STEVIOL GLYCOSIDES

Prepared at the 69th JECFA (2008) and published in FAO JECFA
Monographs 5 (2008), superseding specifications prepared at the 68th
JECFA (2007), published in FAO JECFA Monographs 5 (2008). An
ADI of 0 - 4 mg/kg bw (expressed as steviol) was established at the
69th JECFA (2008).

SYNONYMS

INS no. 960

DEFINITION

The product is obtained from the leaves of Stevia rebaudiana Bertoni.
The leaves are extracted with hot water and the aqueous extract is
passed through an adsorption resin to trap and concentrate the
component steviol glycosides. The resin is washed with a solvent
alcohol to release the glycosides and product is recrystallized from
methanol or aqueous ethanol. Ion exchange resins may be used in the
purification process. The final product may be spray-dried.
Stevioside and rebaudioside A are the component glycosides of
principal interest for their sweetening property Associated glycosides
include rebaudioside C, dulcoside A, rubusoside, steviolbioside, and
rebaudioside B generally present in preparations of steviol glycosides
at levels lower than stevioside or rebaudioside A.

Chemical name

Stevioside:

13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]
kaur-16-en-18-oic acid, β-D-glucopyranosyl ester

Rebaudioside A:

13-[(2-O-β-D-glucopyranosyl-3-O-β-Dglucopyranosyl-
β-D-glucopyranosyl)oxy]kaur-6-en-8-oic acid, β-Dglucopyranosyl ester

C.A.S. number

Stevioside: 57817-89-7
Rebaudioside A: 58543-16-1

Chemical formula

Stevioside: C38H60O18
Rebaudioside A: C44H70O23

Structural Formula

The seven named steviol glycosides:

Formula weight

Stevioside: 804.88
Rebaudioside A: 967.03

Assay

Not less than 95% of the total of the seven named steviol glycosides,
on the dried basis.

DESCRIPTION

White to light yellow powder, odourless or having a slight
characteristic odour. About 200 - 300 times sweeter than sucrose.

FUNCTIONAL USES

Sweetener

CHARACTERISTICS

IDENTIFICATION

Solubility (Vol. 4)

Freely soluble in water

Stevioside and rebaudioside A

The main peak in the chromatogram obtained by following the
procedure in Method of Assay corresponds to either stevioside or
rebaudioside A.

pH (Vol. 4)

Between 4.5 and 7.0 (1 in 100 solution)

PURITY

Total ash (Vol. 4)

Not more than 1%
Loss on drying (Vol. 4) Not more than 6% (105°, 2h)

Residual solvents (Vol. 4)

Not more than 200 mg/kg methanol and not more than 5000 mg/kg
ethanol
(Method I in Vol. 4, General Methods, Organic Components, Residual
Solvents)

Arsenic (Vol. 4)

Not more than 1 mg/kg
Determine by the atomic absorption hydride technique (Use Method II
to prepare the test (sample) solution)

Lead (Vol. 4)

Not more than 1 mg/kg
Determine using an AAS/ICP-AES technique appropriate to the
specified level. The selection of sample size and method of sample
preparation may be based on the principles of the methods described
in Vol. 4 (under “General Methods, Metallic Impurities”).

METHOD OF ASSAY

Determine the percentages of the individual steviol glycosides by high
pressure liquid chromatography (Volume 4).

Standards

Stevioside, >99.0% purity and rebaudioside A, >97% purity (available
from Wako pure Chemical Industries, Ltd. Japan).

Mobile phase

Mix HPLC-grade acetonitrile and water (80:20). Adjust the pH to 3.0
with phosphoric acid (85% reagent grade). Filter through 0.22 μm
Millipore filter or equivalent.

Standard solutions

(a) Accurately weigh 50 mg of dried (105°, 2 h) stevioside standard
into a 100-ml volumetric flask. Dissolve with mobile phase and dilute
to volume with mobile phase.
(b) Repeat with previously dried rebaudioside A standard.

Sample solution

Accurately weigh 60-120 mg of dried (105°, 2 h) sample into a 100-ml
volumetric flask. Dissolve with mobile phase and dilute to volume with
the mobile phase.

Chromatography Conditions

Column: Supelcosil LC-NH2 or equivalent (length: 15-30 cm; inner
diameter: 3.9-4.6 mm)
Mobile phase: A 80:20 mixture of acetonitrile and water (see above)
Flow rate: Adjust so that the retention time of rebaudioside A is
about 21 min.
Injection volume: 5-10 μl
Detector: UV at 210 nm
Column temperature: 40°

Procedure

Equilibrate the instrument by pumping mobile phase through it until a
drift-free baseline is obtained. Record the chromatograms of the
sample solution and of the standard solutions.
The retention times relative to rebaudioside A (1.00) are:

0.45-0.48 for stevioside 0.12-0.16 for rubusoside
0.25-0.30 for dulcoside A 0.35-0.41 for steviolbioside
0.63-0.69 for rebaudioside C 0.73-0.79 for rebaudioside B

Measure the peak areas for the seven steviol glycosides from the
sample solution (the minor components might not be detected).
Measure the peak area for stevioside for the standard solution.
Calculate the percentage of each of the seven steviol glycosides, X, in
the sample from the formula:

%X = [WS/W] x [fXAX/AS] x 100

where

WS is the amount (mg) of stevioside in the standard solution
W is the amount (mg) of sample in the sample solution
AS is the peak area for stevioside from the standard solution
AX is the peak area of X for the sample solution
fX is the ratio of the formula weight of X to the formula weight of
stevioside: 1.00 (stevioside), 0.98 (dulcoside A), 1.20 (rebaudioside A),
1.18 (rebaudioside C), 0.80 (rubusoside), 0.80 (steviolbioside), and
1.00 (rebaudioside B).

Calculate the percentage of total steviol glycosides (sum the seven
percentages).

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