Analysis of Food Sugars in Various Matrices Using UPLC with … · To assess the utility of the method for the quantification of sugars in fruit juices, three fruit juice samples

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Analysis of Food Sugars in Various Matrices Using UPLC with Refractive Index (RI) DetectionMark E. BenvenutiWaters Corporation, Milford, MA, USA

IN T RO DU C T IO N

Sugars are found in a variety of food matrices as either naturally occurring or

artificially added. Fructose, glucose, and sucrose are important constituents of

various fruit juices. Maltose is found in products derived from corn and grain

products. Lactose, also known as milk sugar, exists in dairy products. This set of

sugars is known as the five food sugars.

Analysis of these sugars is important for quality control purposes, or to determine

authenticity or adulteration of food products. Due to the lack of chromophores

on these molecules, typical LC methods use evaporative light scattering (ELS),

electrochemical, or refractive index (RI) detection. In many laboratories, RI

is the detection method of choice because it is simple to use and needs no

equilibration between injections. RI provides excellent repeatability and shows a

linear response for quantification. There is no need for a clean nitrogen supply,

which provides an economic benefit and is ideal for a manufacturing facility.

In laboratories where RI detection is the preferred detection method for these

samples, a UPLC-compatible version was required to take advantage of the

benefits of sub-2-µm particle technology.

This application note shows the efficacy of the Waters® ACQUITY UPLC H-Class

System with the ACQUITY RI Detector to analyze sugars in several different food

matrices with a run time of less than 3.5 minutes.

WAT E R S SO LU T IO NS

ACQUITY UPLC® H-Class System

ACQUITY® Refractive Index Detector

ACQUITY UPLC BEH Amide

Column Chemistry

K E Y W O R D S

Food sugars, BEH Amide column chemistry

A P P L I C AT IO N B E N E F I T S

Using UPLC® with RI detection enables the

detection of the food sugars fructose, glucose,

sucrose, maltose, and lactose in a variety of

matrices resulting in the following benefits:

■■ Run time of less than 3.5 minutes

increases throughput.

■■ Isocratic elution eliminates the need for

column re-equilibration between injections.

■■ Method allows the use of robust

UPLC BEH Amide column chemistry

for sugar separation.

Figure 1. ACQUITY UPLC H-Class System with RI Detector.

2Analysis of Food Sugars in Various Matrices Using UPLC with Refractive Index (RI) Detection

E X P E R IM E N TA L

LC conditions

System: ACQUITY UPLC H-Class

Run time: 3.5 min

Column: ACQUITY BEH Amide

2.1 x 50 mm, 1.7µm

Temp.: 85 °C

Mobile phase: 77:23 acetone/water with

0.05% triethylamine (TEA)

Flow rate: 0.15 mL/min

Injection volume: 1.0 µL

Detection: ACQUITY Refractive

Index Detector

Data acquisition: 20 points/s

Time constant: Normal

Flow cell: 40 °C

Standard preparation

A stock solution of fructose, glucose, sucrose,

maltose, and lactose was prepared by dissolving

~1 gram of each of these sugars in water and

diluting to 100 mL with same. From this stock,

six dilutions were made in 50:50 acetonitrile/water

to create working standards with concentrations

listed in Table 1. The fructose high standard was

~1400 ppm greater than that in the other sugars due

to the amount weighed for the stock, extending the

curve for this analyte compared to the other sugars.

Sample preparation

Individual samples of orange, white grape, and

pineapple juices were centrifuged at 4000 rpm

for 30 min to remove solids. The supernatants

were diluted 1:5 with 50:50 acetonitrile/water,

and injected.

A corn syrup sample was prepared by dissolving

1.024 g syrup in water, and further diluting to

50 mL. 500 µL of this was diluted with the same

amount of 50:50 acetonitrile/water, then injected.

A sample of low fat milk was diluted 1:10 with

50:50 acetonitrile/water, and centrifuged at

4000 rpm for 30 min to remove the precipitated

lipids. A portion of the supernatant was collected

for injection.

Std Fructose Glucose Sucrose Maltose Lactose1.0 6505.0 5125.0 5110.0 5065.0 5165.0

2.0 2602.0 2050.0 2044.0 2026.0 2066.0

3.0 1301.0 1025.0 1022.0 1013.0 1033.0

4.0 650.5 512.5 511.0 506.5 516.5

5.0 520.4 410.0 408.8 405.2 413.2

6.0 260.2 205.0 204.4 202.6 206.6

Table 1. Concentrations of the mixed sugar standards in ppm (mg/kg).

3Analysis of Food Sugars in Various Matrices Using UPLC with Refractive Index (RI) Detection

Figure 2. Overlay of the chromatograms from the six standards of differing concentrations.

µRIU

-6.00-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.0044.0046.0048.0050.00

Minutes

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1- Fructose2- Glucose3- Sucrose4- Maltose5- Lactose

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Figure 3. Calibration curves for the five food sugars.

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FructoseR2= 0.99902

GlucoseR2= 0.99951

SucroseR2= 0.99992

MaltoseR2= 0.99991

LactoseR2= 0.99988

R E SU LT S A N D D IS C U S S IO N

The resulting chromatograms from injections of each of the six standards are shown in Figure 2. Note that all

target analytes elute in less than 3.5 minutes. Figure 3 shows the linear calibration curves for each of the five

food sugars where R2 values are greater than 0.999 for all compounds.

4Analysis of Food Sugars in Various Matrices Using UPLC with Refractive Index (RI) Detection

To assess the utility of the method for the quantification of sugars in fruit juices, three fruit juice samples were

analyzed, and the resulting chromatograms are shown in Figure 4. Differences in sugar content and ratios for

each juice were quite apparent. The ratio of fructose, glucose, and sucrose was ~1:1:2, while the white grape

juice contains fructose and glucose, but negligible sucrose. This is consistent with the literature.1 To assess the

retention time and amount repeatability, each juice was injected three times. The resulting data are shown in

Table 2. The %RSD for retention time and amount for all analytes was less than 0.11 and 1.25, respectively.

Figure 4. Chromatograms of orange, white grape, and pineapple juices.

µRIU

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Minutes1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50

1- Fructose 22.9 g/L2- Glucose 21.4 g/L3- Sucrose 53.4 g/L

1- Fructose 79.5 g/L2- Glucose 77.2 g/L

1- Fructose 32.3 g/L2- Glucose 34.6 g/L3- Sucrose 44.6 g/L

Orange

White grape

Pineapple

1 2

3

1 2 3

1 2

Sugar Orange White grape PineappleRT Amount RT Amount RT Amount

Fructose 0.104 1.218 0.048 0.365 0.066 0.371

Glucose 0.038 0.827 0.035 0.659 0.049 0.321

Sucrose 0.035 0.588 N/A N/A 0.043 0.429

Table 2. Reproducibility data (%RSD) for retention time and amount for three injections of each fruit juice.

5Analysis of Food Sugars in Various Matrices Using UPLC with Refractive Index (RI) Detection

Figure 5. Chromatogram of corn syrup showing calculated concentration of the two sugars; glucose and maltose.

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2- Glucose 154.4 g/L4- Maltose 104.4 g/L

2

4

Figure 6. Chromatogram of low fat milk showing the presence of lactose.

Minutes1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50

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5- Lactose 47.9 g/L

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The chromatograms for corn syrup and low fat milk are shown in Figures 5 and 6. These samples show very

different profiles from those expected for fruit juices. The supermarket corn syrup sample used here consists

primarily of glucose and maltose, as shown in Figure 5. Unlike high fructose corn syrups, where cornstarch

has been enzymatically converted to glucose and then to fructose, this sample still has high levels of maltose.

Lactose is a food sugar found in dairy products and is present in the chromatogram of the milk sample in Figure 6.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Reference

1. Sanz et al. Inositols and Carbohydrates in Different Fresh Fruit Juices. Food Chemistry. 2004; 87: 326.

The use of UPLC Technology enables highly efficient, rapid sugar

separations in less than 3.5 minutes. The BEH Amide column

chemistry allows for the use of basic modifiers and higher column

temperatures to improve peak shape and quantification. The

ACQUITY Refractive Index Detector’s low internal volume delivers

low dispersion that is compatible with narrow UPLC peak volumes,

while still delivering stable baseline performance for reliable

quantitative results, evident in the sugar analysis.

CO N C LU S IO NS

This application note demonstrates a rapid isocratic method for

the analysis of food sugars.

Waters ACQUITY UPLC H-Class System with RI detection

provides the following benefits:

■■ Rapid analysis time with no column equilibration necessary

■■ A reduction in turnaround time resulting in

increased throughput

■■ Low eluent flow rate reduces solvent cost and waste disposal

Waters, ACQUITY UPLC, ACQUITY, and UPLC are registered trademarks of Waters Corporation. T he Science of What’s Possible is a trademark of Waters Corporation. All other trademarks are the property of their respective owners.

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