FOOD & BEVERAGE

Analysis of Artificial Sweeteners
by HILIC–MS Method
Jonas Henschel1, Alexander Schriewer1, Heiko Hayen1, andWen Jiang2, 1Institute of Inorganic and Analytical Chemistry,
University of Münster, Münster, Germany, 2HILICON AB
In recent decades, the use of artificial sweeteners greatly increased.
As sugar substitutes they have become popular in today’s calorieconscious society. The most commonly used artificial sweeteners
in beverages and food are acesulfame, aspartame, cyclamate,neohesperidin dihydrochalcone (NHDC), neotame, saccharin,and sucralose. Some of them show resistance to wastewater
treatment and distribute in the aquatic environment as emerging
contaminants. In addition, concerns about health risks from theartificial sweeteners have been raised as well (1–3).
The analytical challenge is to simultaneously determine the artificial
sweeteners with diverse physicochemical properties. Considering
the polar and hydrophilic nature of these compounds, hydrophilicinteraction liquid chromatography (HILIC) should be considered as analternative to commonly used reversed-phase liquid chromatography.
In this application, we used one iHILIC-Fusion(+) column packedwith charge modulated hydroxyethyl amide silica, which combineshydrophilic partitioning, weak electrostatic interactions, and hydrogenbonding in HILIC separation.

Figure 1: LC–MS–MS chromatograms of seven artificial sweetenerstandards with iHILIC-Fusion(+).

artificial sweeteners in the diet cola of a well-known brand. After asimple and fast sample preparation, we can identify acesulfame,cyclamate, and aspartame in the tested diet cola (Figure 2).

Experimental

LC–MS–MS system: Advance UHPLC with a CTC PAL autosampler
and Bruker EVOQ™ Elite triple quadrupole system withelectrospray ionization; multiple reaction monitoring mode (MRM)
in ESI(+) and ESI(-) was used for selective and sensitive detection,
see details in Table 1.
Column: 150 × 2.1 mm, 3.5-µm 100Å iHILIC-Fusion(+)(P/N 100.152.0310, HILICON AB, Sweden)
Gradient elution: A) acetonitrile; B) 10 mM ammonium formate,
pH = 3.5; gradient elution from (95/5) A/B to (84/16) A/B in 8.5 min.
Flow rate: 0.3 mL/min
Column temperature: 40 °C
Injection volume: 5 µL
Sample preparation: details on request

Results and Conclusion

Seven artificial sweetener standards can be simultaneously
determined by the combination of iHILIC-Fusion(+) HILIC column
and EVOQ™ Elite triple quadrupole MS–MS in a 12 min run, as
shown in Figure 1. The developed method was verified to detect

Table 1: Precursor ions, product ions, and corresponding
collision energy parameters for MS–MS detection of artificial
sweeteners.
AnalytePrecursor ionsm/z [M-H]-Product ions
(P1/P2/P3) m/z

Collision energy[V]-11/-27/-46

Acesulfame

162.2

82.2/78.1/64.2

Cyclamate

178.2

80.1

-24

Saccharin182.2

42.3/106.0-22/-17

NHDC

611.2

303.0/166.0/125.0-43/-59/-45

442.6(+formate)396.9/361.0/359.0-6/-9/-9

Sucralose

Figure 2: LC–MS–MS chromatograms of diluted and degassed dietcoke with iHILIC-Fusion(+).

This work describes an efficient HILIC–MS method for separation anddetection of artificial sweeteners. It can be further developed to a routine
analysis method with a combination of HILIC and aerosol-based detectors(ELSD, NQAD, and CAD) and used in different application areas.

References

M. Scheurer, H.J. Brauch, and F.T. Lange, Anal. Bioanal. Chem. 394,1585–1594 (2009).
(2) F.T. Lange, M. Scheurer, and H.J. Brauch, Anal. Bioanal. Chem. 403,2503–2518 (2012).
(3) R.A. Lorenzo, M.T. Pena, P. Fernandez, P. Gonzalez, and A.M. Carro, FoodControl 47, 43–52 (2015).
(1)

2

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[M-H]+Aspartame

295.3

120.1/180.0/235.0+23/+12/+11

Neotame379.5

172.1/319.1/120.1+21/+14/+31

THE APPLICATIONS BOOK – JULY 2016