KMITL Sci. J. Vol.8 No.

2 (Section B)

July December, 2008

IDENTIFICATION AND DETERMINATION METHODS OF

SYNTHETIC SWEETENER, ASPARTAME

Khesorn Nantachit1 , Somporn Putiyanan1 and Prapart Phoowiang1

1

Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University,

Chiang Mai, 50200.

ABSTRACT
Aspartame is the derivative of dipeptide which is L-aspartyl-L-phenylalanine methyl ester. (-) Aspartame is
a synthetic sweetener which is expensive and it can isomerise to be racemate form rapidly if it is kept
unproperly. Now-a-days there is no report about toxicity of racemate form of aspartame it may be toxic like
racemate form of thalidomide. The aim of this study was to search for identification and determination
methods of synthetic sweetener, aspartame. Identification methods were tested with ninhydrin solution and
tested with alkaline hydroxylamine solution by using thin layer chromatogram (TLC). TLC were
developed with acetonitrile phosphate buffer. Testing with ninhydrin solution was sensitive (limit of
detection was 7.5 g) but was not specific because this method was used to detect amino acid having a free
-amino group. Testing with alkaline hydroxylamine solution (limit of detection was 300 g) was specific
method because it was used to detect carbonyl functional group and formed brown color complex with
ferric chloride. The determination methods were colorimetric method (reaction with ninhydrin solution was
the same as identification method) and high pressure liquid chromatographic method (HPLC). The relative
standard deviation of colorimetric method of simple sugar samples was 5.20-17.29% and HPLC method
was 2.06-11.7% and the percent recovery of these two methods were about 97%. Aspartame was found
lower than the label amount in many samples and the percent relative standard deviations were large
because aspartame was a chiral compound which could be destroyed by air, light, heat and moisture if it
was kept unproperly. These three analytical methods can be used to investigate the use of aspartame in
Thailand in order to stimulate Ministry of Public Heath to remind the use of aspartame.
KEYWORDS : Identification, Determination, Aspartame

1. INTRODUCTION1-11

Synthetic sweeteners were used in diabetes patients or to control body weight in obese persons. They were
also used to reduce manufacturing cost. They gave sweetness but did not have calorie. They may be
harmful to the applicants such as cyclamate which is the carcinogenic agent.
Aspartame is the derivative of dipeptide which is L-aspartyl-L-phenylalanine methyl ester. It has
been used in United States since 1981. Aspartame is metabolized to be phenylalanine, aspartic acid and
methanol.4,5 (as shown in figure 1) It is forbidden for the person who lacked of enzyme phenylalanine
hydroxylase. In this case phenylalanine is changed to be phenylpyruvate and phenylacetate which cause
the mental deterioration. Some people who took aspartame might have neurological complaints such as
headache, depression and insomnia. Aspartame cannot be used in old diabetic patients because these
persons begin to be dementia and have visual deterioration problem.
(-) Aspartame is a synthetic sweetner which is expensive and it can isomerise to be racemate form
rapidly if it is kept unproperly. (as shown in figure 2 and 3) There is no report about toxicity of racemate
form of aspartame it may be toxic like racemate form of thalidomide. ADI (acceptable daily intake) of
aspartame followed FAO/WHO is 40 mg/Kg. The amount of aspartame in simple sugar sample was 20-38
mg/sachet so it may be harmful if it is taken large amount everyday. From this information it is important
to search for identification and determination methods of aspartame to investigate its use in order to
stimulate the Ministry of Public Health to be interested in this problem.

Corresponding Author. Email: khesornn@pharmacy.cmu.ac.th

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KMITL Sci. J. Vol.8 No.2 (Section B)

O
CH3

C
H

H
N

July December, 2008

H NH2
CO2H
O
Aspartame

Metabolise

COO
CO2H

NH3
NH2
Phenylalanine

O
C OH

Aspartic acid

CH3OH
Methanol

Figure 1: Metabolic pathway of aspartame

Figure 2: UV spectrum of standard (-) aspartame 0.1% in 95% ethanol

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KMITL Sci. J. Vol.8 No.2 (Section B)

July December, 2008

Figure 3: UV spectrum of racemate form of aspartame 0.05% in 95% ehtanol

3. MATERIAL AND METHOD

]
Materials
1. Ninhydrin solution 1.0 g of ninhydrin was dissolved in 50 ml of 95% ethanol and 10 ml of
glacial acetic acid
2. Alkaline hydroxylamine solution
Solution A Hydroxylamine hydrochloride 12.5 g were dissolved in methanol and were diluted
to 100 ml with the same solvent.
Solution B Sodium hydroxide 12.5 g were dissolved in methanol and were diluted to 100 ml
with same solvent. Solution A and B were mixed immediately before use.
3. Ferric chloride solution Ferric chloride 10.5 g were dissolved in 95% ethanol and were
diluted to 100 ml with the same solvent.
4. Acetonitrile phosphate buffer 10 volumes of acetonitrile plus 90 volumes of 6.8 g/l solution
of potassium dihydrogen phosphate and were previously adjusted to pH 3.7 with phosphoric acid
Instrument
1. Shimadzu UV 2450
2. HPLC, Agilant 1100 diode array detector, Water Sperisorb 5 m ODS 2 particle size and 4.6 x
250 mm. long.
Methods
1. Identification methods
1.1 Test with ninhydrin solution1,3,4,6
The samples were dissolved in water and spotted on thin layer chromatogram (TLC). TLC was
developed in acetonitrile phosphate buffer, was dried and sprayed with ninhydrin solution and warmed in
hot air oven. Aspartame gave pink spot. Limit of detection of this method was 7.5 g.

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KMITL Sci. J. Vol.8 No.2 (Section B)

O
CH3

H
N

C
H

July December, 2008

H NH2

CO2H

C
C O

O
C
O
Aspartame

Ninhydrin
Heat

O
C

O
CH3

C
H

Figure 4: Chemical reaction of ninhydrin test

H
N

N C
CO2H

C
O

Pink color

1.2 Test with alkaline hydroxylamine solution4,5

Thin layer chromatogram of sample aspartame was developed in the same solvent as ninhydrin
method. TLC was dried, sprayed with alkaline hydroxylamine and followed with hydrochloric acid (1+1)
and dried. Ferric chloride solution was sprayed into TLC. Positive test was brown spot. Limit of detection
of this method was 300 g.

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KMITL Sci. J. Vol.8 No.2 (Section B)

O
CH3

H NH2

H
N

July December, 2008

CO2H
O
Aspartame

Alkaline hydroxylamine
solution

O
H3C

H NH2

H
N

NH
NH

ONa

(1) Spray with HCL (1+1) and dry

(2) Spray with ferric chloride solution

O
H3C

C
H

H NH2

H
N
NH
Fe

HN

OH

Cl
Brown color

Figure 5: Chemical reaction of alkaline hydroxylamine solution test

2.

Determination methods
2.1 Colorimetric method1,3,4,6
Test with simple sugar samples, 1 g of sample was dissolved with 40.0 ml distilled water and was
diluted to 100.0 ml with absolute ethanol. 1.0 ml of acetate buffer was mixed with 1.0 ml sample solution
and 2.0 ml ninhydrin solution. The mixture was heat in boiling water bath for 8 minutes after that the
mixture was diluted to 10.0 ml with absolute ethanol. The absorbance of sample solution was measured at
406 nm.
The reaction time of heating step was at 8-10 minutes. Sample solution gave two maximum
absorption wavelength which were 406 and 557 nm. The maximum absorption wavelength at 406 was
stable so the absorbance of sample was measured at this wavelength. The purple color of sample solution
remained stable for at least one and a half hours. The linearity of calibration curve was 7.5-37.5 g/ml.
2.2 HPLC method5
The used HPLC method was followed British Pharmacopoeial (2008) method. HPLC (Agilant
1100), diode array detector was used at 214 nm, the column used was Water Sperisorb 5 m ODS 2 particle
size and 4.6 x 250 mm long. The mobile phase was acetonitrile in phosphate buffer (the same solvent as in
ninhydrin identification method) and the flow rate was 1.0 ml/min. (Limit of detection was 7 g) 25 l of

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KMITL Sci. J. Vol.8 No.2 (Section B)

July December, 2008

standard and sample solutions were injected in replicates. Retention time was about 6 minutes as shown in
figure 6 and 7.

Figure 6: Chromatogram of standard racemate form of aspartame 200 ppm eluted with acetonitrile
phosphate buffer. (retention time 6.339 minutes, peak area 1767.38135 mAU.)

Figure 7: Chromatogram of aspartame in sugar sample eluted with acetonitrile phosphate buffer (retention
time 6.391 minutes, peak area 1471.91687 mAU.)

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KMITL Sci. J. Vol.8 No.2 (Section B)

July December, 2008

Colorimetric
HPLC
Sample 2
3.8
Colorimetric
HPLC
Sample 3
3.6
Colorimetric
HPLC
Sample 4
0.5
Colorimetric
HPLC
% LA = % label amount

3.03
3.12
2.13
4.09
3.51
3.82
0.37
0.38

2.75
3.04
2.18
4.16
3.35
3.55
0.36
0.36

2.33
3.08
2.08
3.50
3.20
3.74
0.36
0.38

3.23
3.00
2.25
3.17
3.14
3.67
0.42
0.31

2.12
2.94
2.44
3.30
3.14
3.80
0.37
0.29

Table 2: % Recovery of sugar sample No.1

Method
Amount added (ppm)
Colorimetric
200

HPLC

Amount found (ppm)

156.13
226.38
209.20
205.30
179.55
Mean
195.58
194.85
193.84
194.78
192.35
Mean

200

2.69
3.04
2.22
3.64
3.27
3.72
0.38
0.34

0.46
0.06
0.14
0.41
0.17
0.10
0.02
0.04

% RSD

SD

Method

3.8

Calculated % LA

Mean

Sample 1

%LA

SamplesSugar

Results and Discussion

Table 1: Assay results of simple sugar samples

17.29
2.06
6.31
11.17
5.20
2.64
5.26
10.81

% Recovery
78.06
113.19
104.60
102.65
89.77
97.65
97.79
97.43
96.92
97.39
96.18
97.14

Identification method1,6 that tested with ninhydrin solution was sensitive but was not specific for
aspartame because the detection limit was 7.5 g and it was used to detect for amino acids having a free amino group. The method that tested with alkaline hydroxylamine6 solution was not sensitive but it was
specific for aspartame because the detection limit was 300 g and it was used to detect for carbonyl
functional group after that it was formed color complex with ferric chloride solution. Colorimetric
determination method gave large relative standard deviations (RSD) because aspartame was a chiral
compound which was destroyed by heat and the color solution itself was also decomposed by heat. HPLC
determination method also gave large relative standard deviation in some samples because aspartame was a
chiral compound if it was kept unproperly it could be destroyed. The relative standard deviations of simple
sugar samples 1-4 of colorimetric method was 5.20-17.29% while HPLC method was 2.06-11.7% and
the percent recovery of these two methods were about 97% from these results it was shown that
colorimetric method was given the precission lower than HPLC method but these two method were given
the same accuracy. Aspartame was found lower than the label amount in many samples because they were
kept unproperly so the aspartame might be destroyed.

4. CONCLUSION1,3,4,5,
Testing with alkaline hydroxylamine was specific for aspartame identification. Colorimetric method can be
used to determine aspartame in simple sugar samples as well as HPLC method.
The colorimetric method was simple, rapid and inexpensive when compared with HPLC method so the
former method can be used for qualitative and quantitative determination of aspartame in simple sugar
sample for routine work in small laboratory in developing country. These three analytical methods can be
used to investigate the use of aspartame in Thailand in order to stimulate the Ministry of Public Health to
remind the use of aspartame.

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KMITL Sci. J. Vol.8 No.2 (Section B)

July December, 2008

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