SUCROSE ESTERS OF FATTY ACIDS

Prepared at the 53rd JECFA (1999) and published in FNP 52 Add 7 (1999), superseding specifications prepared at the 51st JECFA (1998), published in FNP 52 Add 6 (1998). ADI 0-30 mg/kg bw for sucrose esters of fatty acids and sucroglycerides established at the 49th JECFA in 1997.

SYNONYMS

INS No. 473

DEFINITION

Mono-, di- and triesters of sucrose with food fatty acids, prepared from sucrose and methyl and ethyl esters of food fatty acids or by extraction from sucroglycerides. Only the following solvents may be used for the production: dimethyl formamide, dimethyl sulfoxide, ethyl acetate, isopropanol, propylene glycol, isobutanol and methyl ethyl ketone.

Assay

Not less than 80%

DESCRIPTION

Stiff gels, soft solids or white to slightly greyish white powders

FUNCTIONAL USES

Emulsifier

CHARACTERISTICS

IDENTIFICATION

Solubility (FNP 5)

Sparingly soluble in water; soluble in ethanol

Test for fatty acid

Add 1 ml of ethanol to 0.1 g of the sample, dissolve by warming, add 5 ml of dilute sulfuric acid TS, heat in a water bath for 30 min and cool. A yellowish white solid or oil is formed which is soluble in 3 ml of ether and has no odour of isobutyric acid.

Test for sugar

To 2 ml of the solution separated from the solid and oil in the test for fatty acids, add 1 ml of anthrone TS carefully down the inside of the test tube; the boundary surface of the two layers turns to blue or green.

PURITY

Sulfated ash (FNP 5)

Not more than 2%

Test 1 g of the sample (Method I)

Acid value (FNP 5)

Not more than 6

Free sucrose

Not more than 5%
See description under TESTS

Dimethyl formamide

Not more than 1 mg/kg
See description under TESTS

Dimethyl sulfoxide

Not more than 2 mg/kg
See description under TESTS

Ethyl acetate, isopropanol and
propylene glycol

Not more than 350 mg/kg, singly or in combination

See description under TESTS

Isobutanol

Not more than 10 mg/kg
See description under TESTS

Methanol

Not more than 10 mg/kg
See description under TESTS

Methyl ethyl ketone

Not more than 10 mg/kg
See description under TESTS

Lead (FNP 5)

Not more than 2 mg/kg
Prepare a sample solution as directed for organic compounds in the Limit Test and determine by atomic absorption spectroscopy (FNP 5)

TESTS

PURITY TESTS

Free sucrose

Determine by gas liquid chromatography (FNP5) using the following conditions:

Reagents:

- Internal Standard: 5 mg/ml cholesterol in chloroform or 10 mg/ml

tetracosane in chloroform

- Pyridine (dried over molecular sieve)

- N,O-Bis-(Trimethylsilyl)-acetamide (BSA)

- Trimethylchlorosilane (TMCS)

Procedure: Weigh accurately 20-50 mg of the sample into a silylation vial, add 1 ml internal standard solution, 1 ml pyridine, and 0.5 ml each of BSA and TMCS. Seal vial, and heat at 70� for 30 min. Inject 1 �l into the gas liquid chromatograph.

Conditions:

Column:

- length: 2-3 m

- diameter: 4 mm (i.d.)

- material: glass

- packing: Dexil

Carrier gas: Nitrogen

Flow rate: 40 ml/min

Detector: FID

Temperature programme: Hold for 1 min at 160�, then 160-375� at 15�/min

Measure peak areas for sucrose and internal standard. The response factor (RF) is calculated from a number of gas liquid chromatography runs with standard solutions of sucrose containing internal standard.

Calculation:

and

Dimethyl formamide

Determine by hydrolysis to dimethylamine and analysis by gas liquid chromatography (FNP 5) using the following conditions:

Reagents:

- Dimethyl formamide

- Dimethylamine hydrochloride

- Methanol

- Ethanol

- Hydrochloric acid

- Sodium hydroxide

Standard solutions: Prepare 4.47 mg/ml (equivalent to 4.0 mg/ml of dimethyl formamide) stock solution of dimethylamine hydrochloride in ethanol, and prepare standard solutions equivalent to 4, 2 and 1 �g/ml of dimethyl formamide, respectively, by dilution of the stock solution with 0.1% sodium hydroxide solution in ethanol.

Sample preparation: The apparatus for the hydrolysis is shown in Figure. Weigh accurately about 40 g of the sample into a 1000-ml round-bottomed flask. Add 500 ml of 5% methanolic solution of sodium hydroxide, and attach the flask to the apparatus. Set an Erlenmeyer flask containing 10 ml of 1% methanolic solution of hydrochloric acid to the apparatus. Heat the round-bottomed flask and let the content reflux for 1 hour, then distil to collect about 50 ml of the distillate while cooling water of the reflux condenser is stopped. Evaporate the distillate to almost dryness on a boiling water bath. Dissolve the residue with a small amount of ethanol, add 2.5 ml of 5% ethanolic solution of sodium hydroxide, and dilute to 25 ml with ethanol to prepare a sample solution.

Procedure: Inject 2 �l of the sample solution into the gas liquid chromatograph under the conditions below.

Calibration curve: Prepare a calibration curve by injecting each 2 �l of the standard solutions into the gas chromatograph.

Conditions:

Column:

- length: 2 m

- diameter: 2 mm (i.d.)

- material: Glass

- packing: 10% amine 220 and 10% KOH on 80/100 weak acid washed

Chromosorb W

- conditioning: Heat to 130� overnight with 5 ml/min of nitrogen flow rate

Carrier gas: Nitrogen

Flow rate: 17 ml/min

Detector: FID

Temperatures

- injection port: 198�5�

- column: 60�

Calculation:

where

C_DFA = Concentration of dimethyl formamide

C = Concentration of dimethyl formamide detected

W = weight of sample taken

Dimethyl sulfoxide

Determine by gas liquid chromatography (FNP 5) under the following conditions:

Reagents:

- Tetrahydrofuran

- Dimethyl sulfoxide

Standard solutions (prepared fresh monthly):

- Prepare a 0.25 mg/ml stock solution of dimethyl sulfoxide in tetrahydrofuran

- Prepare a range of solutions containing 0.5, 1 and 5 �g/ml of dimethyl sulfoxide by dilution of the stock solution with tetrahydrofuran.

Procedure: Weigh accurately about 5 g of the sample, dissolve and dilute it with tetrahydrofuran to 25 ml to prepare a sample solution. Inject 3 �l of the sample solution into the gas chromatograph under the conditions below.

Calibration curve (prepared daily): Prepare a calibration curve by injecting each 3 �l of the standard solutions into the gas chromatograph

Conditions:

Column:

- length: 2 m

- diameter: 3 mm (i.d.)

- material: Glass

- packing: 10% PEG 20M and 3% KOH on Gas Chrom Z

- conditioning: Raise the oven temperature to 180� at a rate of 10�/min and let stand for 24 to 48 h with 30 to 40 ml/min of nitrogen

Carrier gas: Nitrogen

Flow rate: 50 ml/min

Detector: Flame photometric (using 394 nm sulfur filter)

Temperatures

- injection port: 210�

- column: 160� (do not exceed 200�)

Calculation

where

C_DMSO = Concentration of dimethyl sulfoxide

C = Concentration of dimethyl sulfoxide detected

W = weight of sample taken

Propylene glycol

Determine by gas liquid chromatography (FNP 5) using the following conditions:

Reagents:

- Propylene glycol

- 1,2-Butylene glycol

- Acetic anhydride

- Toluene

- Acetone

Standard solutions:

- Internal standard solution: Prepare 1000 mg/l solution of 1,2-butylene glycol in acetone. 200 �l of the solution contains 0.2 mg of 1,2-butylene glycol.

- Standard solution of propylene glycol: Prepare 1000 mg/l solution of propylene glycol in acetone.

Sample preparation: Weigh accurately about 2 g of the sample in a 100-ml flat-bottomed flask, and add 10 ml of acetic anhydride and 200 �l of the internal standard solution. Attach a reflux condenser and heat the flask in a boiling water bath for 1 hour. Add 50 ml of water and heat the flask for another 10 min in a boiling water bath. After heating, let it cool to room temperature. Transfer the content to a 100-ml separating funnel and extract with 10 ml of toluene. After separation, discharge the lower layer (aqueous phase). Add 50 ml of water to the separating funnel and wash the extract. The upper layer (toluene phase) is used as a sample solution.

Procedure: Inject 2 �l of the sample solution into the gas chromatograph under the following conditions. (Note: Since there would be a component of high boiling point, which may have a longer retention time, it is necessary at the end of each measurement to raise the oven temperature to 200� and to evacuate it from the column).

Calibration curve: Follow the same procedure using 100, 200 and 500 �l of the standard solution of propylene glycol in place of the sample, and prepare a calibration curve.

Column:

- length: 2 m

- diameter: 3 mm (i.d.)

- material: Glass

- packing: 5% Alkyleneglycol phthalate on 80/100 Chromosorb W

- conditioning: Heat to 150� overnight with approximately 50 ml/min of nitrogen

Carrier gas: Nitrogen

Flow rate: 40 ml/min

Detector: Flame ionization

Temperatures:

- injection port: 230�

- column: 110�

Calculation:

where

C = Concentration of propylene glycol (mg/kg)

A_PG = Peak area of propylene glycol

A_IS = Peak area of internal standard

W_SPL = Weight of sample (g)

C_fPG = Sensitivity correction factor for propylene glycol (slope of the calibration curve)

Methanol, isopropanol, isobutanol,
ethyl acetate and methyl ethyl ketone

Determine by gas chromatography (FNP 5) with a head space sampler using the following conditions:

Reagents:

- Methanol

- Isopropanol

- Isobutanol

- Ethyl acetate

- Methyl ethyl ketone

Standard solutions: Take each 1 g of methanol, isopropanol, isobutanol, methyl ethyl ketone and ethyl acetate in a volumetric flask and add water to total volume of 100 ml, and prepare 0.02-0.4 g/100 ml solutions by dilution of this solution. If necessary, prepare standard solutions containing up to 7 g/100 ml of isopropanol and ethyl acetate.

Procedure: Place 1 g (1.0�0.1 g) of powdered sample in a sample vial. Add 5 �l of water to the sample vial and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the below-mentioned conditions.

Calibration curve: Take 1 g of powdered sucrose esters of fatty acids, solvent free or known residual solvent contents, in a sample vial, add 5 �l of the standard solution and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the following conditions and obtain calibration curves for each solvent.

Column:

- length: 30 m

- diameter: 0.53 mm (i.d.)

- material: Silica capillary

- film: 100% methyl polysiloxane

- conditioning: Heat to 60� for 2-3 h with approximately 10 ml/min of nitrogen

Carrier gas: Nitrogen

Flow rate: 5 ml/min

Detector: Flame ionization

Temperatures

- injection port: 110�

- column: 40�

- detector: 110�

Head space sampler:

- Sample volume: 1.0�0.1 g + 5 �l

- Sample heating temp.: 80�

- Sample heating time: 40 min

- Syringe temperature: 85�

- Sample gas injection: 0.4 ml

Calculation:

where

C_i = Concentration of solvent i (mg/kg)

A_i = Peak area of solvent i (�v.sec.)

Cf_i = Conversion coefficient for solvent i (slope of the calibration curve) (�g/�v.sec)

METHOD OF ASSAY

Determine by high pressure liquid chromatography (FNP5) using the following conditions:

Sample preparation: Add about 250 mg of the sample, accurately weighed to a 50 ml volumetric flask. Dilute to volume with tetrahydrofuran, and mix. Filter through a 0.5-�m membrane filter.

Procedure: Inject 100 �l of the sample into the pre-stabilized high pressure liquid chromatograph.

Conditions:

Column: Styrene-divinylbenzene copolymer for gel permeation

chromatography (TSK-GEL G2000 (Supelco) or equivalent)

Mobile phase: HPLC-grade degassed tetrahydrofuran

Flow rate: 0.7 ml/min

Detector: Refractive index detector

Temperatures:

Column: 38�

Detector: 38�

Record the chromatogram for about 90 min. Calculate the percentage of sucrose ester content in the sample taken by the formula:

100 A/T

where

A = the sum of peak areas for the three main components, the mono-, di- and triesters, eluting at about 65, 68 and 73 min, respectively

T = the sum of all peak areas eluting within 90 min

Figure

Apparatus for hydrolysis

a: Reflux condenser

b: Condenser

c: Round bottomed flask

d: Water bath

e: Erlenmeyer flask

	Prepared at the 53rd JECFA (1999) and published in FNP 52 Add 7 (1999), superseding specifications prepared at the 51st JECFA (1998), published in FNP 52 Add 6 (1998). ADI 0-30 mg/kg bw for sucrose esters of fatty acids and sucroglycerides established at the 49th JECFA in 1997.
SYNONYMS	INS No. 473
DEFINITION	Mono-, di- and triesters of sucrose with food fatty acids, prepared from sucrose and methyl and ethyl esters of food fatty acids or by extraction from sucroglycerides. Only the following solvents may be used for the production: dimethyl formamide, dimethyl sulfoxide, ethyl acetate, isopropanol, propylene glycol, isobutanol and methyl ethyl ketone.
Assay	Not less than 80%
DESCRIPTION	Stiff gels, soft solids or white to slightly greyish white powders
FUNCTIONAL USES	Emulsifier
CHARACTERISTICS
IDENTIFICATION
Solubility (FNP 5)	Sparingly soluble in water; soluble in ethanol
Test for fatty acid	Add 1 ml of ethanol to 0.1 g of the sample, dissolve by warming, add 5 ml of dilute sulfuric acid TS, heat in a water bath for 30 min and cool. A yellowish white solid or oil is formed which is soluble in 3 ml of ether and has no odour of isobutyric acid.
Test for sugar	To 2 ml of the solution separated from the solid and oil in the test for fatty acids, add 1 ml of anthrone TS carefully down the inside of the test tube; the boundary surface of the two layers turns to blue or green.
PURITY
Sulfated ash (FNP 5)	Not more than 2% Test 1 g of the sample (Method I)
Acid value (FNP 5)	Not more than 6
Free sucrose	Not more than 5% See description under TESTS
Dimethyl formamide	Not more than 1 mg/kg See description under TESTS
Dimethyl sulfoxide	Not more than 2 mg/kg See description under TESTS
Ethyl acetate, isopropanol and propylene glycol	Not more than 350 mg/kg, singly or in combination See description under TESTS
Isobutanol	Not more than 10 mg/kg See description under TESTS
Methanol	Not more than 10 mg/kg See description under TESTS
Methyl ethyl ketone	Not more than 10 mg/kg See description under TESTS
Lead (FNP 5)	Not more than 2 mg/kg Prepare a sample solution as directed for organic compounds in the Limit Test and determine by atomic absorption spectroscopy (FNP 5)
TESTS
PURITY TESTS
Free sucrose	Determine by gas liquid chromatography (FNP5) using the following conditions: Reagents: - Internal Standard: 5 mg/ml cholesterol in chloroform or 10 mg/ml tetracosane in chloroform - Pyridine (dried over molecular sieve) - N,O-Bis-(Trimethylsilyl)-acetamide (BSA) - Trimethylchlorosilane (TMCS) Procedure: Weigh accurately 20-50 mg of the sample into a silylation vial, add 1 ml internal standard solution, 1 ml pyridine, and 0.5 ml each of BSA and TMCS. Seal vial, and heat at 70� for 30 min. Inject 1 �l into the gas liquid chromatograph. Conditions: Column: - length: 2-3 m - diameter: 4 mm (i.d.) - material: glass - packing: Dexil Carrier gas: Nitrogen Flow rate: 40 ml/min Detector: FID Temperature programme: Hold for 1 min at 160�, then 160-375� at 15�/min Measure peak areas for sucrose and internal standard. The response factor (RF) is calculated from a number of gas liquid chromatography runs with standard solutions of sucrose containing internal standard. Calculation: and
Dimethyl formamide	Determine by hydrolysis to dimethylamine and analysis by gas liquid chromatography (FNP 5) using the following conditions: Reagents: - Dimethyl formamide - Dimethylamine hydrochloride - Methanol - Ethanol - Hydrochloric acid - Sodium hydroxide Standard solutions: Prepare 4.47 mg/ml (equivalent to 4.0 mg/ml of dimethyl formamide) stock solution of dimethylamine hydrochloride in ethanol, and prepare standard solutions equivalent to 4, 2 and 1 �g/ml of dimethyl formamide, respectively, by dilution of the stock solution with 0.1% sodium hydroxide solution in ethanol. Sample preparation: The apparatus for the hydrolysis is shown in Figure. Weigh accurately about 40 g of the sample into a 1000-ml round-bottomed flask. Add 500 ml of 5% methanolic solution of sodium hydroxide, and attach the flask to the apparatus. Set an Erlenmeyer flask containing 10 ml of 1% methanolic solution of hydrochloric acid to the apparatus. Heat the round-bottomed flask and let the content reflux for 1 hour, then distil to collect about 50 ml of the distillate while cooling water of the reflux condenser is stopped. Evaporate the distillate to almost dryness on a boiling water bath. Dissolve the residue with a small amount of ethanol, add 2.5 ml of 5% ethanolic solution of sodium hydroxide, and dilute to 25 ml with ethanol to prepare a sample solution. Procedure: Inject 2 �l of the sample solution into the gas liquid chromatograph under the conditions below. Calibration curve: Prepare a calibration curve by injecting each 2 �l of the standard solutions into the gas chromatograph. Conditions: Column: - length: 2 m - diameter: 2 mm (i.d.) - material: Glass - packing: 10% amine 220 and 10% KOH on 80/100 weak acid washed Chromosorb W - conditioning: Heat to 130� overnight with 5 ml/min of nitrogen flow rate Carrier gas: Nitrogen Flow rate: 17 ml/min Detector: FID Temperatures - injection port: 198�5� - column: 60� Calculation: where C_DFA = Concentration of dimethyl formamide C = Concentration of dimethyl formamide detected W = weight of sample taken
Dimethyl sulfoxide	Determine by gas liquid chromatography (FNP 5) under the following conditions: Reagents: - Tetrahydrofuran - Dimethyl sulfoxide Standard solutions (prepared fresh monthly): - Prepare a 0.25 mg/ml stock solution of dimethyl sulfoxide in tetrahydrofuran - Prepare a range of solutions containing 0.5, 1 and 5 �g/ml of dimethyl sulfoxide by dilution of the stock solution with tetrahydrofuran. Procedure: Weigh accurately about 5 g of the sample, dissolve and dilute it with tetrahydrofuran to 25 ml to prepare a sample solution. Inject 3 �l of the sample solution into the gas chromatograph under the conditions below. Calibration curve (prepared daily): Prepare a calibration curve by injecting each 3 �l of the standard solutions into the gas chromatograph Conditions: Column: - length: 2 m - diameter: 3 mm (i.d.) - material: Glass - packing: 10% PEG 20M and 3% KOH on Gas Chrom Z - conditioning: Raise the oven temperature to 180� at a rate of 10�/min and let stand for 24 to 48 h with 30 to 40 ml/min of nitrogen Carrier gas: Nitrogen Flow rate: 50 ml/min Detector: Flame photometric (using 394 nm sulfur filter) Temperatures - injection port: 210� - column: 160� (do not exceed 200�) Calculation where C_DMSO = Concentration of dimethyl sulfoxide C = Concentration of dimethyl sulfoxide detected W = weight of sample taken
Propylene glycol	Determine by gas liquid chromatography (FNP 5) using the following conditions: Reagents: - Propylene glycol - 1,2-Butylene glycol - Acetic anhydride - Toluene - Acetone Standard solutions: - Internal standard solution: Prepare 1000 mg/l solution of 1,2-butylene glycol in acetone. 200 �l of the solution contains 0.2 mg of 1,2-butylene glycol. - Standard solution of propylene glycol: Prepare 1000 mg/l solution of propylene glycol in acetone. Sample preparation: Weigh accurately about 2 g of the sample in a 100-ml flat-bottomed flask, and add 10 ml of acetic anhydride and 200 �l of the internal standard solution. Attach a reflux condenser and heat the flask in a boiling water bath for 1 hour. Add 50 ml of water and heat the flask for another 10 min in a boiling water bath. After heating, let it cool to room temperature. Transfer the content to a 100-ml separating funnel and extract with 10 ml of toluene. After separation, discharge the lower layer (aqueous phase). Add 50 ml of water to the separating funnel and wash the extract. The upper layer (toluene phase) is used as a sample solution. Procedure: Inject 2 �l of the sample solution into the gas chromatograph under the following conditions. (Note: Since there would be a component of high boiling point, which may have a longer retention time, it is necessary at the end of each measurement to raise the oven temperature to 200� and to evacuate it from the column). Calibration curve: Follow the same procedure using 100, 200 and 500 �l of the standard solution of propylene glycol in place of the sample, and prepare a calibration curve. Column: - length: 2 m - diameter: 3 mm (i.d.) - material: Glass - packing: 5% Alkyleneglycol phthalate on 80/100 Chromosorb W - conditioning: Heat to 150� overnight with approximately 50 ml/min of nitrogen Carrier gas: Nitrogen Flow rate: 40 ml/min Detector: Flame ionization Temperatures: - injection port: 230� - column: 110� Calculation: where C = Concentration of propylene glycol (mg/kg) A_PG = Peak area of propylene glycol A_IS = Peak area of internal standard W_SPL = Weight of sample (g) C_fPG = Sensitivity correction factor for propylene glycol (slope of the calibration curve)
Methanol, isopropanol, isobutanol, ethyl acetate and methyl ethyl ketone	Determine by gas chromatography (FNP 5) with a head space sampler using the following conditions: Reagents: - Methanol - Isopropanol - Isobutanol - Ethyl acetate - Methyl ethyl ketone Standard solutions: Take each 1 g of methanol, isopropanol, isobutanol, methyl ethyl ketone and ethyl acetate in a volumetric flask and add water to total volume of 100 ml, and prepare 0.02-0.4 g/100 ml solutions by dilution of this solution. If necessary, prepare standard solutions containing up to 7 g/100 ml of isopropanol and ethyl acetate. Procedure: Place 1 g (1.0�0.1 g) of powdered sample in a sample vial. Add 5 �l of water to the sample vial and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the below-mentioned conditions. Calibration curve: Take 1 g of powdered sucrose esters of fatty acids, solvent free or known residual solvent contents, in a sample vial, add 5 �l of the standard solution and seal it quickly with a septum. Set the sample vial in a pre-conditioned gas chromatograph and start the analysis under the following conditions and obtain calibration curves for each solvent. Column: - length: 30 m - diameter: 0.53 mm (i.d.) - material: Silica capillary - film: 100% methyl polysiloxane - conditioning: Heat to 60� for 2-3 h with approximately 10 ml/min of nitrogen Carrier gas: Nitrogen Flow rate: 5 ml/min Detector: Flame ionization Temperatures - injection port: 110� - column: 40� - detector: 110� Head space sampler: - Sample volume: 1.0�0.1 g + 5 �l - Sample heating temp.: 80� - Sample heating time: 40 min - Syringe temperature: 85� - Sample gas injection: 0.4 ml Calculation: where C_i = Concentration of solvent i (mg/kg) A_i = Peak area of solvent i (�v.sec.) Cf_i = Conversion coefficient for solvent i (slope of the calibration curve) (�g/�v.sec)
METHOD OF ASSAY	Determine by high pressure liquid chromatography (FNP5) using the following conditions: Sample preparation: Add about 250 mg of the sample, accurately weighed to a 50 ml volumetric flask. Dilute to volume with tetrahydrofuran, and mix. Filter through a 0.5-�m membrane filter. Procedure: Inject 100 �l of the sample into the pre-stabilized high pressure liquid chromatograph. Conditions: Column: Styrene-divinylbenzene copolymer for gel permeation chromatography (TSK-GEL G2000 (Supelco) or equivalent) Mobile phase: HPLC-grade degassed tetrahydrofuran Flow rate: 0.7 ml/min Detector: Refractive index detector Temperatures: Column: 38� Detector: 38� Record the chromatogram for about 90 min. Calculate the percentage of sucrose ester content in the sample taken by the formula: 100 A/T where A = the sum of peak areas for the three main components, the mono-, di- and triesters, eluting at about 65, 68 and 73 min, respectively T = the sum of all peak areas eluting within 90 min Figure Apparatus for hydrolysis a: Reflux condenser b: Condenser c: Round bottomed flask d: Water bath e: Erlenmeyer flask