1

WAT E R S SO LU T IO NS

ACQUITY UPLC BEH Amide Column

ACQUITY UPLC H-Class System

ACQUITY QDa Detector

K E Y W O R D S

Sugars, glucose, fructose, polyols,

carbohydrate, maltose, inositol, cider,

wine, beer, QDa

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

When analyzing non-UV active sugars in

beverages, coupling the ACQUITY UPLC®

H-Class System with the ACQUITY® QDa®

Detector provides a number of benefits such as:

■■ Use of both retention time and

mass-to-charge (m/z) to increase

analytical specificity.

■■ Obtain information-rich data from

different sugars and sugar alcohols

present in food products.

■■ Provides a complementary detection method

to Refractive Index (RI) and Evaporative

Light Scattering (ELS) detectors that are

commonly used for carbohydrate analysis.

IN T RO DU C T IO N

Sugars and sugar alcohols (or sugar polyols) are classes of carbohydrates that

are important in human nutrition and natural constituents of foods. Some sugars

are added to processed foods in order to enhance flavor, or to mimic fresh food

products. With the increasing incidence of obesity and diabetes across the

developed world, interest in monitoring sugar intake has vastly increased in

recent years. Consequently there are now requirements to provide accurate

information on product labeling in order to comply with the increasingly stringent

regulatory demands.

The analysis of sugars and sugar alcohols remains a challenging application due

to the lack of chromophores within their compound structure and the similarity

between their molecules, many of which are simply isomers of one another.

Because of its separation power, accuracy, and speed of analysis, HPLC has

become the method of choice for the analysis of sugars.1,2 In order to detect these

compounds, HPLC techniques have employed Refractive Index (RI) or Evaporative

Light Scattering (ELS) detection. RI detection requires careful control of the

mobile phase to avoid any changes through the analysis and therefore demands

isocratic elution. It is also challenging to change the mobile phase composition

from one analysis to the next with RI detection as it may take hours to equilibrate the

detector when a different mobile phase composition is introduced. Even when a

new batch of the same mobile phase is introduced, small changes can be detected

by RI, resulting in baseline variation. ELS detection is more robust for mobile

phase composition changes but like RI, it often does not meet the sensitivity and

selectivity demands for the detection of sugars in complex food matrices.

An alternative to RI and ELS detection is the use of mass detection with

electrospray ionization (ESI). Mass detection complements traditional detectors

used for LC. It offers the opportunity to decrease detection limits and also to

obtain mass spectral information on the components in the sample. By combining both

chromatographic retention time and mass information, increased selectivity for

the analysis of sugars and sugar alcohols can be achieved in a much shorter time.

Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider Using the ACQUITY UPLC H-Class System and the ACQUITY QDa DetectorMark E. Benvenuti, Dimple Shah, and Jennifer A. BurgessWaters Corporation, Milford, MA, USA

2Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider

E X P E R IM E N TA L

UPLC conditions

LC system: ACQUITY UPLC H-Class

Runtime: 12.0 min with 15.0 min

equilibration

Column: ACQUITY UPLC BEH Amide

1.7 µm, 2.1 x 100 mm

Column temp.: 85 °C

Mobile phase A: Water, 0.1% NH4OH

Mobile phase B: Acetonitrile, 0.1% NH4OH

Flow rate: 0.25 mL/min

Injection volume: 0.7 µL

Gradient table:

Time %A %B Curve

0 10 90 6

12.0 40 60 6

12.01 10 90 6

MS conditions

MS system: ACQUITY QDa Detector

Ionization mode: ESI-

Capillary voltage: 0.8 V

Cone voltage 4.0 V

Probe temp.: 600 °C

Acquisition rate: 1 Hz

SIR masses: see Table 2

Full scan range: 50 to 850 m/z

The ACQUITY QDa is the only mass detector that has been completely designed

to be incorporated into a liquid chromatography system. It fits onto the LC stack,

occupying the same space as a PDA detector. Extensive training is not required so

users already familiar with HPLC can immediately make use of the selectivity and

sensitivity that mass detection affords. In this application note we describe the

profiling of sugars in orange juice, wine, beer, and cider using the ACQUITY QDa

Detector coupled to ACQUITY UPLC H-Class System.

Table 1. SIR m/z used for the monosaccharides, disaccharides, and myo-inositol.

Compound SIR (m/z)

Fructose 215.1 [M+Cl–]–

Glucose 215.1 [M+Cl–]–

Myo-Inositol 179.2 [M-H+]–

Lactose 377.2 [M+Cl–]–

Maltose 377.2 [M+Cl–]–

Sucrose 341.3 [M-H+]–

Standard preparation

Individual 1000 mg/L stocks of the food sugars, fructose, glucose, sucrose, and

maltose, along with myo-inositol were made in water. From these, a 50 mg/L

mixed stock was prepared in 50:50 water:acetonitrile.

This stock was further diluted as necessary in 50:50 water:acetonitrile to

determine retention times for the analytes.

Sample preparation

Samples of orange juice, hard cider, wine, and beer were purchased locally.

The orange juice was centrifuged at an rcf of 2465 g for 30 mins. at room

temperature to remove pulp. Two dilutions of the supernatant (1:100, 1:1000)

were made in 50:50 water-acetonitrile. The beer samples were degassed by

sonication, and filtered through a 0.2 micron PVDF filter, along with the hard cider

and wine. Different dilutions were made in 50:50 water:acetonitrile. The hard

cider and wine were diluted 1:100, and the beer was diluted 1:2.

3Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider

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

The separation of common food sugars including two monosaccharides (glucose and fructose), three

disaccharides (sucrose, maltose, and lactose), and a sugar alcohol (myo-inositol) is shown in Figure 1. Multiple

masses were monitored for each of the carbohydrates. Fructose, glucose, and myo-inositol all have a molecular

mass of 180. Using ESI, myo-inositol forms a deprotonated molecular ion with m/z 179. The most abundant

ion for fructose and glucose is the chloride adduct [M+Cl–]– at m/z 215. Such chloride adducts have been

previously reported to be used for MS analysis of some sugars as the intensities of the chloride adducts can

exceed the [M-H+]– of these analytes.3 The chloride adduct is also present for myo-inositol but at a lower

response than m/z 179. Sucrose shows two abundant ions, the deprotonated molecular ion at m/z 341 and

chloride adduct at m/z 377. Maltose has a dominant chloride adduct ion at m/z 377.

As can be observed in Figure 1, sucrose and myo-inositol show partial co-elution, but their different masses

allow them to be easily distinguished using mass detection. Sucrose (m/z 341) elutes slightly earlier than

myo-inositol (m/z 179 and m/z 215). For other, less selective detectors, further method development would

be required in order to separate these analytes. With the ACQUITY QDa Detector, sucrose and myo-inositol

can easily be distinguished without further method development.

1- Fructose 2- Glucose 3- Sucrose 4- Myo-inositol 5- Maltose 6- Lactose

5,6

4 3

2

1

3.0 2.0 5.0 7.0 9.0 4.0 6.0 8.0 Minutes

m/z 215

m/z 215 m/z 341 m/z 377

m/z 179

m/z 377 m/z 215

Figure 1. Chromatogram showing an overlay of multiple SIR channels (m/z 179, 215, 341, and 377) for the analysis of mono- and disaccharides and sugar alcohols using a gradient separation.

4Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider

This method was used to analyze the sugars present in a number of different beverage samples. The sugar

profile of an orange juice sample is shown in Figure 2. Here we show that there are five major peaks that eluted

with the SIR channels used to monitor the sugars and sugar alcohol. Peaks 1 and 2 correspond to fructose

and glucose, respectively (apparent in SIR m/z 215, with a small response at m/z 179). Peak 4 (m/z 341)

corresponds to sucrose, and peak 5 corresponds to myo-inositol (m/z 179). Peak 3 does not correspond to the

retention time of any of the standards used in this application.

i2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

1 2

3 5

4

Peak #

Analyte RT

1 Fructose 3.2

2 Glucose 4.3

3 Unknown 6.0

4 Sucrose 6.7

5 Myo-Inositol 6.8

B. m/z 215

A. m/z 179

B. m/z 341

Figure 2. SIR chromatograms of: A. m/z 179, B. m/z 215, and C. m/z 341 for the analysis of mono- and disaccharides and sugar alcohols in an orange juice sample. The vertical axis has been scaled to show responses relative to the largest peak (sucrose m/z 341). Labeled peaks and their identity and retention time are displayed in the table inset.

5Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider

Wine, beer, and cider samples were analyzed using the gradient method and the profiles of monosaccharides

(using SIR channels of m/z 179 and 215), shown in Figure 3. The main sugars present in wine were glucose

and fructose. Residual amounts were detected in the finished wines, with higher levels of fructose in many

cases.4 Both fructose and glucose were detected in the two moscato wines (white and pink) and the cabernet

sauvignon. Myo-Inositol was also detected in the wine samples. Sucrose and maltose were not detected in the

wines (data not shown).

Figure 3: Overlay of SIR chromatograms at m/z 215 (A-G) and m/z 179 (H-N) for the beverage samples and a mixed standard at 5 ppm.

Fructose, glucose Myo-inositol

H. Pink moscato

L. Black lager

K. Pale ale

M. Hard cider

J. Cabernet sauvignon

I. White moscato

N. 5 ppm standard

A. Pink moscato

E. Black lager

D. Pale ale

F. Hard cider

C. Cabernet sauvignon

B. White moscato

G. 5ppm standard

During the beer brewing process, soluble sugars such as maltose, glucose, and maltotriose are enzymatically

produced from the malt starches. These sugars are subsequently converted to alcohol through the yeast

fermentation process.5 Carbohydrates that are detected in finished beers either come from residual sugars that

were not consumed in the fermentation process, or those that were added during a secondary fermentation

process to produce additional CO2.5 These residual sugars contribute to the sweetness of beer and they

typically include combinations of fructose, glucose, sucrose, maltose, and maltotriose.

As shown in Figure 3, there were no detectable levels of glucose in the beer samples, and levels of fructose

were extremely low. An example of a residual disaccharide detection is shown in Figure 4 for sucrose in the

pale ale sample. The SIR chromatogram shows the sucrose peak (m/z 341) and spectrum from the simultaneous,

full-spectrum analysis, which is shown with the spectrum from the sucrose standard. The black lager did not

show an equivalent peak. Myo-Inositol was also detected in the beers and cider.

6Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider

341

342 377

341

342377 404

A. standard

B. Pale ale

C. standard

D. Pale ale

Figure 4. SIR chromatogram of m/z 341 in a mixed standard (A), and the pale ale beer sample (B). Spectra summed at the retention time of the major peak are shown in C and D for the standard and beer, respectively. The major ion in the spectrum (m/z 341) corresponds to the [M-H+] –.

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

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

©2014 Waters Corporation. Produced in the U.S.A. July 2014 720005102EN AG-PDF

CO N C LU S IO NS

The analysis of carbohydrates in food samples can be a challenging

task given the mix of closely related UV-transparent compounds.

With its high separation power, LC has become the method

of choice, offering improvements in speed over other techniques.

RI and ELS detection have been widely adopted in some

laboratories. The combination of the ACQUITY UPLC H-Class

System with the ACQUITY QDa Detector offers scientists a

complementary detection system with the additional

advantages of:

■■ Improved analytical selectivity by combining both retention

time and mass analysis for compound identification.

■■ Detection of UV-transparent molecules using a sensitive and

selective detector.

■■ The ability to discriminate between co-eluting components

using their mass-to-charge ratios.

■■ Reduced burden for method development since baseline

separation of all components is not required.

■■ The ability to deploy multiple methods on a single system that

can rapidly change depending on method conditions.

References

1. LC Nogueiraa, F Silvab, I M P L V O Ferreirab, LC Trugoa. Separation and quantification of beer carbohydrates by high-performance liquid Chromatography with evaporative light scattering detection. J Chrom A. (2005) 1065, 2: 207–210.

2. IMPLVO Ferreira, AMP Gomes, MA Ferreira. Determination of sugars, and some other compounds in infant formulae, follow-up milks and human milk by HPLC-UV/RI. Carbohydr Polym. (1998) 37, 3: 225–229.

3. E Rogatsky, H Jayatillake, G Goswami, V Tomuta, D Stein. Sensitive LC-MS Quantitative Analysis of Carbohydrates by Cs+ Attachment. JASMS. (2005) 16, 11: 1805–1811.

4. NJ Berthels, RR Cordero Otero, FF Bauer, JM Thevelein, and IS Pretorius. Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains. FEMS Yeast Research. (2004) 4, 7: 683–689.

5. ED Baxter and PS Hughes. Beer: Quality, Safety and Nutritional Aspects. The Royal Society of Chemistry. (2001).