APPLICATION NOTE

Liquid Chromatography
Author:Wilhad M. Reuter
PerkinElmer, Inc.
Shelton, CT

The Qualitative andQuantitative Analysis of Sugars
and Erythritol by LC-ELSD
in Energy/Vitamin Drinks

Introduction

With the abundance of energy/
vitamin drinks to choose from,combined with an emphasis on
lower calories, more and moreindividuals are focusing on their
daily sugar intake before
consuming these drinks. With this consideration, this application highlights both the LC
separation of various sugars and erythritol (sugar alcohol) as well as the analysis ofthese components in a selection of three energy/vitamin drinks. As both sugars andsugar alcohols are inherently poor UV absorbers, refractive index (RI) has historically
been the detector of choice for these compounds. However, when using an RI detector,
scientists are limited by both sensitivity and the fact that it cannot be used with any
solvent gradients. By comparison, when using an Evaporative Light Scattering Detector(ELSD) for detection, researchers are afforded both greater sensitivity and the ability torun solvent gradients. This application takes particular advantage of the ELSD’s solvent
gradient flexibility. As the seven sugars and sugar alcohol to be analyzed have widelydiffering column retention, a solvent gradient was targeted to provide the optimal
separation in the least amount of time.

Experimental
Hardware/Software
A PerkinElmer Flexar Binary HPLC System (binary pump,autosampler, vacuum degasser, and column oven with anImtakt® 3 µm 250 x 3 mm Unison UK-Amino column) wasused for all separations. Detection was accomplished with
PerkinElmer's ELSD TSII. All instrument control, analysis, anddata processing was done via Chromera™ software.

Method Parameters
Table 1. HPLC Conditions
HPLC Conditions
ColumnImtakt 3 µm 250 x 3 mm Unison UK-Amino

Mobile Phase
Gradient

Equilibrate for 6 min. at 3:97 water/acetonitrile
Step 1: Hold for 7 min. at 3:97 water/acetonitrile
Step 2: Linear gradient to 25:75 water/acetonitrile in 10 min.
Step 3: Hold for 3 min. at 25:75 water/acetonitrile

Flow Rate

0.8 mL/min.

Oven Temp.

60 ºC

ELSD Conditions

Spray Chamber Temp.: 35 ºC
Drift Tube Temp.: 60 ºC

Injection Volume5 µL

Solvents, Standards, and Samples
All solvents and diluents used were HPLC grade.
All sugar and erythritol standards were obtained from SigmaAldrich®. Sugars included arabinose, xylose, fructose, mannose,glucose, sucrose, and maltose. All standard dilutions were madeusing 10:90 water/methanol. Methanol was used instead ofacetonitrile due to the solubility of the sugars. Even withmethanol, for the starting high concentration stock solution, thestandards were first dissolved in 5 mL of water before adding
45 mL of methanol.

Samples included three different energy/vitamin drinks purchased
at a local grocery store. All samples were first diluted 1/40 with
10:90 water/methanol and then filtered via 0.2 µm filters toremove small particles.

Results and Discussion
Figure 1 shows a 400 ppm standard (Std) chromatogram ofseven common sugars plus erythritol, a sugar alcohol, run under
the optimized conditions described above.
Using glucose and sucrose as examples, Figure 2 shows thecalculated linearity within a 25 – 400 ppm and 50 – 800 ppm

Figure 1. Chromatogram of standard solution containing erythritol and sevencommon sugars

concentration range, respectively. Both sugars were found to bequite linear within their ranges. It should be noted that the upper
concentrations were chosen to accommodate the expected highend sugar concentrations within the analyzed samples and that theactual upper limit of linearity may be higher.

Figure 2. Linearity plots of glucose (25 - 400 ppm concentration range) and sucrose (50 - 800 ppm concentration range)

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Subsequently, using the same chromatographic conditions, threeenergy/vitamin drinks were analyzed: Drink X, Drink Y, and Drink Z.
The results for Drinks X and Y are shown in Figure 3, eachoverlaid upon the 400 ppm standard chromatogram. Comparing
the chromatograms of these drinks, (highlighted in Figures 3a and3b), it can be observed that Drinks X and Y contain basically thesame three sugars, namely fructose, glucose (also known as
dextrose), and sucrose, with some minor differences in proportion.

Adding up the individual corrected sugar amounts for each drink
and converting the total from ppm to percent, the overall sugar
content for Drinks X and Y was determined to be 5.27 % and5.32 %, respectively. Both of these results were consistent withthe label claim of overall grams of sugar per bottle. Per label
claim, Drink X was calculated to contain 5.35 % sugar and Drink
Y was calculated to contain 5.41 % sugar.
The results for Drink Z are shown in Figure 4. The upper
chromatogram shows the drink injected by itself, with only one
prominent peak being observed at about five minutes. The lower
chromatogram shows the chromatographic results of 1/40 diluted
Drink Z spiked 1:1 with the 400 ppm sugar standard. The solitarypeak that appears in the 1/40 diluted Drink Z is seen to perfectly
overlay the erythritol peak in the standard. It should be noted that
although ribose elutes very close to erythritol, it was observed thatan erythritol standard spiked with an equal concentration ofribose resulted in a prominent shoulder on the upslope of theerythritol peak (data not shown). As the spiked Drink Z did notdisplay such a shoulder, this supported the identification of thecomponent in Drink Z as erythritol. This was also supported byDrink Z’s label claim, claiming erythritol as a significantcomponent. Interestingly, regarding Drink Z’s zero-calorie label
claim, as opposed to true sugars, a sugar alcohol, such as
erythritol, contributes no significant calories and qualifies as botha sugar-free and calorie-free component in foods and beverages.
Also, it should be pointed out that per label claim, Drink Z isreported to contain Reb A, a stevia plant extract, now being
increasingly introduced into products as an effective zero-calorie
sugar substitute. Reb A was not analyzed in this study.

Figure 3. a) Overlaid chromatogram of Drink X (in black) with 400 ppm standardchromatogram b) Overlaid chromatogram of Drink Y(in black) with 400 ppm
standard chromatogram

Based on standard calibrations, the quantitative results per injected
sample are shown in Table 2.
Table 2. Quantitative Results

Drink X:
Area

Amount (ppm)

Corrected Amounts
for 1/40 dilution(ppm)

Fructose

376704.35

231.139245.2

Glucose265239.31

213.748549.6

Sucrose706187.45

873.2334929.2

Area

Amount (ppm)

Corrected Amounts
for 1/40 dilution(ppm)

Fructose

167061.81

111.044441.6

Glucose228723.71

186.96

7478.4

Sucrose835912.45

1031.6141264.4

Component

Drink Y:
Component

Figure 4. Upper chromatogram: Drink Z, diluted 1/40 with 10% water in methanol;
Lower chromatogram: 1/40 diluted Drink Z spiked 1:1 with 400 ppm sugar standard

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Conclusion

References

This work has demonstrated the effective chromatographic
separation of seven sugars and erythritol using a PerkinElmer
Flexar/Chromera LC-ELSD system. The results exhibited very good
linearity over the tested concentration ranges. By choosing an
ELSD as the detector, advantage was taken of the ELSD’s inherent
compatibility with running solvent gradients, allowing for theoptimal separation of erythritol and all seven sugars in under18 minutes.

1. Unison UK-Amino application brochure (Imtakt, Anoka,MN, USA)

From a food quality perspective, there is an ever growing
emphasis on food monitoring. With this in mind, this work also
focused on the sugar/erythritol analysis of three energy/vitamin
drinks, identifying the sugars/sugar alcohol contained in eachdrink, as well as comparing the three drinks’ similarities anddifferences, both chromatographically and quantitatively.

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