ANNEX 2

SPECIFICATIONS\r\nAND TESTS FOR ACESULFAME POTASSIUM

Acesulfame\r\n potassium; Acesulfame K;

INS\r\n 950

ADI\r\n = 0 - 15 mg/kg bw

Potassium\r\n salt of 6-methyl-1,2,3-oxathiazine-4(3H)-one-2,2-dioxide; potassium salt of\r\n 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide

55589-62-3

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201,24

 Odourless, white crystalline powder_\r\n

Sweetener,\r\n flavour enhancer

Freely\r\n soluble in water, very slightly soluble in ethanol.

Dissolve\r\n 10 mg of the sample in 1,000 ml of water. The solution shows an absorbance\r\n maximum at 227±2 nm

Passes\r\n test.

(Test\r\n the residue obtained by igniting 2 g of the sample).

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Not\r\n more than 1.0% (105^oC, 2 h).

5.5\r\n - 7.5 (1% soln).

Passes\r\n test for 20 mg/kg of UV active components.

See\r\n description under Tests.

Not\r\n more than 3 mg/kg.

(Method\r\n III; using an appropriate sample size and appropriate volumes of the standard\r\n solution for construction of the calibration curve)

Not\r\n more than 1 mg/kg.\r\n

Not\r\n less than 99.0% and not more than 101.0% on the dried basis.

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Proceed\r\n as directed under the method for Chromatography (High Performance Liquid\r\n Chromatography, FNP 5) using the following conditions and using\r\n 4-hydroxybenzoic acid ethyl ester as the reference substance:

Column: 25 cm x 4.6 mm stainless steel

Stationary phase: Reversed phase (C18 silica gel, 3 - 5 µm)

Elution: Isocratic

Mobile phase: Acetonitrile/0.01 mol/l tetrabutyl ammonium\r\n hydrogen sulfate (TBAHS) in water; 40/60 v/v

Flow: About 1 ml/min

Detector type: UV or Diode array, 227 nm

Sample size: 20 µl of a 10 g/l solution of the sample in\r\n deionized water

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If\r\n peaks other than that due to acesulfame K appear within three times the\r\n elution time of acesulfame K, carry out a second run using 20 µl of a 0.2\r\n mg/l solution of the sample.

The\r\n sum of the areas of all peaks eluted in the first run within 3 times the\r\n elution time of acesulfame K elution time, except for the acesulfame K peak,\r\n does not exceed the peak area of acesulfame K in the second run.

Tested\r\n under JECFA monograph 1 - Vol.4. Determine using an atomic absorption\r\n technique appropriate to the specified level.The selection of sample size and\r\n method of sample preparation may be based on the principles of the method\r\n described in JECFA monograph 1 - Vol.4 “Instrumental Methods.”

Dissolve\r\n about 0.15 g of the dried sample (dissolution may be slow), accurately\r\n weighed, in 50.0 ml glacial acetic acid and titrate potentiometrically with\r\n 0.1 N perchloric acid, or add two drops of crystal violet TS and titrate with\r\n 0.1 N perchloric acid, to a blue-green end-point which persists for at least\r\n 30 sec. Perform a blank determination and make any necessary correction.

Each ml of 0.1 N perchloric acid is equivalent to 20.12 mg of C₄H₄KNO₄S.

ANNEX 3

SPECIFICATIONS\r\nAND TESTS FOR ISOMALT

Hydrogenated\r\n isomaltulose

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ADI\r\n “Not limited”

A\r\n mixture of hydrogenated mono- and disaccharides whose principal components\r\n are the disaccharides:

6-O-a-D-Glucopyranosyl-D-sorbitol\r\n (1,6-GPS) and

1-O-a-D-Glucopyranosyl-D-mannitol\r\n dihydrat (1,1-GPM)

64519-82-0

6-O-a-D-Glucopyranosyl-D-sorbitol:\r\n C₁₂H₂₄O₁₁

1-O-a-D-Glucopyranosyl-D-mannitol\r\n dihydrate: C₁₂H₂₄O₁₁.H₂O

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1,1-GPM (without molecules of crystal water)

6-O-a-D-Glucopyranosyl-D-sorbitol:\r\n 344,32

1-O-a-D-Glucopyranosyl-D-mannitol\r\n dihydrat: 380,32

Odourless,\r\n white, crystalline slightly hygroscopic substance.

Sweetener,\r\n bulking agent, anticaking agent, glazing agent

Freely\r\n soluble in water, very slightly soluble in ethanol.

Passes\r\n test (See description under Tests).

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Not\r\n more than 0,05%. (Test 5 g of the sample).

Not\r\n more than 3%.

Not\r\n more than 6%.

Not\r\n more than 0,3%.

Not\r\n more than 2 mg/kg.

Not\r\n more than 1 mg/kg.\r\n

Not\r\n less than 98% of hydrogenated mono- and disaccharides and not less than 86%\r\n of the mixture of 6-O-alpha-D-glucopyranosyl-D-sorbitol and\r\n 1-O-alpha-D-glucopyranosyl-D-mannitol on the anhydrous basis.

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TLC aluminium foils or plates of approx. 12 cm length and coated\r\n with a layer of approx. 0.2 mm, Kieselgel 60 F254, Art. 5554, Merck, or\r\n equivalent.

Standard solution:

Dissolve 500 mg of each of the following sugar alcohols in 100\r\n ml of water: Sorbitol, mannitol, lactitol, maltitol,\r\n 1-O-alpha-D-gluco-pyranosylD-mannitol (1,1-GPM), and\r\n 6-O-alpha-D-glucopyranosyl-D-sorbitol (1,6- GPS)

Test solution:

Dissolve 500 mg of sample in 100 ml of water.

Solvent A:

Isopropanol:n-butanol:aqueous boric acid solution (25\r\n mg/ml):acetic acid:propionic acid (50:30:20:2:16;v/v)

Solvent B :

Ethylacetate:pyridine:water:acetic acid:propionic acid\r\n (50:50:10:5:5;v/v).

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I: 0.1% Na-metaperiodate in water (w/w).

II: ethanol:sulfuric acid:anisaldehyde:acetic acid\r\n (90:5:1:1;v/v).

Procedure

Apply\r\n approximately 0.3 µl each of the reference and test solution to the bottom of\r\n the TLC plate. Dry the spots in warm air. Develop the plate to a height of 10\r\n cm in a developing chamber containing either solvent A or solvent B. Allow\r\n the plate to dry in warm air and dip the plate for up to 3 sec into Detecting\r\n solution I.

 Dry\r\n the plate in hot air. Note: The plate should be completely dry on both\r\n sides. Dip the plate in Detecting solution II up to 3 sec and dry in hot\r\n air until coloured spots become visible. Optionally, the background colour\r\n may be brightened in warm steam.

The\r\n approximate R_f values and colours of the spots on the TLC-plate of\r\n the substances are described as follows:

Substance

color

R_f (in solvent A)

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...

Mannitol

reddish (light)

0,36

0,40

Sorbitol

brown

0,36

0,36

GPM

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...

0,28

0,16

GPS

blue-grey

0,25

0,13

Maltitol

green

0,26

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Lactilol

olive-green

0,23

0,14

The\r\n R_f values may vary slightly depending on the commercial source of\r\n the silica gel plates.

The\r\n principal spots in the chromatogram obtained from a test solution of isomalt\r\n are similar in R_f value and colour to GPM and GPS.

Tested\r\n under JECFA monograph 1 - Vol.4- Proceed as directed under\r\n Reducing Substances (as glucose), Method II. The weight of cuprous oxide\r\n shall not exceed 50 mg.

Test\r\n solution:

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Standard\r\n solutions:

Prepare\r\n three Standard solutions in the same manner as the Sample solution but adding\r\n 0.5 ml, 1.0 ml, and 1.5 ml, respectively, of a standard nickel solution\r\n containing 10 mg/kg Ni, in addition to the 20.0 g of the sample.

Procedure:

Zero\r\n the instrument by measuring the methyl isobutyl ketone solution (Prepare in\r\n the same manner as the Sample solution, but omit the sample).  Measure the\r\n absorbances of the methyl isobutyl ketone solution extracted at 232.0 nm, use\r\n a nickel hollow cathode lamp and an air–acetylene flame.

Tested\r\n under JECFA monograph 1 - Vol.4. Determine using an atomic absorption\r\n technique appropriate to the specified level. The selection of sample size\r\n and method of sample preparation may be based on the principles of the method\r\n described in JECFA monograph 1 - Vol.4 “Instrumental Methods.”

Internal\r\n standard solution

Dissolve\r\n suitable quantities of phenyl-ß-D-glucopyranoside and maltitol in water to\r\n obtain a solution of about 1 mg phenyl-ß-D-glucopyranoside and 50 mg maltitol\r\n per g water.

Standard\r\n solutions

Dissolve\r\n accurately weighed quantities of 1-O-alpha-D-glucopyranosylD-mannitol\r\n (1,1-GPM) and 6-O-alpha-D-glucopyranosyl-D-sorbitol (1,6- GPS), calculated as\r\n dry substance, in water to obtain two separate solutions having a\r\n concentration of about 50 mg per g each. Also prepare an aqueous standard\r\n solution containing approx. 1 mg mannitol and 1 mg sorbitol per g.

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Dissolve an accurately weighed quantity of the sample (approx. 1\r\n g) in water to obtain a concentration of about 10 g per 100 g.

Procedure

Pipet 100.0 mg of standard solution or sample solution into a\r\n glass tube fitted with a screw cap and add 100.0 mg of internal standard\r\n solution. Remove the water by lyophilization and dissolve the residue in 1.0\r\n ml of pyridine. Add 4 mg O-benzyl-hydroxylamine hydrochloride, and cap the\r\n tube and set it aside for 12 h at room temperature. Then, add 1 ml of\r\n Nmethyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) and heat to 80º for 12 h\r\n shaking occasionally and allow to cool. Inject 1 µl portions of these\r\n solutions directly into a gas chromatograph under the following operating\r\n conditions:

Carrier gas: Helium (initial flow rate: approx. 1 ml/min at 80ºC\r\n and 1 atm; split flow: 25 ml/min)

Column: Fused silica HT-8 (25 m x 0.22 mm x 0.25 µm), or\r\n equivalent;

Injector: Programmed temperature vaporizer: 30ºC; 270º/min to\r\n 300ºC (49 min)

Detector: Flame ionization detector; 360ºC;

Temperature program: 80º (3 min); 10º/min to 210º; 5º/min to\r\n 300º (6 min)

Approximate retention times:

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Mannitol 19.5 min

Sorbitol 19.6 min

Internal standards:

Phenyl-ß-D-glucopyranoside 26.8 min

Maltitol 33.5 min

Hydrogenated disaccharides (32 - 36 min)

1,1-GPS 33.9 min

1,1-GPM 34.5 min

1,6-GPS 34.6 min

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Where:

a_I = peak area of component I (µV·s)

a_S = peak area of internal standard (µV·s)

m_S = mass of internal standard used for\r\n derivatization (mg d.s.)

m_ISOMALT = mass of sample used for derivatization (mg\r\n d.s.)

F_I = relative response factor fI/fS

f_I = response factor of component I: fI\r\n =(aI/mI)x(100/% purity)

f_S = response factor of internal standard: fS\r\n =(aS/mS)x(100/% purity)

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NOTE: Use maltitol as internal standard for the calculation of\r\n hydrogenated disaccharides (e.g. 1,1-GPM, 1,6-GPS) and phenyl-ß-Dglucoside\r\n for the calculation of hydrogenated monosaccharides (mannitol, sorbitol). For\r\n the total of other saccharides (hydrogenated or not), subtract the sum of\r\n 1,1-GPM, 1,6-GPS, sorbitol and mannitol from 100%.)

ANNEX 4

SPECIFICATIONS\r\nAND TESTS FOR SACCHARIN

INS\r\n 954

ADI\r\n = 0 - 5 mg/kg bw

1,2-Benzisothiazole-3(2H)-one-1,1-dioxide;\r\n

3-oxo-2,3-\r\n dihydrobenzo[d]isothiazol-1,1-dioxide

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C₇H₅NO₃S

183,18

White\r\n crystals or a white, crystalline powder, odourless or with a faint, aromatic\r\n odour

Sweetener

Slightly\r\n soluble in water; soluble in basic solutions; sparingly soluble in ethanol.

A\r\n saturated aqueous solution is acidic.

Passes\r\n test (See description under Tests).

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Not\r\n more than 1% (105^oC , 2 h)

226\r\n - 230^oC.

Not\r\n more than 0.2% (Test 2 g of the sample).

Passes\r\n test (See description under Tests).

Passes\r\n test (See description under Tests).

Not\r\n more than 25 mg/kg.

Not\r\n more than 30 mg/kg.

Not\r\n more than 1 mg/kg.\r\n

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Dissolve\r\n about 0.1 g of the sample in 5 ml of 5% sodium hydroxide solution. Evaporate\r\n to dryness and gently fuse the residue over a small flame until it no longer\r\n evolves ammonia. After the residue has cooled, dissolve it in 20 ml of water,\r\n neutralize the solution with dilute hydrochloric acid TS and filter. The\r\n addition of a drop of ferric chloride TS to the filtrate produces a violet\r\n colour.

Mix\r\n 20 mg of the sample with 40 mg of resorcinol, add 10 drops of sulfuric acid,\r\n and heat the mixture in a liquid bath at 200º for 3 min. After cooling, add\r\n 10 ml of water and an excess of sodium hydroxide TS. A fluorescent green\r\n liquid is produced.

Add\r\n ferric chloride TS dropwise to 10 ml of a hot, saturated solution of the\r\n sample. No precipitate or violet colour appears.

Dissolve\r\n 0.2 g of the sample in 5 ml of sulfuric acid TS. Keep at 48^o to 50^o\r\n for 10 min. The colour should not be darker than a very light brownish-yellow\r\n (Matching Fluid A).

Tested\r\n under JECFA monograph 1 - Vol.4- Proceed as directed under monograph Selenium\r\n Limit Test, Method I, Test 0.2 g of the sample.

Tested\r\n under JECFA monograph 1 - Vol.4. Determine using an atomic absorption\r\n technique appropriate to the specified level. The selection of sample size\r\n and method of sample preparation may be based on the principles of the method\r\n described in JECFA monograph 1 - Vol.4 “Instrumental Methods.”

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Each ml of 0.1 N sodium hydroxide is equivalent to 18.32 mg of C₇H₅NO₃S.

ANNEX 5

SPECIFICATIONS\r\nAND TESTS FOR SORBITOL

D-Glucitol,\r\n D-Sorbitol, Sorbol

INS\r\n 420

ADI\r\n “Not limited”

D-Sorbitol

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C₆H₁₄O₆

182,17

White\r\n hygroscopic powder, crystalline powder, flakes or granules

Sweetener,\r\n humectant, texturizer, stabilizer, bulking agent

Very\r\n soluble in water, slightly soluble in ethanol.

88\r\n – 102 ^oC.

Passes test (See description under Tests).

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Not\r\n more than 1% (Karl Fischer Titrimetric Method)

Not\r\n more than 0.1%

Chlorides

Not\r\n more than 50 mg/kg

Not\r\n more than 100 mg/kg

Nickel

Not\r\n more than 2 mg/kg

Not\r\n more than 0.3%

Not\r\n more than 1.0% (as glucose)

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Not\r\n less than 97.0% of C₆H₁₄O₆ of total\r\n glycitols and not less than 91.0% of D-sorbitol on the anhydrous basis. The\r\n term glycitols refers to compounds with the structural formula CH₂OH-(CHOH)_n-CH₂OH,\r\n n £\r\n 4.

Proceed as directed under Thin Layer Chromatography of Polyols\r\n under JECFA monograph 1 - Vol.4.

Use the following:

Standard solution

Dissolve 50 mg of reference standard sorbitol (USP) in 20 ml\r\n water

Test solution

Dissolve 50 mg of the sample in 20 ml of water

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Tested\r\n under JECFA monograph 1 - Vol.4- Test 10 g of sample by the Limit Test using\r\n 1.5 ml of 0.01N hydrochloric acid in the control.

Tested\r\n under JECFA monograph 1 - Vol.4- Test 10 g of sample by the Limit Test using\r\n 2.0 ml of 0.01N sulfuric acid in the control.

Tested\r\n under JECFA monograph 1 - Vol.4- Reducing Substances (as glucose), Method II.\r\n The weight of cuprous oxide shall not exceed 50 mg.

Transfer\r\n 2.1 g of the sample into a 250 ml flask fitted with a ground glass joint, add\r\n 40 ml of 0.1N hydrochloric acid, attach a reflux condenser, and reflux for 4\r\n h. Transfer the\r\n solution to a 400 ml beaker, rinsing the flask with about 10 ml of water,\r\n neutralize with 6N sodium hydroxide and proceed as directed under Reducing\r\n Substances (as Glucose) Method II. The weight of cuprous oxide shall not\r\n exceed 50 mg.

Tested\r\n under JECFA monograph 1 - Vol.4.Determine using an atomic absorption\r\n technique appropriate to the specified level. The selection of sample size\r\n and method of sample preparation may be based on the principles of the method\r\n described in JECFA monograph 1 - Vol.4 “Instrumental Methods.”

Determine\r\n the polyol content of the sample using liquid chromatography (see JECFA\r\n monograph 1 - Vol.4).

Apparatus

Liquid chromatograph (HPLC)

Detection: differential refractometer maintained at constant\r\n temperature Integrator recorder

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Column: AMINEX HPX 87 C (resin in calcium form), length 30 cm,\r\n internal diameter 9 mm

Eluent: double distilled degassed water (filtered through\r\n Millipore membrane filter 0.45 µm)

Chromatographic\r\n conditions

Column temperature: 85±0.5^o

Eluent flow rate: 0.5 ml/min

Standard\r\n solution

Dissolve an accurately weighed quantity of sorbitol in water to\r\n obtain a solution having known concentration of about 10.0 mg of sorbitol per\r\n ml.

Test\r\n solution

Transfer about 1 g of the sample accurately weighed to a 50 ml\r\n volumetric flask, dilute with water to volume and mix.

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Separately inject equal volumes (about 20 µl) of the sample\r\n solution and the standard solution into the chromatograph. Record the\r\n chromatograms and measure the responses of each peak. Calculate separately\r\n the quantities, in mg, of sorbitol in the portion of sample taken by the\r\n following formula:

Where:

C = s the concentration, in mg per ml, of sorbitol in the\r\n standard solution

A_t = the peak response of the sample solution

A_c\r\n = the peak response of the standard solution.