Introduction to Artificial Sweeteners & Method of Analysis, Chemical Structures and Basic Characteristics of Artificial Sweeteners: Presented by M.Sudheeshna Pharmaceutical Analysis

1. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
ARTIFICIAL SWEETENERS
& METHOD OF ANALYSIS
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
M.SUDHEESHNA
(Reg. No.20L81S0713)
Under the guidance/Mentorship of
Dr. P. Vinod Kumar, M.Pharm, Ph.D
Professor of Pharmaceutical Analysis
Head of department - Pharmaceutical Analysis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
 Introduction
 Chemical Structures and Basic Characteristics of Artificial
Sweeteners:
 Toxic Potential of Artificial Sweeteners
 Advantages & Disadvantages
 Qualitative Detection
 Quantitative Determination
 Conclusion
 References
Contents:

3. RIPER
AUTONOMOUS
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
ARTIFICIAL SWEETENERS:
• These are also known as sugar substitutes, alternative sweeteners,
non – sugar sweeteners.
• They are low calorie, synthetic sugar substitutes but may be derived
from naturally occuring substances, including herbs or sugar itself.
• These are the ingredients added to yogurt, foods, medicinal
preparations, candy, jams, soft drinks, beverages to provide sweetness
without adding a calorie.
• Eg: Saccharin, Aspartame, Sucralose, Neotame, Acesulfame - K
Introduction:

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4

5. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
1. Acesulfame potassium:
• Potassium salt of 6-methyl-1,2,3- oxathiazine-4(3H)-one 2,2-dioxide.
• It has a slightly bitter after-taste, especially at high concentrations.
• Its water solubility is very good, whereas most synthetic sweeteners
have unsatisfactory water solubility.
• It is stable at high cooking and
baking temperatures.
Chemical structures and basic characteristic of
artificial sweeteners:

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• 1,2-benzisothiazol-3(2H)-on-1,1-dioxide.
• It is the oldest sweetener on the market.
• It is commercially available in three forms (i.e. acid saccharin,
sodium saccharin, and calcium saccharin).
• It has high solubility and stability.
• However, at low pH, it can slowly hydrolyze to 2-sulfobenzoic acid
and 2-sulfoamylobenzoic acid.
2. Sodium Saccharine:

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• N-L-α-aspartyl-L-phenylalanine-1-methyl ester.
• Although it is relatively stable in its dry form, the compound
undergoes pH- and temperature- dependent degradation in solution,
what makes aspartame undesirable as a baking sweetening agent.
• Below pH 3 aspartame is unstable and hydrolyzes to produce
aspartylphenylalanine and above pH 6, it changes to form 5-benzyl-
3,6-dioxo-2-piperazineacetic acid.
3. Aspartame:

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• 4-chloro-4-deoxy-α-D-galactopyranosyl-1,6-dichloro-1,6-
dideoxy-β-D-fructofuranoside.
• The sweetener is derived from ordinary sugar through a multistep
manufacturing process, in which three of the hydroxyl groups on the
sugar molecule are selectively replaced with three atoms of chlorine.
• excellent solubility, and high compatibility
4. Sucralose:

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• N-[N-(3,3-dimethylbutyl)-L-a-aspartyl]-L-phenylalanine
1-methyl ester, is a derivative of dipeptide, which is made from the
amino acids aspartic acid and phenylalanine.
• It has extensive shelf life in dry conditions.
• In aqueous solutions, it is approximately as stable as aspartame in the
acidic pH range, but it is significantly more stable in the neutral pH
range.
5. Neotame:

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11. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
Toxic Potential of Artificial Sweeteners:

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Advantages:
• Better weight control
• Tooth decay reduction
• Blood sugar control
• Contain zero calories
Disadvantages:
• Obesity, mineral depletion
• Certain artificial sweeteners are listed as anticipated human
carcinogens.
• Common side effects: dizziness, mood swings, headache etc
• Many tests concerning artificial sweeteners have been proven to be
poorly conducted.

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PROCEDURE:
Qualitative detection of acesulfame, saccharin and
cyclamate by thin-layer chromatographic method:
Extract the sweetener from acidified food product with water or take acidified
aqueous extract and pass through the ion-exchanger and wash with water
Elute the sweeteners with dilute ammonia solution.
Evaporate the ammonical solution under vacuum to dryness and take up the
residue in 1 ml of 50% methanol
Alternatively extract these sweeteners from acidified sample, pH 0.6, with
ethyl acetate and use concentrated ethylacetate for TLC.

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Apply 2-10 µl of sample solution along with standards on LC
plates coated with polyamide.
Develop the plate to about 15 cm height with a developing solvent
consisting of xylol: n- propanol: formic acid (5:5:1).
Dry the plates in a current of air and spray with 0.2% solution of
dichlorofluoresein
After being dried, examine under UV light.
To identify the spots in day light, place the plate in chamber
containing bromine and then expose to ammonia vapour
Spots appear on a reddish background.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
PRINCIPLE:
• It operates on the principle of hydrophobic interactions which result from
repulsive forces between a relatively polar solvent, non -polar analyte and
non-polar stationary phase.
IDENTIFICATION:
• Identify the intense sweeteners by comparing the retention times of the
analyte concerned in the sample solution with that of the standard substance
or by simultaneous injection of the standard solution and the sample
solution.
Determination of Acesulphame – K,
Aspartame and Saccharin by RP - HPLC

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PROCEDURE:
Preparation of sample test solution:
1) Bevarages:
2) Juices , flavoured milk drinks:
Dilute 20 ml of the liquid in a 100 ml volumetric flask with water.
Filter the solution through a membrane filter of pore size 0.2 um
before injection.
Dilute 20 ml sample with 50 ml water in a 100 ml volumetric
flask.
Add 2 ml Carrez solution 1 , mix and 2 ml of Carrez solution 2 ,
dilute to mark with water and filter

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Jams and related products:
Wash the settled matter twice with water and centrifuge again
Collect each of the supernatant in the 100 ml vol flask and then
dilute the solution with water
If the fat free insoluble matter in the initial sample mass exceeds
approx 3 gm, centrifuge the clarified solution for 10 minutes
before filtering it quantitatively into a 100 ml volumetric flask
Weigh to the nearest 1 mg, 20 gm of homogenized sample in a
100 ml volumetric flask
Add about 60 ml water and place the flask in an ultrasonic bath
at 400C for 20 minutes

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The temperature should not exceed 400 C since aspartame can get
degraded. Cool to room temperature
Add 2 ml Carrez solution 1 , mix followed by
2 ml carrez solution 2
Shake vigorously and allow to stand for 10 minutes.
Dilute to mark with water, filter
If the fat free insoluble matter in the initial mass exceeds 3 gm, it
is advisable to centrifuge the clarified sample solution for 10
minutes at 1400 r.p.m
Wash with water and centrifuge again as in case of cloudy liquid
samples

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Semisolid and solid products :
Weigh 10 – 20 gm of thoroughly homogenized sample in a 100 ml
volumetric flask.
Add about 50 ml water and place the vol flask in an ultra sonic bath at
40 0 C for 20 minutes
Cool to room temperature, add 2 ml Carrez solution 1 , mix and add 2
ml of Carrez solution 2, dilute with water and filter
In case of very complex matrices, additional purification using the
solid phase extraction column
In this case add 2 ml of clarified filterate to the cartridge, previously
activated with 3 ml of methanol and 20 ml water and elute with about
20 ml of mobile phase

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Chromatographic conditions:
reversed phase HPLC
column lengths 100mm for 3 μm, 300mm for 10μm
Internal diameter = 4 mm
Flow rate 0.8 ml /min to 1 ml / min
Injection volume 10 µl upto 20 µl
stationary phase C 18 of 5μm particle size
Mobile phase phosphate buffer = 0.02mol
/pH=4.3, 3.5,6.5, acetonitrile ,
Methanol
detection UV at a wavelength of:
217 nm for aspartame
227 for Acesulphame – K
265 for saccharin

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
• Calculate the mass fraction w expressed in mg / kg or mass
concentration p in mg / litre of the intense sweetener as under
A1 x V1 x m1 x F1
Where,
• A 1 = peak area of the intense sweetener concerned obtained with
sample test solution
• A2 = peak area of the intense sweetener concerned obtained with
the standard test solution
Calculation:
A2 x V2 x m0
x1000
W or P =

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• V1 = total volume of sample test solution in milliliters
• V2 = total volume of the standard test solution in milliliters
• m1 = mass of the intense sweetener concerned in standard test
solution
• m0 = initial sample mass in gms or mls
• F1 = dilution factor for the purification method used

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
Determination :
• Integrate the peak areas or determine the peak heights and compare
the results with the corresponding values for the standard substance
with the nearest peak area / height or use a calibration graph. Check
the linearity of the calibration graph.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
• The number of people suffering from diabetes, obesity, hypertension,
and heart disease is increasing every year.
• So nowadays artificial sweetener are receiving much more attention.
But it gets bad reputation due to their safety issue.
• On the other hand in spite of demand for rare sugars, their
commercial availability, application and usefulness is negligible as
they are expensive to prepare and unavailable in nature.
• So research is required to make natural sugars having the desired
quantities of sweetness, low caloric value, and least observed
physiological effects.
Conclusion:

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References:
• L.M.L. Nollet, Food Analysis by HPLC, Marcel Dekker, New York,
USA, 2000.
• European Commission, Directive 94/35/EC of European Parliament
and of the Council of 30 June 1994 on sweeteners for use in
foodstuffs, Off. J. Eur. Un. L237 (1994) 13.
• A. Herrmann, E. Damawandi, M. Wagmann, J. Chromatogr., A 282
(1983) 85.

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