Artificial Sweeteners Analysis

A METHOD FOR THE SIMULTANEOUS
DETERMINATION OF SWEETENERS IN
FOODS
Steve Appleton
Durham Scientific Services

FOOD STANDARDS AGENCY
Proposal: Develop a fully validated
method for the simultaneous
determination of sweeteners in
foods

Permitted sweeteners
Sweeteners in Food Regulations 1995
• Acesulfame K (E950) ~ 25-2000ppm
• Aspartame (E951) ~ 25-6000ppm
• Saccharin (E954) ~ 80-3000ppm
• Sucralose (E955) ~ 10-3000ppm
• Neohesperidine dihydrochalcone (NHDC)
(E959) ~ 10-400ppm

Permitted sweeteners
Sweeteners in Food Regulations 1995
• Cyclamate (E952)
• Thaumatin (E957)
• Aspartame-Acesulfame salt (E962)

Methodology
Sweeteners are a group of chemically unrelated
compounds.
• High Performance Liquid Chromatography
• UV detection is suitable for compounds
possessng a chromophore such as acesulfame
K, saccharin, aspartame and NHDC.
• Refractive index detection is suitable for
sucralose.
• Cyclamate can be derivatised to make it UV
active or can undergo postcolumn reaction with
fluorescence detection.

Other methods
Methodology
• Ion chromatography with pulsed
amperometric detection
• Capillary electrophoresis
• Enzymatic
• Ion selective electrode
• Spectrophotometry
• Flow injection analysis

Methodology
• The European Organisation for
Standardisation (CEN) has published six
validated methods with respect to intense
sweetener determination.
• Standard method BS EN 12856, Foodstuffs
– Determination of acesulfame K,
aspartame and saccharin – High
performance liquid chromatographic
method.

Methodology
Sample preparation
• Extraction into water, aqueous solutions or
organic solvents.
• Clean samples can be analysed directly or filtered
before analysis.
• More complicated matrices:
• clarified using Carrez solutions to precipitate
proteins and fatty acids.
• use of solid phase extraction (SPE), typically
C18 since colourings, flavourings and fat cannot
be separated by Carrez clarification.

Evaporative Light Scattering
Detector (ELSD)
Gaining popularity as a universal detector.
Simple three step process
• Nebulisation – column effluent forms a dispersion
of droplets
• Evaporation – mobile phase is evaporated, leaving
a fine mist of dried sample.
• Detection – laser light scattered by sample particles
is detected, generating an electrical signal.

Pro
Evaporative Light Scattering
Detector (ELSD)
• since all particles scatter light, all sample
components are detected, regardless of their
structure or optical properties.
Con
• method limitations as it can only be used with
volatile mobile phases and mobile phase
modifiers

ELSD

ELSD plus UV

Food matrices examined
• Yogurt – natural, low fat
Development
• Jam – raspberry, seedless
• Mayonnaise
• Chocolate cake
• Cola drink
• Samples spiked with sucralose, acesulfame K,
saccharin, aspartame and NHDC, each at 100ppm.

Variables
Development
• Extraction solvent
Variation of water: IDA ratio
Too much IDA gives poor peak shape
Too little IDA gives poor recoveries for
NHDC and aspartame
Optimum 75:25 (by volume) water: IDA

Development
• Clarification using Carrez reagents
• pH
Use has little effect on chromatography.
Use gives much lower NHDC recoveries.
Little variation in recoveries over the
range 3-8 except for aspartame.
Aspartame most stable at pH 4, at pH 7
decomposition is rapid.

Working Method
Extraction
Liquid samples
• Measure 50mL of sample into a 100mL
volumetric flask and add 20mL of IDA. Make up
to volume with water and mix well.
Solid samples
• Weigh 20g of sample into a jar. Add 80mL of
75:25 water: IDA and mix well by hand. Place the
jar on a mechanical shaker and shake vigorously
for 30 minutes.

Working Method
Solid samples (continued)
• Transfer the solution to a centrifuge tube and
centrifuge at 3000 rpm for 15 minutes. Filter the
supernatant liquid through a Whatman GF/A filter
paper or similar. Take a portion of the filtrate and
pass through a syringe filter into a 2mL glass vial
ready for HPLC analysis.

Working Method
HPLC with UV Diode Array Detection
Acesulfame K, Saccharin, Aspartame, NHDC
Column : ACE 5 C18 250 x 4 mm 5µm
Guard column : ACE 5 C18 10 x 4 mm 5µm
Column temperature : 30°C,
Flow :
1.0 mL/min
Stop time : 40 mins
Solvent : Time (min) 0 25 30 35
A : Water 88% 50% 30% 88%
B : Acetonitrile 2% 40% 60% 2%
C : Methanol 0% 0% 0% 0%
D :1% TFA 10% 10% 10% 10%
Injection volume : 5µL
Detector : Acesulfame K 231nm, Saccharin 231nm, Aspartame 215nm,
NHDC 280nm.

Working Method
HPLC with Refractive Index Detection
Sucralose
Column : Hypersil ODS 250 x 4 mm 5µm
Guard column : Hypersil ODS 4 x 4 mm 5µm
Column temperature : 30°C
Flow :
1.5 mL/min
Stop time :
15 mins
Solvent :
Premixed 20:80 methanol:water
Injection volume : 100µL

Mean % Recoveries
Yogurt Jam Mayo Cake Cola
Sucralose 95 108 108 86 101
Ace K 93 108 110 108 105
Saccharin 86 107 109 88 105
Aspartame 90 125 113 55 104
NHDC 72 108 109 75 107