Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider
Using the ACQUITY UPLC H-Class System and the ACQUITY QDa Detector
Mark E. Benvenuti, Dimple Shah, and Jennifer A. Burgess
Waters Corporation, Milford, MA, USA
A P P L I C AT I O N B E N E F I T S
INT RODUC T ION
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:
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.
■■
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.
WAT E R S S O LU T I O N S
ACQUITY UPLC BEH Amide Column
ACQUITY UPLC H-Class System
ACQUITY QDa Detector
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.
KEY WORDS
Sugars, glucose, fructose, polyols,
carbohydrate, maltose, inositol, cider,
wine, beer, QDa
1
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% NH 4OH
Mobile phase B:
Acetonitrile, 0.1% NH 4OH
Flow rate: Injection volume: 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 –] –
0.25 mL/min
Myo-Inositol
179.2 [M-H +] –
0.7 µL
Lactose
377.2 [M+Cl –] –
Maltose
377.2 [M+Cl –] –
Sucrose
341.3 [M-H +] –
Gradient table:
Time %A
%B
Curve
01090 6
12.040 60
6
12.0110
90
6
MS conditions
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.
MS system: ACQUITY QDa Detector
Ionization mode: ESI-
Capillary voltage: 0.8 V
Cone voltage
4.0 V
Sample preparation
Probe temp.: 600 °C
Acquisition rate:
1 Hz
SIR masses: see Table 2
Full scan range: 50 to 850 m/z
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.
This stock was further diluted as necessary in 50:50 water:acetonitrile to
determine retention times for the analytes.
Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider
2
R E S U LT S A N D D I S C U S S I O 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
123456-
m/z 215
3 4
Fructose
Glucose
Sucrose
Myo-inositol
Maltose
Lactose
m/z 341
m/z 377
m/z 179
2
5,6
m/z 215
m/z 215
m/z 377
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
Minutes
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.
Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider
3
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.
Peak
#
Analyte
RT
1
Fructose
3.2
2
Glucose
4.3
3
Unknown
6.0
4
Sucrose
6.7
5
Myo-Inositol
6.8
2.00
3.00
4.00
A. m/z 179
3
5.00
6.00
5
7.00
8.00
9.00
B. m/z 215
2
1
2.00
3.00
4.00
5.00
6.00
i
7.00
4
2.00
3.00
4.00
5.00
6.00
Minutes
7.00
8.00
9.00
B. m/z 341
8.00
9.00
Figure 2. SIR chromatograms of: A. m/z 179, B. m/z 215, and C. m/z 341 for the analysis of monoand 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.
Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider
4
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).
Fructose, glucose
Myo-inositol
A. Pink moscato
B. White moscato
C. Cabernet sauvignon
D. Pale ale
H. Pink moscato
I. White moscato
J. Cabernet sauvignon
K. Pale ale
E. Black lager
L. Black lager
F. Hard cider
M. Hard cider
G. 5ppm standard
N. 5 ppm standard
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.
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 CO 2.5 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.
Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider
5
A. standard
C. standard
341
342
B. Pale ale
D. Pale ale
377 404
341
342 377
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 +] –.
Profiling Mono and Disaccharides in Juice, Wine, Beer, and Cider
6
C O N C LU S I O N S
References
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:
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.
■■
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.
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).
Waters, ACQUITY, QDa, ACQUITY UPLC, and T he Science of W hat’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
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com