equation: Y = 8.88e-003 X^2 + 4.57e+001 X - 4.23e+003; R^2: 0.999918; Weighting: None; Equation:
Normalized Intercept/Slope: ; RSD(E): 1.252103Y = 5.30e-003 X^2 + 2.79e+001 X - 2.54e+003;
Normalized Intercept/Slope: ; RSD(E): 1.220112 Normalized Intercept/Slope: ; RSD(E): 1.148411
50000.0 30000.0

50000.0 Sucrose Maltose

25000.0
40000.0
Reported by User: System Project Name: Sugars in Honey with RI
40000.0
Report
20000.0 Method: LC Calibration Report Date Printed:
30000.0 Report Method ID: 2927 2/4/2015
15000.0
12:38:27 PM US/Eastern
30000.0
A r ea
A r Ae ra e a

20000.0
10000.0
20000.0

10000.0 5000.0
50000.0
10000.0
0.0
0.0 R2 = 0.999952 R2 = 0.999940
0.0 40000.0
-5000.0
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
0.00 200.00 400.00 Concentration
600.00 800.00 1000.00 1200.00 Concentration
Concentration 30000.0
Peak Name: Sucrose; RT: 4.464; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3505; R: 0.999952; Peak Name: Maltose; RT: 5.155; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3506; R: 0.999940;
Peak Name: Glucose;
R^2: 0.999903; RT: 3.772;
Weighting: None; Fit Type: Quadratic
Equation: (2nd Order);
Y = 5.14e-003 Cal CurveXId:
X^2 + 2.66e+001 3504; R: 0.999959;
- 2.48e+003; R^2: 0.999881; Weighting: None; Equation: Y = 3.27e-003 X^2 + 1.56e+001 X - 1.42e+003;
A r ea

Figure 3. Results of 5-level calibration sets for fructose, glucose, maltose and sucrose.
R^2: 0.999918;
Normalized Weighting: ;None;
Intercept/Slope: Equation:
RSD(E): 1.252103Y = 5.30e-003 X^2 + 2.79e+001 X - 2.54e+003;
20000.0
Normalized Intercept/Slope: ; RSD(E): 1.389827
Normalized Intercept/Slope: ; RSD(E): 1.148411
30000.0

10000.0
25000.0
Reported by User: System Project Name: Sugars in Honey with RI
Reported by User: System Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed:
Report
20000.0 Method: LC Calibration Report Date Printed: 0.0 Method ID: 2927
Report 2/4/2015
3
Report Method ID: 2927 2/4/2015 12:38:27 PM US/Eastern
12:38:27 PM US/Eastern
15000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
a
 Using the same chromatographic conditions, four honey samples chromatograms of these honey samples with the sugar standards,
were analyzed. The chromatographic results for Honey X, it can be observed that all three honey samples contain the same
Honey Y and Honey Z are shown in Figure 4. Comparing the three sugars: fructose, glucose and small amounts of sucrose.

45.00

40.00

35.00

30.00

25.00
µRIU

20.00 Sucrose

15.00
Fructose

10.00

Glucose
5.00

0.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

4.00 4.50
Figure 4. Overlaid chromatograms of Honey X (green), Honey Y (black) and Honey Z (blue).

Based on standard calibration, the quantitative results for each Table 2. Quantitative Results.
honey sample are shown in Table 2. Combining the fructose and Honey X:
glucose percentages for each honey sample, the overall fructose Component Amount (mg) Percent Sugar (w/w)
and glucose content for Honey X, Y, and Z was determined to Fructose 556.05 22.24
be 50.90%, Reported
57.13%,byand
User: System
53.60%, respectively. These results Glucose Project Name:
716.48 Sugars in Honey with RI
28.66
Report Method: Overlay Report
are consistent with the accepted overall content of fructose and Date Printed:
Sucrose 79.875 3.20
Report Method ID:
2232
2232
glucose in honey, expected to be somewhere around 60%.1 The 1/30/2015
Page: 1 of 1 4:03:09 PM US/Eastern
sucrose content for each honey sample was determined to be Honey Y:
3.20%, 3.26% and 3.90%, respectively. These values are all Component Amount (mg) Percent Sugar (w/w)
below the 5% mass ratio limit for sucrose that is allowed in Fructose 610.23 24.41
unadultered honey. Based on the data presented, the three Glucose 817.95 32.72
store-bought honey samples do not appear to be adultered Sucrose 81.525 3.26
with cheaper sweeteners.
Honey Z:
Upon closer examination of the chromatogram of Honey W, a
smaller but significant peak was observed at about 5.10 minutes Component Amount (mg) Percent Sugar (w/w)
(Figure 5). This matched the elution time for maltose in the Fructose 602.30 24.09
standard mix. The amount of maltose was calculated to be Glucose 737.78 29.51
43.85 mg, and the percent sugar was calculated to be 1.75% Sucrose 97.525 3.90
(w/w). Considering the 5% (by weight) limit that is allowed in
commericially available “pure”-labeled honeys, the resulting
maltose level found in Honey W suggests it was not adultered.

4
 30.00

25.00

20.00 Fructose
µRIU

15.00

10.00 Sucrose
1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Minutes Glucose
5.00

Maltose

0.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Figure 5. Overlay chromatograms of Honey W (red) and the 133 ppm sugar standard (black), zooming in on last eluting peak.

Conclusion References
This work has demonstrated the effective chromatographic 1. W. Guo, Y. Liu, X. Zhu and S. Wang, "Dielectric properties of
separation of four sugars using a PerkinElmer Altus HPLC System honey adulterated with surcrose syrup", Journal of Food
with RI detection. The results exhibited very good retention time Engineering, pp. 1-7, 2011.
Reported
repeatability as well asbyexcellent
User: System
linearity over the tested Project Name: Sugars in Honey with RI
2. A. Moussa, D. Noureddine, A. Saad and S. Douichene, "The
Report
concentration Method: Overlay Report
ranges. Date Printed:
Report Method ID:2232
2232 Relationship between Fructose, Glucose and Maltose Content
1/30/2015
From a food quality
Page: 1 ofperspective,
1 there is an ever growing emphasis Project Name: Sugars in Honey
with Diastase Number and Anti-Pseudomonal Activity
3:28:50 PM US/Eastern of Natural
on food monitoring. This is especially the case pertaining to the Honey Combined with Potato Starch", Organic Chemistry
eport adulteration of honey. With this in mind, this work focused on the Current Research, vol. 1, no. 5, pp. 1-5, 2012. Date
sugar analysis of four store bought honeys, identifying the particular
analytes contained in each of the honey samples, as well as
3. Codex Alimentarius Commission, 2001; GB18796-2005, 2005
1/3
comparing the sugar profiles, both chromatographically and
quantitatively. 3:28:50 PM US/

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