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WAT E R S SO LU T IO NS

ACQUITY UPLC® H-Class System

ACQUITY QDa Detector

ACQUITY UPLC BEH Amide Column

Sep-Pak® C18 Cartridge

K E Y W O R D S

QDa, milk, infant formula, sugar,

carbohydrate, maltose, lactose,

fructose, glucose, sucrose,

myo-inositol, monosaccharide

A P P L I C AT IO N B E N E F I T S■■ The ACQUITY QDa™ Detector provides

improved analytical selectivity by

combining both retention time and mass

analysis for compound identification.

■■ Information-rich data from different sugars

and sugar alcohols present in food products.

■■ The ACQUITY QDa Detector provides

complementary detection to Refractive

Index (RI) or Evaporative Light Scattering

(ELS) detectors that are commonly employed

for carbohydrate analysis.

IN T RO DU C T IO N

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.

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

2Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY UPLC H-Class System and ACQUITY QDa Detector

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 NH4HCO3,

0.1% in NH4OH

Flow rate: 0.13 mL/min

Injection volume: 0.7 µL

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.

Standard preparation

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 al3 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.

3Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY UPLC H-Class System and ACQUITY QDa Detector

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

Fructose [M+Cl-]- m/z 215.1

Sucrose [M-H+]- m/z 341.3

Myo-inositol [M-H+]-

m/z 179.2

Myo-inositol [M+Cl-]- m/z 215.1

8.0 10.0 12.0 14.0 16.0 7.0 9.0 11.0 13.0 15.0 17.0 Minutes

Sucrose [M+Cl-]- m/z 377

Fructose [M-H+]- m/z 179.2

Glucose [M-H+]- m/z 179.2

Glucose [M+Cl-]- m/z 215.1

Maltose [M+Cl-]- m/z 377.2

Maltose [M-H+]- m/z 341.3

Lactose [M+Cl-]- m/z 377.2

Lactose [M-H+]- m/z 341.3

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.

4Profiling Mono and Disaccharides in Milk and Infant Formula Using the ACQUITY UPLC H-Class System and ACQUITY QDa Detector

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.

2B. m/z 179

2A. m/z 377 Sucrose

Maltose Lactose

Myo-inositol

8.0 10.0 12.0 14.0 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.

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.

Sucrose

Maltose Lactose

A. Mixed standard

B. Dairy-based infant formula

C. Soy-based infant formula

Lactose

Maltose

Sucrose

8.0 10.0 12.0 14.0 16.0

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

©2014 Waters Corporation. Produced in the U.S.A. April 2014 720005035EN AG-PDF

CO N C LU S IO NS

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:

■■ 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 the baseline

separation of all components is not required.

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

can rapidly change between different method conditions.

References

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.

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.

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.

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.