Profiling Mono and Disaccharides in Milk and Infant Formula
Using the ACQUITY UPLC H-Class System and 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
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The ACQUITY QDa™ Detector provides
improved analytical selectivity by
combining both retention time and mass
analysis for compound identification.
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Information-rich data from different sugars
and sugar alcohols present in food products.
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The ACQUITY QDa Detector provides
complementary detection to Refractive
Index (RI) or Evaporative Light Scattering
(ELS) detectors that are commonly employed
for carbohydrate analysis.
WAT E R S S O LU T I O N S
ACQUITY UPLC H-Class System
®
ACQUITY QDa Detector
ACQUITY UPLC BEH Amide Column
Sep-Pak® C18 Cartridge
KEY WORDS
QDa, milk, infant formula, sugar,
carbohydrate, maltose, lactose,
fructose, glucose, sucrose,
myo-inositol, monosaccharide
INT RODUC T ION
Sugars and sugar alcohols (or sugar polyols) are classes of carbohydrates that are
natural constituents of foods and provide important nutritional benefits. 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, the need to better monitor sugar intake has grown in recent
years. Consequently there are now requirements to provide accurate information
about sugar content on food product labels in order to comply with increasingly
stringent regulatory demands.
The analysis of sugars and sugar alcohols is challenging because they lack
chromophores within their compound structures, and because of the close
similarity between the various molecules, many of which are simply isomers of
one another. Due to its separation power, accuracy, and speed of analysis, HPLC
has become the method of choice for the analysis of sugars.1,2 HPLC techniques
employ RI or ELS detection. RI detection requires careful control of the mobile phase
to avoid any changes through the analysis and therefore it requires isocratic
elution. With RI detection it is also difficult to change the mobile phase composition
from one analysis to the next because the RI detector may require several hours to
equilibrate 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 ELS often does not meet the sensitivity and selectivity
demands for the detection of sugars in complex food matrices.
An alternative is the use of a mass detector with electrospray ionization (ESI). The
Waters® ACQUITY QDa Detector offers the opportunity to decrease detection limits
as well as the ability to obtain mass spectral information on components in the
sample. This combination of chromatographic retention time and mass information
can provide improved selectivity for the profiling of sugars and sugar alcohols.
The ACQUITY QDa Detector is the only mass detector that has been completely
designed to be incorporated with an LC system. It fits in the LC stack, occupying
the same amount of space as a PDA detector. Extensive training is not required,
so users already familiar with HPLC can quickly take advantage of the improved
selectivity and sensitivity that mass detection affords. In this application note
we describe the use of the ACQUITY QDa Detector coupled to the ACQUITY UPLC
H-Class System for the profiling of sugars in milk and infant formulas.
1
Standard preparation
E X P E R IM E N TA L
UPLC conditions
System: ACQUITY UPLC H-Class
Runtime: 17.0 min
Column: ACQUITY UPLC BEH Amide
1.7 µm 2.1 X 150 mm
Column temp.:
35 °C
Mobile phase: 75:25 Acetonitrile: water
10mM in NH 4 HCO 3,
0.1% in NH 4OH
Flow rate: 0.13 mL/min
Injection volume: 0.7 µL
Individual 1000 mg/L stocks of the five food sugars: fructose, glucose, sucrose,
maltose, and lactose, along with myo-inositol were prepared 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 whole milk, a dairy-based infant formula, and a soy-based formula
were prepared as described by Chavez-Servin et al.3 A portion of the resulting
supernatants were subjected to a pass-through cleanup step described by
Chavez-Servin et al 3 using a Sep-Pak C18 Cartridge. A second portion was
also analyzed without the cleanup step and found to give equivalent results
(data not shown). Dilutions of all extracts were made (1:500 and 1:20) in
50:50 water:acetonitrile prior to injection.
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
Full scan:
50 to 450 m/z
SIR masses: See Table 1
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 +] -
Table 1. SIR m/z used for the monosaccharides,
disaccharides and myo-inositol.
Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY UPLC H-Class System and ACQUITY QDa Detector
2
R E S U LT S A N D D I S C U S S I O N
The separation of five common food sugars including two monosaccharides (glucose and fructose), three
disaccharides (sucrose, lactose, and maltose), and a sugar alcohol (myo-inositol) is shown in Figure 1. In order
to separate the disaccharides (lactose and maltose), an isocratic method was employed. 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 reportedly been used for
MS analysis of some sugars as the intensities of the chloride adducts can exceed the [M-H +] - of these analytes.4
The presence of chloride adducts in this work was from background chloride, which is ubiquitous in the
environment. The chloride adduct was also present for myo-inositol but at a lower response than m/z 179.
Sucrose showed two abundant ions: the deprotonated molecular ion at m/z 341 and a chloride adduct at
m/z 377. Lactose and maltose have dominant chloride adduct ions at m/z 377. The SIR traces at a concentration
of 20 ppm are shown as an overlay in Figure 1, with multiple responses apparent for the compounds that have
both the [M-H +] - and [M+Cl -] -.
Myo-inositol
[M-H+]m/z 179.2
Sucrose [M-H+]m/z 341.3
Fructose
[M+Cl-]m/z 215.1
Sucrose [M+Cl-]m/z 377
Glucose
[M+Cl-]m/z 215.1
Myo-inositol
[M+Cl-]m/z 215.1
Lactose
[M+Cl-]m/z 377.2
Maltose
[M+Cl-]m/z 377.2
Glucose
[M-H+]m/z 179.2
Fructose
[M-H+]m/z 179.2
7.0
8.0
9.0
10.0
Lactose
[M-H+]m/z 341.3
Maltose
[M-H+]m/z 341.3
11.0
12.0
13.0
14.0
15.0
16.0
17.0
Minutes
Figure 1. Chromatogram showing an overlay of multiple SIR channels (m/z 179, 215, 341, and 377) of a 20-ppm mixed standard
using an isocratic separation for the analysis of mono- and di-saccharides and sugar alcohols.
Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY UPLC H-Class System and ACQUITY QDa Detector
3
Myo-inositol is an important sugar alcohol that
is present at high levels in human breast milk.
Myo-inositol is supplemented into infant formula
to ensure that infants are able to receive equivalent
amounts from formula as compared to human
breast milk.5 Using this isocratic method,
myo-inositol shows a partial co-elution with lactose
at approximately 15 minutes. For RI or ELS detection
this would impact the accurate detection of both
lactose and myo-inositol. With mass detection,
however, this did not cause an issue as lactose and
myo-inositol have different molecular weights
which allowed us to assess them separately. This
is demonstrated in Figure 2 where the separate
SIR channels for myo-inositol and lactose are shown.
The separation of maltose and lactose is important
in some foods where lactose-free products are
manufactured as alternatives for consumers with
dairy intolerance or allergies. Maltose is also a
major carbohydrate in soy,6 which is often used as
a dairy substitute in lactose-free products. In order
to determine the presence or absence of lactose, it
must be separated from maltose, which has the same
molecular mass. Therefore, it is the combination of
the complementary selectivities of LC and MS that
enables compound identification. Figure 3 shows
the profiles of lactose and maltose in two different
infant formulas, along with a standard that contains
both lactose and maltose. As the chromatograms
show, lactose was present in the dairy-based infant
formula, with no detectable level of maltose. The
converse was true for the soy-based infant formula,
with no detectable level of lactose.
Sucrose
2A. m/z 377
Maltose
Myo-inositol
8.0
10.0
12.0
14.0
Lactose
2B. m/z 179
16.0
Figure 2A. SIR chromatogram of m/z 377 showing elution of
sucrose, maltose and lactose. 2B. SIR chromatogram of m/z 179
showing the elution of myo-inositol. Because it has a different
mass, myo-inositol can still be selectively analyzed using mass
detection, even though it partially co-elutes with lactose.
A. Mixed standard
Sucrose
Maltose
Lactose
B. Dairy-based infant formula
C. Soy-based infant formula
Lactose
Sucrose
Maltose
8.0
10.0
12.0
14.0
16.0
Figure 3. SIR chromatograms of m/z 377 in: A. Sugar standard at 20 ppm; B. Dairy-based
powdered infant formula, and C. Soy-based powdered infant formula.
Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY UPLC H-Class System and ACQUITY QDa Detector
4
C O N C LU S I O N S
References
The analysis of carbohydrates in food samples can be challenging
because of the mix of closely related UV-transparent compounds.
The combination of the ACQUITY UPLC H-Class System and the
ACQUITY QDa Detector offers scientists the advanced performance
expected of UPLC ® separations – high resolution, sensitivity, and
improved throughput, along with a complementary mass detector
to RI and ELS that provides the additional advantages of:
1. L C Nogueiraa, F Silvab, I M P L V O Ferreirab, L C Trugoa. Separation
and quantification of beer carbohydrates by high-performance liquid
chromatography with evaporative light scattering detection. J Chrom A.
1065 (2): 207-210, February 2005.
2. I M P L V O Ferreira, A M P Gomes, M A Ferreira. Determination of sugars,
and some other compounds in infant formulae, follow-up milks and
human milk by HPLC-UV/RI. Carbohydr Polym. 37 (3): 225-229, 1998.
3. J L Chávez-Servín, A I Castellote, M C López-Sabater. Analysis of
mono- and disaccharides in milk-based formulae by high-performance
liquid chromatography with refractive index detection. J Chrom A.
1043 (2): 211-215, 2004.
■■
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.
5. H E Indyk, D C Woollard. Determination of free myo-inositol in milk
and infant formula by high-performance liquid chromatography. Analyst.
119: 397-402, 1994.
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Reduced burden for method development since the baseline
separation of all components is not required.
6. M Kizito, E Iheanacho. Comparative Studies of the Nutritional Composition of
Soy Bean (glycine max) and Lima Bean (phaseolus lunatus). Scientia Africana.
9 (2): 29-35, 2010.
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The ability to deploy multiple methods on a single system that
can rapidly change between different method conditions.
4. E Rogatsky, H Jayatillake, G Goswami, V Tomuta, D Stein. Sensitive LC-MS
Quantitative Analysis of Carbohydrates by Cs+ Attachment. JASMS. 16 (11):
1805-1811, 2005.
Waters, ACQUITY UPLC, UPLC, Sep-Pak, and T he Science of W hat’s Possible are registered trademarks of Waters Corporation.
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