APPLICATION NOTE

Liquid Chromatography

Author:
Jamie Foss

PerkinElmer, Inc.
Shelton, CT

Analysis of Sugars in Maple Syrup

by Hydrophilic Interaction Introduction
Maple syrup is a popular natural
Chromatography (HILIC) sweetener that the U.S. Food and

with Refractive Index Detection Drug Administration defines as the

liquid food derived by concentrating
and heat-treating sap from the
maple tree. Sucrose is the primary sugar found in maple syrup, along with lesser amounts
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of glucose, fructose and complex carbohydrates.2 Often, the total sugar content of maple
syrup is based solely on the amount of sucrose, and does not consider the other sugar
sources.2 Maple syrup also contains various other components including minerals, vitamins,
amino acids, organic acids, and phytohormones.3 The sugar composition of maple syrup
differs from honey, which is dominated by both glucose and fructose, and can contain other
sugars such as sucrose and maltose, in much smaller concentrations.3
The analysis of simple sugars is commonly performed using high performance liquid
chromatography (HPLC). Often, hydrophilic interaction chromatography (HILIC) is used with
refractive index (RI) detection owing to the polarity of the sugars, and the lack of a suitable
chromophore for UV detection. Therefore, this work describes a simple HILIC-RI method
for the analysis of four common sugars found in maple syrup. The structures of these four
sugars are shown in Figure 1.
 Fructose Glucose Sucrose Maltose
Figure 1. Chemical structures of the four sugars analyzed in this study.

Experimental Table 1. LC Parameters.

Hardware and Software PerkinElmer Brownlee Analytical Amino, 3 µm,

Column
150 x 4.6 mm (Part# N9303501)
Chromatographic separation was achieved using a PerkinElmer LC
300 HPLC system, consisting of an LC 300 10K psi pump and an Solvent A: 75:25 Acetonitrile:Water
Solvent Program: Isocratic
LC 300 autosampler equipped with an integrated column oven.
Detection was achieved using an LC 300 Refractive Index (RI) Mobile Phase Time Flow Rate
Step %A %B
(min.) (mL/min.)
detector. Instrument control, analysis, and data processing were 1 0.00 1.5 100 0
performed using the Simplicity™Chrom CDS software platform. 2 6.00 1.5 100 0

Method Parameters Data Collection

5 pts/sec (Hz)
The LC parameters are shown in Table 1. Rate
Analysis Time 6 min.
Solvents, Standards and Samples
All solvents and diluents used were HPLC grade. Unless otherwise Pressure 2100 psi/145 bar maximum
specified, standard and sample extract dilutions were prepared RI Flow
35 ºC
using 75:25 acetonitrile:water. Cell Temp.
Oven Temp. 35 ºC
The four sugar standards were obtained from Sigma-Aldrich®, Inc
(St. Louis, MO) as powders. These included: fructose, glucose, Injection Vol. 10 μL (Partial Loop)
sucrose, and maltose. Approximately 0.75 mL of syrup was pipetted into a tared 50 mL
A 5 mg/mL stock standard mix solution was prepared by dissolving centrifuge tube to obtain an exact sample weight. 25 mL of water
250 mg of each standard into a 50-mL volumetric flask using 1:1 was added, and the samples were sonicated for 25 minutes. After
methanol:water as the diluent to ensure complete dissolution. This sonication, the samples were filtered using 0.45-µm nylon filters
also served as the Level 7 calibration standard. (Part# 02542880). Samples were then diluted 10-fold with mobile
phase prior to injecting 10 µL for analysis.
Additional calibrants were prepared by serially diluting the standard
mix to concentration levels of 2.5, 1.25, 0.625, 0.3125, 0.15625,
Results and Discussion
and 0.078125 mg/mL, using mobile phase (75:25 acetonitrile:
The chromatogram of the 0.625-mg/mL standard is shown in
water) to give a 7-level calibration set.
Figure 2, with all four sugars eluting in under five minutes.
Two different brands of Grade A Dark Amber maple syrup were
obtained from a local grocery store. One spiced apple syrup was
also obtained for analysis from a local farmer’s market.

Figure 2. Chromatogram of the the 0.625-mg/mL sugar standard.

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Figure 3 shows the overlay of 10 replicate 0.625 mg/mL sugar standard injections, demonstrating exceptional reproducibility. Retention time
%RSDs were less than 0.07%, and peak area %RSDs were less than 0.5% for all analytes.

Figure 3. Chromatographic overlay of 10 replicates of the 0.625-mg/mL sugar standard.

Figure 4 shows the calibration results for all four sugars over a concentration range of 0.078125 to 5 mg/mL. All four sugars followed a linear
(1st order) fit and had R2 coefficients above 0.999 (n=3 at each level).

Figure 4. Results of the 7-level calibration sets for fructose, glucose, sucrose, and maltose.

As listed in Table 2, the limit of detection (LOD) and limit of Table 2. LODs and LOQs for the four analytes, in order of elution.
quantitation (LOQ) were obtained for each analyte, and were Analyte Calculated LOD (mg/mL) Calculated LOQ (mg/mL)
calculated from the standard deviation of the response and the Fructose 0.006 0.022
slope of the calibration curve for each analyte, based on
Glucose 0.039 0.131
triplicate injections of the Level 1 standard.
Sucrose 0.015 0.050
Using the same chromatographic conditions, the three syrup
Maltose 0.015 0.050
samples were analyzed. The chromatographic results for Syrups
A, B, and C can be see in Figures 5, 6, and 7, respectively.

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 Figure 5. Chromatogram of Syrup A.

Figure 6. Chromatogram of Syrup B.

Figure 7. Chromatogram of Syrup C.

Syrups A and B were the two Grade A Dark Amber maple syrups purchased for this study. As expected, the primary constituent is sucrose,
shown around 3.45 minutes. For Syrup C (the spiced apple syrup), illustrated in Figure 7, the chromatographic profile is different, with
two peaks in addition to the sucrose peak observed in Syrups A and B. Upon closer inspection via a chromatographic overlay with the
0.625 mg/mL sugar standard, shown in Figure 8, the additional two peaks can clearly be identified as fructose and glucose.

Figure 8. Overlay chromatograms of Syrup C (blue) and 0.625 mg/mL standard (black).
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Table 3. Amount sugar (mg) and percent sugar (w/w) for each of the three syrups
analyzed. Amounts are based on the average of three injections. Conclusion
This work has demonstrated the fast and robust chromatographic
SYRUP A; SAMPLE WEIGHT: 637.1 mg
separation and quantitation of four sugars using a PerkinElmer
Analyte Amount (mg) Percent Sugar (w/w) LC 300 HPLC system with RI detection. The results exhibited very
Fructose Trace n/a good retention time repeatability, as well as excellent linearity over
Sucrose Trace n/a the tested concentration ranges. The method also affords LOQs of
Glucose 338.19 53.08 ≤ 0.1 mg/mL for most analytes.

SYRUP B; SAMPLE WEIGHT: 886.6 mg This work specifically focused on the sugar analysis of three different
Analyte Amount (mg) Percent Sugar (w/w) syrups, two maple syrups from a local grocery store and one spiced
apple syrup from a local farmer’s market. The method allowed
Fructose Trace n/a
for the identification of specific analytes contained in each of the
Sucrose Trace n/a
syrup samples, as well as comparing their sugar profiles, both
Glucose 477.71 53.88 chromatographically and quantitatively.
SYRUP C; SAMPLE WEIGHT: 831.41 mg
Analyte Amount (mg) Percent Sugar (w/w) References
Fructose 53.46 6.43 1. USDA United States Standards for Grades of Maple Syrup,
Sucrose 45.95 5.53 March 2015.

Glucose 350.91 42.21 2. Storz, G.; Darvill, A. G.; Albersheim, P. Characterization of

polysaccharides isolated from maple syrup. Phytochemistry 1986,
Based on standard calibration, the quantitative results for each 25, 437−441.
syrup sample are shown in Table 3. When analyzed, Syrups A and
3. Ball, D. W. The chemical composition of maple syrup. J. Chem.
B both only contained a quantitative amount of sucrose. Although
Educ. 2007, 84, 1647−1650.
trace amounts of both fructose and glucose were observed, they
were below the limit of detection, and were not considered in any
of the calculations. Syrup C contained much higher concentrations
of fructose and glucose compared to Syrups A and B. This is
most likely due to the apples used in the production of the syrup.
Interestingly, the total percent sugar by weight for Syrup C was
54.17%, closely matching the other two maple syrups which were
based only on the amount of sucrose.

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