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Section B: Specifications of certain food additives (uses other than as flavouring agents) and other substances


Aspartame-acesulfame salt

7



Beeswax

11



Calcium L-5-methyltetrahydrofolate

15



Candelilla wax

21



Ethyl maltol

23



Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae

25



Maltol

29



Phospholipase A1 from Fusarium venenatum expressed in Aspergillus oryzae

31



Pullulan

35



Quillaia extract (Type 1)

39



Quillaia extract (Type 2)

43



Sucrose esters of fatty acids

47

Aspartame-acesulfame salt


Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 55th JECFA (2000) and published in FNP 52 Add 8 (2000). The ADI for aspartame (0-40 mg/kg bw) established at the 25th JECFA (1981) and the ADI for acesulfame K (0-15 mg/kg bw) established at the 37th JECFA (1990) cover the aspartame and acesulfame moieties of the salt.



SYNONYMS

Aspartame-acesulfame, INS No. 962



DEFINITION

The salt is prepared by heating an approximately 2:1 ratio (w:w) of aspartame and acesulfame K in solution at acidic pH and allowing crystallization to occur. The potassium and moisture are eliminated. The product is more stable than aspartame alone.



Chemical names

6-methyl-1,2,3-oxathiazine-4(3H)-one-2,2-dioxide salt of L-phenylalanyl-2-methyl-L-á-aspartic acid.
[2-carboxy-â-(N-(b-methoxycarbonyl-2-phenyl)ethylcarbamoyl)]ethanaminium-6-methyl-4-oxo-1,2,3-oxathiazin-3-ide-2,2-dioxide.



C.A.S. number

106372-55-8



Chemical formula

C18H23O9N3S



Structural formula



Formula weight

457.46



Assay

63.0% to 66.0% aspartame (dried basis) and 34.0% to 37.0% acesulfame (acid form on a dried basis).



DESCRIPTION

A white, odourless, crystalline powder



FUNCTIONAL USES

Sweetening agent, flavour enhancer



CHARACTERISTICS




IDENTIFICATION




Solubility (FNP 5)

Sparingly soluble in water, and slightly soluble in ethanol.



PURITY




Loss on drying (FNP 5)

No more than 0.5% (105°, 4h)



Transmittance (FNP 5)

The transmittance of a 1% solution in water determined in a 1 cm cell at 430 nm with a suitable spectrophotometer using water as a reference, is not less than 0.95, equivalent to an absorbance of not more than approximately 0.022.



Specific rotation (FNP 5)

[a] D 20+14.5 to +16.5.
After preparing a solution of 6.2 g of sample in 100 ml formic acid (15N), make the measurement within 30 min of preparation of the solution. Divide the calculated specific rotation by 0.646 to correct for the aspartame content of the aspartame-acesulfame salt.



5-Benzyl-3,6-dioxo-2-piperazineacetic acid

Not more than 0.5%
See description under TESTS



Lead (FNP 5)

Not more than 1 mg/kg.
Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in FNP 5, "Instrumental Methods."



TESTS




PURITY TESTS




5-Benzyl-3,6-dioxo- 2-piperazine acetic acid

Principle
5-benzyl-3,6-dioxo- 2-piperazine acetic acid is determined in aspartame-acesulfame salt dissolved in methanol-water by comparison to an authentic standard after separation by HPLC.

Apparatus
Use a suitable high-pressure liquid chromatograph equipped with UV detector for measuring absorbance at 210 nm and a 250 x 4.6 mm column packed with octyldecyl silanized silica (10-µm Partisil ODS-3 or equivalent) and operated under isocratic conditions at 40°.

Mobile phase
Dissolve 5.6 g of potassium phosphate monobasic into 820 ml of water in a 1-l flask and adjust the pH to 4.3 with phosphoric acid. Add 180 ml of methanol and mix. Filter through a 0.45 µm filter and de-gas.

Standard
Accurately weigh approximately 25 mg of authentic 5-benzyl-3,6-dioxo- 2-piperazine acetic acid into a 100-ml volumetric flask; add 10 ml of methanol to dissolve the material and dilute to volume with water and mix. Accurately transfer 15 ml of this solution to a 50-ml flask and dilute to volume with a 1:9 (v:v) mixture of methanol:water prepared on the day of use.

Sample
Accurately weigh approximately 50 mg of sample into a 10 ml volumetric flask and dilute to volume with a 1:9 (v:v) mixture of methanol:water prepared on the day of use.

Procedure
Separately inject 20 µl portions of the standard and the sample into the chromatograph (set the flow rate of the mobile phase at 2 ml/min.) and record the peak areas in standard and sample chromatograms (under the conditions described, the retention time of 5-benzyl-3,6-dioxo-2-piperazine acetic acid and aspartame are approximately 4 and 11 min, respectively). Measure the peak area response of 5-benzyl-3,6-dioxo-2-piperazine acetic acid in each chromatogram and calculate the percentage of 5-benzyl-3,6-dioxo-2-piperazine acetic acid as follows:

% = 1000(AUCS)/(ASWu)

Where AU and AS are the peak areas of 5-benzyl-3,6-dioxo-2-piperazine acetic acid in the sample and standard, respectively, CS is the concentration of 5-benzyl-3,6-dioxo-2-piperazine acetic acid in the standard in mg/ml and Wu is the weight, in mg, of aspartame-acesulfame salt taken in the sample preparation.



METHOD OF ASSAY

Principle
Aspartame-acesulfame salt is dissolved in methanol and potentiometrically titrated with tetrabutylammonium hydroxide.

Apparatus
Use a suitable autotitrator (e.g., Metrohm 670, or equivalent) equipped with a glass pH electrode and a silver-silver chloride double liquid junction reference electrode (e.g., Yokogawa pH electrode SM 21-AL4 or equivalent and reference electrode SR 20-AS52 or equivalent).

Standard solution
Prepare a 0.1 M standard solution of tetrabutylammonium hydroxide in a 1:1 (v:v) mixture of 2-propanol:methanol. Weigh 24 and 98 mg benzoic acid with0.01 mg accuracy and dissolve each into two 50-ml volumetric flasks and dilute to volume with 2-propanol. Titrate both solutions with the 0.1 M tetrabutylammonium hydroxide and record the volume required to reach the equivalence point with 0.001 ml accuracy. Perform a blank titration on 50 ml of2-propanol. Determine the standard factor (F) for each titration. and average the two factors as follows:

F = [(WSx 1000)/(122 x (VS-VO))]




Where:

WS = weight of primary benzoic acid (g)
VS = volume of equivalence point (ml)
VO = volume of equivalence for the blank (ml)
122 = molecular weight of benzoic acid




Procedure
Weigh accurately 100 to 150 mg of sample and dissolve it in 50 ml methanol. Titrate with the standardized 0.1 M tetrabutylammonium hydroxide. Determine the volume (ml) of the standard solution needed to reach the first (V1) and second(V2) equivalency points. Perform a blank titration on the methanol. Calculate the acesulfame and aspartame content as follows:

Acesulfame content (% m/m) = [(V1-VB) x N x 163/(10 x W)]
Aspartame content (% m/m) = [(V2-V1) x N x 294/(10 x W)]




Where:

W = Weight of sample (g)
V1 = volume of first equivalence point (ml)
V2 = volume of second equivalence point (ml)
VB = volume of equivalence point of blank (m)l
N = normality of the standard 0.1 M tetrabutylammonium hydroxide
163 = formula weight of acesulfame moiety
294 = formula weight of aspartame moiety
10 = conversion of g to % (m/m)

Beeswax


Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 39th JECFA (1992) and published in FNP 52 Add 1 (1992), and incorporating the decisions on the metals and arsenic specifications agreed at the 63rd JECFA (2004) and published in FNP 52 Add 12 (2004). The 65th JECFA (2005) considered the additive to be of no toxicological concern for the functional uses listed.



SYNONYMS

INS No. 901



DEFINITION

Beeswax is obtained from the honeycombs of bees (Fam. Apidae,e.g. Apis melliferaL) after the honey has been removed by draining or centrifuging. The combs are melted with hot water, steam or solar heat; the melted product is filtered and cast into cakes of yellow beeswax. White beeswax is obtained by bleaching the yellow beeswax with oxidizing agents (e.g. hydrogen peroxide, sulfuric acid) or sunlight. Beeswax consists of a mixture of esters of fatty acids and fatty alcohols, hydro-carbons and free fatty acids; minor amounts of free fatty alcohols are also present.



C.A.S. number

8006-40-4 (yellow beeswax)
8012-89-3 (white beeswax)



DESCRIPTION

Yellow beeswax: yellow or light-brown solid that is somewhat brittle when cold and presents a dull, granular, non-crystalline fracture when broken; it becomes pliable at about 35°. It has a characteristic odour of honey.

White beeswax: white or yellowish white solid (thin layers are translucent) having a faint and characteristic odour of honey



FUNCTIONAL USES

Glazing agent; release agent; stabilizer; texturizer for chewing gum base; carrier for food additives (including flavours and colours); clouding agent



CHARACTERISTICS




IDENTIFICATION




Solubility (FNP 5)

Insoluble in water; sparingly soluble in alcohol; very soluble in ether



PURITY




Melting range (FNP 5)

62 - 65°



Acid value (FNP 5)

17 - 24



Peroxide value

Not more than 5
See description under TESTS



Saponification value (FNP 5)

87 -104



Carnauba wax

Passes test
See description under TESTS



Ceresin, paraffins, and certain other waxes

Passes test
See description under TESTS



Fats, Japan wax, rosin and soap

Passes test
See description under TESTS



Glycerol and other polyols

Not more than 0.5 % (calculated as glycerol)
See description under TESTS



Lead (FNP 5)

Not more than 2 mg/kg
Determine using an atomic absorption 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 FNP 5, "Instrumental Methods".



TESTS




PURITY TESTS




Peroxide value

Weigh accurately 5 g of the sample into a 200-ml conical flask. Add 30 ml of a 2:3 solution of chloroform and acetic acid TS and close the flask with a stopper. Heat with warm water and swirl to dissolve the sample. Cool to room temperature and add 0.5 ml of saturated potassium iodide solution. Close the flask with the stopper and shake vigorously for 60±5 sec. Add 30 ml of water and titrate imme- diately with 0.01 N sodium thiosulfate using starch TS as indicator. Carry out a blank determination.




Peroxide value = (a-b) x N x 1000/W




where




a = volume (ml) of sodium thiosulfate used for the sample
b = volume (ml) of sodium thiosulfate used for the blank
N = normality of the sodium thiosulfate
W = weight of sample (g)



Carnauba wax

Transfer 100 mg of the sample into a test tube, and add 20 ml of n-butanol. Immerse the test tube in boiling water, and shake the mixture gently until the sample dissolves completely. Transfer the test tube to a beaker of water at 60°, and allow the water to cool to room temperature. A loose mass of fine, needle- like crystals separates from clear mother liquor. Under the microscope, the crystals appear as loose needles or stellate clusters, and no amorphous masses are observed, indicating the absence of carnauba wax.



Ceresins, paraffins and certain other waxes

Transfer 3.0 g of the sample to a 100 ml round-bottomed flask, add 30 ml of a 4% w/v solution of potassium hydroxide in aldehyde-free ethanol and boil gently under a reflux condenser for 2h. Remove the condenser and immediately insert a thermometer. Place the flask in water at 80° and allow to cool, swirling the solution continuously. No precipitate is formed before the temperature reaches65°, although the solution may be opalescent.



Fats, Japan wax, rosin and soap

Boil 1 g of the sample for 30 min with 35 ml of a 1 in 7 solution of sodium hydroxide, maintaining the volume by the occasional addition of water, and cool the mixture. The wax separates and the liquid remains clear. Filter the cold mixture and acidify the filtrate with hydrochloric acid. No precipitate is formed.



Glycerol and other polyols

To 0.20 g of the sample in a round-bottom flask, add 10 ml of ethanolic potassium hydroxide TS, attach a reflux condenser to the flask and heat in a water bath for 30 min. Add 50 ml of dilute sulfuric acid TS, cool and filter. Rinse the flask and filter with dilute sulfuric acid TS. Combine the filtrate and washings and dilute to 100.0 ml with dilute sulfuric acid TS. Place 1.0 ml of the solution in a tube, add 0.5 ml of a 1.07 % (w/v) solution of sodium periodate, mix and allow to stand for 5 min. Add 1.0 ml of decolourized fuchsin solution (see below) and mix. Any precipitate disappears. Place the tube in a beaker containing water at 40°. Allow to cool while observing for 10 to 15 min. Any bluish-violet colour in the solution is not more intense than a standard prepared at the same time in the same manner using 1.0 ml of a 0.001 % (w/v) solution of glycerol in dilute sulfuric acid TS.

Decolourized fuchsin solution: Dissolve 0.1 g of basic fuchsin in 60 ml of water. Add a solution of 1 g of anhydrous sodium sulfite (Reagent grade) in 10 ml of water. Slowly and with continuous shaking of the solution add 2 ml of hydrochloric acid. Dilute to 100 ml with water. Allow to stand protected from light for at least 12 h, decolourize with activated charcoal and filter. If the solution becomes cloudy, filter before use. If on standing the solution becomes violet, decolourize again by adding activated charcoal. Store protected from light.

Calcium L5-methyltetrahydrofolate


New specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005). At the 65th JECFA (2005) the Committee had no safety concerns for the use of the substance in dry crystalline or microencapsulated form as an alternative to folic acid used in dietary supplements, foods for special dietary uses and other foods.



SYNONYMS

L-5-Methyltetrahydrofolic acid, calcium salt
L-Methyltetrahydrofolate, calcium salt
L-Methylfolate, calcium
L-5-MTHF-Ca



DEFINITION

Calcium L-5-methyltetrahydrofolate is the calcium salt of L-5-methyltetrahydrofolic acid, which is the predominant, naturally occurring form of folate. It is synthesized by reduction of folic acid to tetrahydrofolic acid followed by methylation and diastereoselective crystallization (in water) of L-5-methyltetrahydrofolic acid as its calcium salt. The product contains variable amounts of water of crystallization.



Chemical name

N-{4-[[((6S)-2-amino-3,4,5,6,7,8-hexahydro-5-methyl-4-oxo-6-pteridinyl)methyl]amino]benzoyl}-L-glutamic acid, calcium salt



C.A.S. number

151533-22-1



Chemical formula

C20H23CaN7O6 (anhydrous form)



Structural formula

(anhydrous form)



Formula weight

497.5 (anhydrous form)



Assay

95.0 - 102.0% (anhydrous basis)





DESCRIPTION

White to light yellowish, almost odourless, crystalline powder



FUNCTIONAL USES

Nutritional supplement



CHARACTERISTICS




IDENTIFICATION




Solubility (FNP 5)

Sparingly soluble in water and very slightly soluble or insoluble in most organic solvents; soluble in alkaline solutions



Infrared absorption

The infrared absorption spectrum of a potassium bromide dispersion of the sample corresponds to that of a Calcium L-5-methyltetrahydrofolate standard (see Appendix).



Calcium

Dilute 30 g of acetic acid (glacial) to 100 ml with water. Dissolve 5.3 g of K4Fe(CN)6 in 100 ml of water. To 5 ml of the acetic acid solution, add 20 mg of the sample and then 0.5 ml of the potassium ferrocyanide solution. Mix and add 50 mg of ammonium chloride. A white crystalline precipitate is formed.



Liquid chromatography

Retention time matches that of a reference standard (see under TESTS)



PURITY




Water (FNP 5)

Not more than 17.0% (Karl Fischer method)
Note: Allow sufficient time (15 min) for release of bound water.



Calcium

7.0 - 8.5% (anhydrous basis)
Accurately weigh 500 mg of sample and transfer to a 500-ml conical flask. Add150 ml of water to dissolve the sample and 20 ml of a pH 10 buffer (NH3/NH4Cl). Using eriochrome black T as indicator, titrate (continuous stirring) with standardized 0.1 M EDTA until the colour changes from violet to blue/green. Each 1.0 ml of 0.1 M EDTA corresponds to 4.008 mg of calcium. Calculate the




calcium content on the anhydrous basis.

Other folates and related substances

Not more than 2.5%
See description under TESTS



D-5-Methylfolate

Not more than 1.0%


See description under TESTS

Total viable aerobic count (FNP 5)

Not more than 1000 CFU/g



Lead (FNP 5)

Not more than 2 mg/kg
Determine using an atomic absorption 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 FNP 5, "Instrumental methods".



TESTS




PURITY TESTS




Other folates and related substances

Principle
Using a reference standard for L-5-Methyltetrahydrofolic acid, calcium salt, Other folates and related substances are quantitated by HPLC. The suitability of the applied HPLC system is checked daily by a system suitability test.

Reference standard solution
Accurately weigh 50 mg of L-5-methyltetrahydrofolic acid, calcium salt (Merck Eprova AG, CH-8200 Schaffhausen, Switzerland) into a 100-ml volumetric flask. Dissolve in a small quantity of water and dilute to volume.

Sample solution
Prepare as for the reference standard using 50 mg of the sample.

Mobile phase solutions
A: Dissolve 7.80 g of NaH2PO4 · 2H2O (0.05 mol) in 1000 ml of water and adjust the pH to 6.5 with 32% NaOH. Filter and degas the solution.
B: Dissolve 5.07 g of NaH2PO4 · 2H2O (0.03 mol) in 650 ml of water and 350 ml of methanol (chromatography grade) and adjust the pH to 8.0 with 32% NaOH. Filter and degas the solution.




Chromatography conditions


Column:

Hypersil-ODS, 5 µm; 250 x 4 mm (Thermo Hypersil Keystone or equivalent)


Flow rate:

1.1 ml/min


Gradient:

Time (min)

% Mobile

% Mobile

Remark




phase A

phase B




0

100

0

Start



0 - 14

100 - 45

0 - 55

Linear gradient



14 - 17

45 - 0

100

Linear gradient



17 - 22

0

100

Hold



22 - 31

100

0

Reconditioning




Temperature: Room temperature
Injection volume: 10 µl
Detection: UV (280 nm)
Run time: 22 min




Retention times given below are approximate:




Folates and related substances

Retention time (min)


4-Aminobenzoylglutamic acid (ABGA)

3.1


4a-Hydroxy-5-methyltetrahydrofolic acid (HOMeTHFA)

4.3


D-Pyrazino-s-triazine derivative (D-Mefox)

6.1


L-Pyrazino-s-triazine derivative (L-Mefox)

6.3


Tetrahydrofolic acid (THFA)

8.5


7,8-Dihydrofolic acid (DHFA)

11.2


Folic acid (FA)

11.4


5,10-Methylenetetrahydrofolic acid (CH2THFA)

11.7


5-Methyltetrahydrofolic acid (5-MTHF)

13.6


5-Methyltetrahydropteroic acid (MeTHPA)

15.1


N2-Methylamino-5-methyltetrahydrofolic acid (DiMeTHFA)

17.6




Sample analysis
Inject the reference standard solution and the sample solutions immediately after preparation, using the conditions described above. (Note: After analysis, flush the column with methanol/water 85:15 and store it under the same conditions.) Calculate the content of each folate (other than 5-MTHF) and related substance, Xi (%), listed in the above table according to the following formula:

Xi (%) = Ai ×WS × S × (RF)i/AS × W




where

Ai = the peak area for each folate (other than 5-MTHF) and related substance
AS = the peak area for the L-5-MTHF-Ca Standard
WS = the weight (mg) of L-5-MTHF-Ca Standard
W = the weight (mg) of the sample
S = the percent of L-5-MTHF in the L-5-MTHF-Ca Standard, calculated as free acid
(RF)i = Response Factor for the i-th substance (absorbance at 280 nm in the applied eluent system relative to that of L-5-MTHF) as follows:




Folates and related substances

RF


ABGA

0.93


HOMeTHFA

1.11


L-Mefox and D-Mefox

1.11


DHFA

0.98


FA

0.86


MeTHPA

0.68


THFA

1.00


CH2THFA

1.00


DiMeTHFA

1.00


If there are any unidentified impurities, apply a RF of 1.00

Calculate the total amount of "Other folates and related substances" by summing the Xi for all impurities.

System suitability test
Mixed folates solution: Weigh 25 mg each of ABGA, HOMeTHFA, L-Mefox, DHFA, FA and MeTHPA (all available from Merck Eprova AG) into a 100-ml volumetric flask. Add a small quantity of water to dissolve the mixture; add some sodium hydrogen carbonate and sodium carbonate to aid the dissolution, and fill to the mark with water.

System suitability test solution (SST solution): Weigh accurately 50 mg of a L-5-MTHF-Ca sample containing DiMeTHFA into a 100-ml volumetric flask. (Available from Merck Eprova AG). Add 1 ml of the Mixed folates solution and a small quantity of water to dissolve, mix and dilute to volume with water.

Procedure: Inject 10 µl of the SST solution immediately. The resolution between L-5-MTHF and MeTHPA must be at least 5.



D-5-Methylfolate

Principle
D-5-Methylfolate is quantitated by HPLC using a chromatographic system which allows separation of the D- from the L-stereoisomer. The suitability of the applied HPLC system is checked daily by a "system suitability test".

Sample preparation
Accurately weigh 50 mg of the sample into a 100 ml volumetric flask. Dissolve in water and dilute to volume with water.

Mobile phase
Mix 970 ml of 0.03 M NaH2PO4(obtained by dissolving 4.68 g of NaH2PO4 · 2H2O in water and diluting with water to 1000 ml) with 30 ml of acetonitrile(chromatography grade) and adjust the pH to 6.8 with 32% NaOH. Filter and degas the solution.




Chromatography Conditions


Column:

Chiral Protein HSA, 5 µm, 150 x 4 mm (ChromTech or equivalent)


Flow rate:

1 ml/min


Temperature:

40°


Injection volume:

10 µl


Detection:

UV (280 nm)


Run time:

22 min


Solvent:

Water




Sample analysis
Inject the sample solution immediately after preparation using the conditions described above. Determine the areas under peak for L-5-MTHF (retention time: ca. 11 min) and D-5-MTHF (retention time: ca. 15 min).

Calculation
Determine the ratio of the peak area for the D-isomer (AD) to the sum of the peak areas for the D- and L-isomers (AT), and calculate the D-5-MTHF content as follows:

D-5-MTHF (%) = 100AD/AT

System suitability test
System suitability test solution (SST solution): Weigh and transfer into a 200-ml volumetric flask the following: 1.0 mg of HOMeTHFA, 1.5 mg ABGA, 2.0 mg each of L-Mefox and MeTHPA, 3.0 mg of FA, 4.0 mg of DHFA, 10 mg of L,D-5-MTHF and D,D-5-MTHF (L-5-MTHF and D-5-MTHF carrying D-glutamic acid substitution), and 50 mg of racemic 5-MTHF-Ca (L-5-MTHF and D-5-MTHF carrying L-glutamic acid substitution) (all available from Merck Eprova AG). Add a small quantity of water to dissolve the mixture; add some sodium hydrogen carbonate to aid the dissolution, and fill to the mark with water. Immediately inject into the HPLC system. The resolution between L-5-MTHF and D-5-MTHF must be at least 2.



METHOD OF ASSAY

Calculate the percentage of L-5-MTHF-Ca in the sample from the content of 5-MTHF-Ca (L- and D-diastereoisomers), determined in the test for "Other folates and related substances", and the content of D-5-MTHF-Ca, determined in the test for D-5-Methylfolate, and correcting for water content, as follows:

L-5-MTHF-Ca (%) =100 × AT× WS× S × (100 - D) × 1.083/AS × W × (100 - %H2O)




where


AT is taken from the calculation for the D-5-Methylfolate analysis
D = the percentage of D-5-Methylfolate in the sample
AS, W, WS, and S are taken from the determination of Other folates and related substances
%H2O = water content (%)
1.083 is the ratio of the formula weight of 5-MTHF-Ca to that of 5-MTHF.

Candelilla wax


Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 39th JECFA (1992) and published in FNP 52 Add 1 (1992), and incorporating the decisions on the metals and arsenic specifications agreed at the 63rd JECFA (2004) and published in FNP 52 Add 12 (2004). The 65th JECFA (2005) considered the additive to be of no toxicological concern for the functional uses listed.



SYNONYMS

INS no. 902



DEFINITION

Crude candelilla wax is obtained by first boiling the dried stalks of the candelilla plant (Euphorbia antisyphilitica) in water acidified with sulfuric acid to release the wax. The molten wax is then skimmed off and allowed to solidify and refined by further treatment with sulfuric acid and subsequent passage through filter-presses.
Candelilla wax consists primarily of odd-numbered n-alkanes (C29 to C33), together with esters of acids and alcohols with even-numbered carbon chains (C28 to C34). Free acids, free alcohols, sterols, neutral resins, and mineral matter are also present.



C.A.S. number

8006-44-8



DESCRIPTION

Yellowish-brown hard, brittle, lustrous solid with an aromatic odour when heated



FUNCTIONAL USES

Glazing agent, texturizer for chewing gum base, surface-finishing agent, carrier for food additives (including flavours and colours), clouding agent



CHARACTERISTICS




IDENTIFICATION




Solubility (FNP 5)

Insoluble in water; soluble in toluene



Infrared absorption

The infrared spectrum of the sample, melted and prepared for analysis on a potassium bromide plate, corresponds to that of a candelilla wax standard (see Appendix).



PURITY




Melting range (FNP 5)

68.5 - 72.5°



Acid value (FNP 5)

Between 12 and 22



Saponification value (FNP 5)

Between 43 and 65



Lead (FNP 5)

Not more than 2 mg/kg
Determine using an atomic absorption 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 FNP 5, "Instrumental Methods".

APPENDIX

Infrared spectrum of candelilla wax

Ethyl maltol


(Tentative)

Information on functional uses and method of assay required

Revised tentative specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2006), superseding specifications prepared at the 14th JECFA (1970) and published in NMRS 48B (1971) and in FNP 52 (1992), and incorporating the decisions on metals and arsenic specifications agreed at the 57th JECFA (2001) and published in FNP 52 Add 9 (2001). An ADI of 0-2 mg/kg bw was established at the 18th JECFA (1974)



SYNONYMS

INS No. 637



DEFINITION




Chemical names

2-Ethyl-3-hydroxy-4-pyrone



C.A.S. number

4940-11-8



Chemical formula

C7H8O3



Structural formula



Formula weight

140.14



Assay

Not less than 98%



DESCRIPTION

White, crystalline powder having a characteristic odour



FUNCTIONAL USES

Flavour enhancer, stabilizer, flavouring agent (See 'Flavouring agents' monograph No. 1481)



CHARACTERISTICS




IDENTIFICATION




Solubility

Sparingly soluble in water; soluble in alcohol



Melting range

89 - 93°



PURITY




Water (FNP 5)

Not more than 0.5 % w/w (Karl Fischer Method)



Sulfated ash (FNP5)

Not more than 0.2 % w/w



Lead (FNP 5)

Not more than 1mg/kg Determine using an atomic absorption 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 FNP 5, "Instrumental Methods".



METHOD OF ASSAY

Prepare a solution of ethyl maltol in 0.1 N hydrochloric acid containing 10 µg/ml, and determine the extinction at 276 mm. E (1%, 1 cm): 276 nm: 655-675

Calculation

Ethyl maltol (%) = 100 x E(sample)/E(standard)

where

E = E (1%, 1 cm)

Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae


Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 61st JECFA (2003) and published in FNP 52 Add 11 (2003). An ADI "not specified" was established at the 61st JECFA (2003).



SOURCES

Produced by submerged fed-batch pure culture fermentation of a genetically modified strain of Aspergillus oryzaecontaining the laccase gene derived from Myceliophthora thermophila,using recombinant DNA techniques and traditional mutagenesis. The enzyme is isolated from the fermentation broth by filtration to remove the biomass and concentrated by ultrafiltration and/or evaporation. Residual production microorganisms are removed from the enzyme concentrate by germ filtration. The final product is formulated using food-grade stabilizing and preserving agents.



ACTIVE PRINCIPLES

Laccase (synonyms: urishiol oxidase; p-diphenol oxidase)



SYSTEMATIC NAMES AND NUMBERS

Benzenediol:oxygen oxidoreductase; EC 1.10.3.2; CAS No. 80498-15-3



REACTIONS CATALYSED

Oxidation of a range of phenolic substances with concomitant reduction of oxygen to water.



DESCRIPTION

Brown liquid



FUNCTIONAL USES

Enzyme preparation. Used in the brewing of beer to prevent the formation of off-flavour compounds such as trans-2-nonenal. Scavenges oxygen that otherwise would react with fatty acids, amino acids, proteins, and alcohols to form off-flavour precursors.



GENERAL SPECIFICATIONS

Must conform to the latest edition of the JECFA General Specifications and Considerations for Enzyme Preparations used in Food Processing,



CHARACTERISTICS




IDENTIFICATION




Laccase activity

The sample shows laccase activity See description under TESTS



TESTS




Laccase activity

Principle
Laccase catalyses the oxidation of syringaldazine to tetramethoxy-azo-bis (methylene quinone) that is measured spectrophotometrically at 530 nm. Laccase activity is expressed in Laccase Myceliophthora Units (LAMU). One LAMU is defined as the amount of enzyme that oxidizes 1 micromole of syringaldazine per minute under standard conditions (pH 7.5; 30°).
(Note: The method can be adapted for manual execution; any suitable spectrophotometer may be used in place of a centrifugal analyser.)

Apparatus
Centrifugal analyser (Cobas Fara, Roche, or equivalent)
Diluter (Hamilton Microlab or equivalent)

Reagents
(Note: Use only deionised water)
Laccase standard (available from Novozymes A/S)
TRIS (Tris(hydroxymethyl)aminomethane)
Maleic acid
Triton X-100 (polyethylene glycol tert-octylphenyl ether)
Ethanol 96%
Syringaldazine
PEG 6000
Glucose
Glycine

Reagent Solutions
TRIS, 1 M stock solution: Dissolve 121.1 g of TRIS in approximately 900 ml of water in a 1-litre volumetric flask. Make to volume and mix.

Maleic acid, 1 M solution: Dissolve 23.2 g of maleic acid in approximately 150 ml water in a 200-ml volumetric flask. Make to volume and mix.

Triton X-100, 10% stock solution: In a beaker, add 25.0 g of Triton X-100 to approximately 200 ml water; stir to dissolve. Transfer to a 250-ml volumetric flask and make up to volume with water.

TRIS buffer, 25 mM (pH 7.50): Add 5 ml of 1 M TRIS, 2 ml of 1 M maleic acid solution, and 1 ml of Triton X-100 10% solution to a 200-ml volumetric flask. Add 150 ml water and adjust pH to 7.50±0.05 using 1 M maleic acid solution. Make up to volume with water. (Note: do not adjust the pH with hydrochloric acid because chloride inhibits laccase activity.)

PEG 6000, 50 g/l solution: Weigh 250 g of PEG 6000 in a beaker, transfer to a 5000 ml volumetric flask, add water and stir until dissolved. Add water to volume.

Syringaldazine, 0.56 mM stock solution: Rinse a 50-ml volumetric flask with water and ethanol to remove any soapy residues. Weigh 10.0 mg of syringaldazine in a weighing boat and transfer to the volumetric flask. Add 96% ethanol to the mark and stir until the syringaldazine is dissolved (approximately 3 h). The solution must be stored in a dark bottle in a refrigerator.

Syringaldazine, 0.22 mM working solution: Rinse a 10-ml volumetric flask with water and ethanol to remove any soapy residues. Transfer 4.0 ml of syringaldazine stock solution to the flask and add water to volume. The solution can be kept in a dark bottle for up to two hours at room temperature.

Glycine buffer, 1.5%: Dissolve 75 g glycine, 150 g glucose, and 250 g PEG 6000 in approximately 4.5 litre water in a 5-l volumetric flask. Adjust pH to 9.20±0.05 using NaOH or 1 M maleic acid. Add water to volume. (Note: Do not adjust pH with hydrochloric acid because chloride inhibits laccase activity.)

Standard and sample solutions
Laccase standard stock solution: Weigh the amount of laccase standard needed to obtain a laccase activity of 0.350 LAMU/ml and transfer the laccase to a 500-ml volumetric flask. Add 300 ml PEG 6000 solution and stir on a magnetic stirrer for 15 min to dissolve the laccase. Add PEG 6000 solution to volume. The laccase stock solution should be prepared on the day of the experiment.

Laccase working standard solutions (for the construction of the standard curve): Prepare six solutions by diluting the laccase stock solution with PEG 6000 solution as shown in the table below. Use the diluter and vials compatible with the centrifugal analyser.




Sample No.

Dilution factor

Laccase stock solution (µl)

PEG 6000 solution (µl)

Activity, LAMU/ml


1

30

20

580

0.01167


2

24

25

575

0.01458


3

20

30

570

0.01750


4

15

40

560

0.02333


5

12

50

550

0.02917


6

10

60

540

0.03500




Laccase control: Use a laccase preparation with known activity. Accurately weigh the amount of the preparation sufficient to obtain laccase activity of approximately 0.70 LAMU/ml in a 200-ml volumetric flask. Place the preparation in the flask and add the PEG 6000 solution to volume. Stir on the magnetic stirrer for 15 min. This is a stock solution. It should be prepared daily. Dilute the stock solution with the PEG 6000 solution 30 times using diluter. Place the diluted solution in a vial.
Analyse the control sample in each run to test the method's performance. A result within 8 percent of the nominal activity is acceptable.

Test samples: Dilute test samples on the basis of the anticipated enzyme content to obtain activity between 0.0117 and 0.0350 LAMU/ml.
Example: accurately weigh 0.6 g sample and dissolve in the PEG 6000 solution in a 250 ml volumetric flask. Stir the solution for 15 min on the magnetic stirrer. If necessary, dilute the sample solution again with the PEG 6000 solution. Place the solutions in vials.

Procedure
1. Pour the syringaldazine working solution (0.22 mM) into a 4-ml reagent container placed in the reagent rack of the centrifugal analyser.
2. Pour the TRIS buffer into a 15-ml reagent container placed in the reagent rack.
3. Place the vials containing standard solutions and the control sample in the calibration rack.
4. Place the vials containing the test samples in the sample rack.
5. Set up the analysis program and start the analysis.

Analysis
The analysis is performed automatically by the centrifugal analyser. The empty rotor of the analyser rotates until the temperature in the cuvette container reaches 30o. Twenty five microliters of the standard solution, control sample or test sample, 20 microliters of water, and 325 microliters of glycine buffer are pipetted into cavites in the rotor. The rotor accelerates and centrifuges and mixes the buffer and samples in the cuvettes. Subsequently, 30 microliters of the substrate is pipetted into each cuvette. The rotor accelerates and centrifuges and mixes the substrate with samples in the cuvettes. The first absorbance reading is taken five seconds later. A total of 25 readings are taken from each cuvette at 5-second intervals. Readings 12 to 24 are used to calculate the increase of absorbance per minute (DAbs/min).

Calculations
The analyser creates a standard curve and uses it to convert the.Abs/min from each cuvette containing the test sample into activity expressed in LAMU/ml. The activity of test samples expressed in LAMU/g is then calculated using the following formula:

Where

A = DAbs/min converted to activity (LAMU/ml)
Vol = volume of the volumetric flask used to dilute the test sample (ml)
D = additional dilution of the sample (ml/ml)
W = weight of the sample (g)

Maltol


(Tentative)
Information on functional uses and method of assay required

Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add13 (2005), superseding specifications prepared at the 25th JECFA (1981) and published in FNP 52 (1992). An ADI of 0-1 mg/kg bw was established at the 25th JECFA (1981)



SYNONYMS

INS No. 636



DEFINITION




Chemical names

3-Hydroxy-2-methyl-4-pyrone



C.A.S. number

118-71-8



Chemical formula

C6H6O3



Structural formula



Formula weight

126.11



Assay

Not less than 99%



DESCRIPTION

White to off-white crystalline powder having a characteristic caramel-butterscotch odour



FUNCTIONAL USES CHARACTERISTICS

Flavour enhancer, stabilizer, flavouring agent (see Flavouring agents monograph No. 1480)



IDENTIFICATION




Solubility (FNP 5)

Soluble in water and ethanol



Melting range (FNP 5)

160 - 164°



Test for phenol

Dissolve 0.1 g of the sample in 10 ml of ethanol and add 3 drops of ferric chloride TS. A reddish violet colour is produced.



Precipitation test

Dissolve 0.5 g of the sample in 10 ml of sodium hydroxide TS and pass carbon dioxide through the solution. White crystals are formed; collect and recrystallize from dilute ethanol. The crystals melt between 160 - 164°



Iodoform reaction

Dissolve 0.1 g of the sample in 5 ml dioxane, add 1 ml of sodium hydroxide TS, and add sufficiently iodine-potassium iodide TS (Iodine TS) with shaking until the colour remains. Heat on a water bath for 5 min. Yellow crystals are formed.



PURITY


Lead (FNP 5)

Not more than 1 mg/kg
Determine using an atomic absorption 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 FNP 5, "Instrumental Methods".



METHOD OF ASSAY

Standard Solution
Transfer about 50 mg of Maltol Reference Standard (available from the United States Pharmacopeia, 12601 Twinbrook Parkway, Rockeville, Md. 20852, USA), accurately weighed, into a 250-ml flask, dilute to volume with 0.1 N hydrochloric acid, and mix. Pipet 5 ml of this solution into a 100-ml volumetric flask, dilute to volume with 0.1 N hydrochloric acid, and mix.

Assay Solution
Transfer about 50 mg of the sample, accurately weighed, into a 250-ml flask, dilute to volume with 0.1 N hydrochloric acid. Pipet 5 ml of this solution into a100-ml volumetric flask, dilute to volume with 0.1 N hydrochloric acid, and mix.

Procedure
Determine the absorbance of each solution in a 1-cm quartz cell at 274 nm using0.1 N hydrochloric acid as the blank.

Calculate the percent of Maltol in the sample by the formula:

% of Maltol = 100 × WS × AA/AS × WA

where

AA = absorbance of the Assay Solution
AS = absorbance of the Reference Standard Solution
WA = weight in mg of the Assay solution (sample)
WS = weight in mg of the Reference Standard

Phospholipase A1 from Fusarium venenatumexpressed in Aspergillus oryzae


New specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005). The 65th JECFA (2005) was unable to assess the safety of the additive.



SYNONYMS

Phospholipase A1



SOURCES

Produced by submerged fed-batch pure culture fermentation of a genetically modified strain of Aspergillus oryzaecontaining the phospholipase A1 gene derived from Fusarium venenatum. The enzyme is isolated from the fermentation broth by filtration to remove the biomass and concentrated by ultrafiltration and/or evaporation. Residual production microorganisms are removed from the enzyme concentrate by germ filtration. The final product is formulated using food-grade stabilizing and preserving agents.



ACTIVE PRINCIPLES

Phospholipase A1



SYSTEMATIC NAMES AND NUMBERS

Phosphatidylcholine 1-acylhydrolase; EC 3.1.1.32; CAS No. 9043-29-2



REACTIONS CATALYSED

Hydrolysis of the sn-1 ester bond of diacylphospholipids to form 2-acyl-1-lysophospholipids and free fatty acids



DESCRIPTION

Brown liquid.



FUNCTIONAL USES

Enzyme preparation. Used in cheese production to reduce the loss of fat and milk solids and increase cheese yield.



GENERAL SPECIFICATIONS

Must conform to the latest edition of the JECFA General Specifications and Considerations for Enzyme Preparations used in Food Processing



CHARACTERISTICS




IDENTIFICATION




Phospholipase A1 activity

The sample shows phospholipase A1 activity See description under TESTS



TESTS




Phospholipase A1 activity

Principle
Phospholipase A1 activity is measured relative to a phospholipase standard using lecithin as a substrate. Phospholipase A1 catalyses the hydrolysis of lecithin to lyso-lecithin and a free fatty acid. The liberated fatty acid is titrated with 0.1 N sodium hydroxide under standard conditions (pH=8.0; 40° ± 0.5). The activity of phospholipase A1 is determined as the rate of sodium hydroxide consumption during neutralization of the fatty acid and is expressed in Lecitase units (LEU) relative to a Lecitase (phospholipase) standard.

1 LEU is defined as the amount of enzyme that under standard conditions (pH=8.0; 40° ±0.5) results in the same rate of sodium hydroxide consumption (in microeq/min) as the Lecitase standard diluted to a nominal activity of 1 LEU/g. The quantification limit of the method is approximately 1.5 LEU/ml. (Note: The method can be carried out using either an automated system or standard laboratory equipment for carrying out titration experiments. Procedures and calculations for both the automated and manual versions are described.)




Automated method
Apparatus
Printer
Computer
pH-Stat Titration Manager (analytical robot; Novo Nordisk Engineering A/S), consisting of the following elements:

- PHM290 pH-Stat Controller (Radiometer)
- ABU901 Autoburette (Radiometer)
- Liquid Handler (Gilson)
- Temperature Regulator (Gilson)
- Syringe Pump (Gilson)
- Silverson Homogenizer L4R
- pH electrode (Radiometer)

Reagents and solutions
(Note: use only deionized water)
Titrant, sodium hydroxide 0.1 N: Use NaOH solution standardized for preparation of 1000 ml of 0.1 N NaOH, for example, one ampoule of Merck Titrisol No. 1.09959 or equivalent. Transfer quantitatively the NaOH solution into a 1000-ml volumetric flask containing approximately 500 ml of water. Add water to volume and mix. The solution is stable for up to 2 months at room temperature.

Calcium chloride, 0.32 M: Weigh 4.70 g calcium chloride (CaCl2 2H2O, Merck 2382 or equivalent). Dissolve in water in a 100 ml volumetric flask. Add water to volume and mix. The solution is stable for up to one week at room temperature.

Sodium deoxycholate, 0.016 M: Weigh 6.7 g sodium deoxycholate (C24H39NaO4). Dissolve in water in a 1000-ml volumetric flask. Add water to volume and mix. The solution is stable for up to one week at room temperature.

Lecithin substrate: Use lecithin (L-á-phosphatidylcholine) Sigma P-5638 or equivalent. Mix lecithin with a spoon and weigh exactly 20.0g. Transfer to a 1000-ml beaker, add 400 ml of water and stir until the lecithin is dissolved. Add 20 ml of 0.32 M calcium chloride and stir for 1-2 min until calcium chloride is dissolved. Add 200 ml of 0.016 M sodium deoxycholate and 400 ml of water. Stir for about 0.5 hour and homogenize for 10 min on a Silverson L4R homogenizer. The solution is stable for up to one day at room temperature.

Hydrochloric acid, 1 N: Use HCl solution standardized for preparation of 1000 ml of 1 N HCl, for example, one ampoule of Merck Titrisol No. 1.09970 or equivalent. Transfer the HCl solution quantitatively to a 1000-ml volumetric flask. Add water to volume. The solution is stable for up to 6 months at room temperature.

Hydrochloric acid, 0.001 N: Transfer 1 ml of 1 N HCl to a 1000-ml volumetric flask. Add water to volume. The solution is stable for up to one week at room temperature.

Standard and sample solutions
Standard stock solution: Use a phospholipase standard (Lecitase standard from Novozymes A/S or equivalent) with known activity, for example, 103160 LEU/g. Accurately weigh (to 4 decimal places) approximately 0.73 g of the standard. Transfer to a 250-ml volumetric flask and add 0.001 N HCl to volume. Stir for approximately 30 min. The solution can be stored for 3 weeks at 5°.

Standard working solutions: Transfer quantitatively 5 ml of the standard stock solution to a 200-ml volumetric flask. Add 0.001 N HCl to volume. This solution contains approximately 7.5 LEU/ml and is referred to as 100% standard. Using the 100% standard, prepare additional standard solutions in 25-ml volumetric flasks according to the following table:




Standard solution

Approximate strength (LEU/ml)

Volume of 100% standard (ml)

Volume of 0.001 N HCl


80%

6.0

20

5


60%

4.5

15

10


40%

3.0

10

15


20%

1.5

5

20


The standard solutions are used by the automated system to calculate a 2nd degree polynomial standard curve. An example of the standard (calibration) curve is shown below

Calibration Curve

Control sample: A phospholipase product with known activity, for example, 10500 LEU/ml, is used to prepare the control sample. Transfer 1 ml of the control solution to a 50-ml volumetric flask and add 0.001 HCl to volume to obtain a stock solution. Transfer 1 ml of the stock solution to a 50-ml volumetric flask and add 0.001 N HCl to volume. Analyse the control sample in each run. A result within 10 percent of the nominal activity is acceptable.

Blank sample: Use 0.001 N HCl as the blank sample.

Test samples: Remove the test sample from a refrigerator or freezer and keep it at room temperature for approximately 2 h before analysis. Accurately weigh the sample into a measuring flask and add 0.001 N HCl to volume. Repeat dilution if necessary to obtain the activity of approximately 6 LEU/ml. The dilution should be performed within one hour of the analysis. Enter the sample weight (in grams) and the dilution volume (in milliliters) into the calculation program for the automated method or into the calculation formula for the manual method.

Procedure
The method uses a pH-stat Titration Manager at pH=8.0 and 400 ± 0.5. The pHstat Titration Manager automatically runs a two-point calibration of the pH electrode and then analyses the blank, the standard solutions, the control sample and the test samples, each in duplicate.
Start the reaction when 800 µl of the sample is added to 20 µl of the lecithin substrate. Titrate the liberated fatty acid with 0.1 N sodium hydroxide under standard conditions (pH=8.0; 400 ± 0.5). Record the rate of sodium hydroxide consumption (microeq/min) over 2 min, starting 90 sec and ending 210 sec from the start of the reaction and use to calculate the mean slope of the titration curve. The mean slopes of the titration curves for the control sample, standard solutions, and test samples are automatically transferred to the calculation program.

Calculations
All results are calculated automatically by the calculation program. A 2nd degree polynomial standard curve is calculated based on the mean slopes (microeq/min) for the standard solutions. Based on the standard curve, the calculation program calculates the results for the control and test samples in Lecitase activity units per 1 ml (LEU/ml). Subsequently, the program calculates the activity of the test samples in Lecitase activity units per one gram of the phospholipase A1 preparation (LEU/g).

Manual method
Procedure
The method is carried out using a titrator that measures the titrant consumption rate as a function of time (e.g., TitraLab 854 from Radiometer). The titrator must be programmed to maintain pH=8 and measure the NaOH consumption rate in microequivalents per minute (microeq/min). The following procedure is followed:
Calibrate the pH electrode at pH 7 and 10. Transfer 20 ml of the lecithin substrate to a beaker and place in a water bath at 40° ± 0.5. Adjust the substrate to pH 8.0 using 0.1 N NaOH and start the titration by the addition of either 0.8 ml of the standard solution, the test sample, or the control sample. Measure the NaOH consumption rate for 4 min. Determine the NaOH consumption rate between 90 and 120 sec from the reaction start and use this information to construct a standard curve and for activity calculations.

Calculation
Construct a standard curve by plotting the NaOH consumption rate (in microeq/min) against the enzyme activity (in LEU/ml). The activity of the control sample and test samples are read from the calibration curve (in LEU/ml). Note: a 2nd degree polynomial standard curve can be plotted using suitable software, and the activity of the test sample and control sample can be calculated from the standard curve using the same software.

The activity of the test samples in LEU/g is calculated according to the following equation:


where:

W(g) is the sample weight and
V(ml) is the volume of the volumetric flask in which the sample was diluted. For example, if the sample is weighed into a 50 ml volumetric flask and diluted to volume, V=50 ml.

Pullulan


New specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005). An ADI "not specified" was established at the 65th JECFA (2005).



SYNONYMS

INS No. 1204



DEFINITION

Linear, neutral glucan consisting mainly of maltotriose units connected by á-1,6 glycosidic bonds. It is produced by fermentation from a food grade hydrolysed starch using a non-toxin producing strain of Aureobasidium pullulans. After completion of the fermentation, the fungal cells are removed by microfiltration, the filtrate is heat-sterilized and pigments and other impurities are removed by adsorption and ion exchange chromatography.



C.A.S. number

9057-02-7



Chemical formula

(C6H10O5)x



Structural formula



Assay

Not less than 90% of glucan on the dried basis



DESCRIPTION

White to off-white odourless powder



FUNCTIONAL USES

Glazing agent, film-forming agent, thickener



CHARACTERISTICS




IDENTIFICATION




Solubility (FNP 5)

Soluble in water, practically insoluble in ethanol



pH (FNP 5)

5.0 - 7.0 (10% solution)



Precipitation with polyethylene glycol 600

Add 2 ml of polyethylene glycol 600 to 10 ml of a 2% aqueous solution of pullulan. A white precipitate is formed.



Depolymerisation with pullulanase

Prepare two test tubes each with 10 ml of a 10% pullulan solution. Add 0.1 ml pullulanase solution having activity 10 units/g (refer to pullulanase activity, under Methods for enzyme preparations in FNP 5) to one test tube, and 0.1 ml water to the other. After incubation at about 25° for 20 min, the viscosity of the pullulanase-treated solution is visibly lower than that of the untreated solution.



PURITY




Loss on drying (FNP 5)

Not more than 6% (90°, pressure not more than 50 mm Hg, 6h)



Mono-, di- and oligosaccharides

Not more than 10% (expressed as glucose) See description under TESTS



Viscosity

100-180 mm2/s (10% w/w aqueous solution at 30°)
See description under TESTS



Lead (FNP 5)

Not more than 1 mg/kg
Determine using an atomic absorption 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 FNP 5, "Instrumental Methods".



Microbiological criteria (FNP 5)

Yeast and moulds:

Not more than 100 CFU/g


Coliforms:

Negative in 25 g


Salmonella:

Negative in 25 g



TESTS




PURITY TESTS




Mono-, di- and oligosaccharides

Principle
The soluble mono-, di- and oligosaccharides of pullulan are measured using the anthrone-sulfuric acid method after pullulan has been precipitated with methanol and KCl.

Equipment
Spectrophotometer capable of measuring absorbance at 620 nm

Procedure
Preparation of standard: Weigh accurately 0.2 g glucose, dissolve in water and make up to 1l.

Measurement of mono-, di- and oligosaccharides: Weigh accurately 0.8 g sample and dissolve in water to make 100 ml (stock solution). Place 1 ml of the stock solution in a centrifuge tube. Add 0.1 ml saturated potassium chloride solution. Add 3 ml methanol and mix vigorously for 20 sec. Centrifuge at 11000 rpm for10 min. Add 0.2 ml of the supernatant to 5 ml modified anthrone solution (0.2 g anthrone in 100 g 75% (v/v) sulfuric acid, freshly prepared). Add 0.2 ml of glucose standard solution and 0.2 ml water (blank control) to separate 5 ml portions of modified anthrone solution. Mix rapidly. Place samples in a 90º water bath and incubate for 15 min. Measure absorbance of the test solution at 620 nm.

Calculate the percent of mono-, di- and oligosaccharides expressed as glucose, C, in the sample:

C (%)= [(At - Ab) × 0.41 × G × 100]/(As - AbW

where

At is absorbance of the test solution
Ab is absorbance of the water blank
As is absorbance of the standard solution
G is weight of the glucose (g)
Wis weight of the sample (g)



Viscosity

Dry the sample for 6 h at 90º under reduced pressure (50 mm Hg). Weigh 10.0 g of the sample and dissolve in water to yield 100 g of solution. Use an Ubbelohde-type (falling-ball) viscometer. Charge the viscometer with sample in the manner dictated by the design of the instrument. Immerse the viscometer vertically in the thermostatic tank at 30 ± 0.1º and allow to stand for 20 min so that the sample equilibrates with the temperature in the tank. Adjust the meniscus of the column of liquid in the capillary tube to a position about 5 mm above of the first mark. With the sample flowing freely, measure, in seconds, the time required for the meniscus to pass from the first to the second mark. Calculate the viscosity, V:

V(mm2/s) = C× t

where

C = calibration constant of the viscometer (mm2/s2)
t = flow time (s)



METHOD OF ASSAY

Calculate the percentage of pullulan on dried basis, P, as the difference between100% and the sum of the percentages of known impurities (mono-, di- and oligosaccharides and water).

P(%) = 100 - (L+C)

where

L is loss on drying
C is is taken from the calculation for mono-, di- and oligosaccharides

Quillaia extract (Type 1)


Specifications prepared at the 61st JECFA (2003), published in FNP 52 Add 11 (2003) and republished in FNP 52 Add 13 (2005). A group ADI of 0.1 mg quillaia saponins per kg bw was established at the 65th JECFA (2005) for Quillaia Extract (Type 1) and Quillaia Extract (Type 2).



SYNONYMS

Quillaja extract, Soapbark extract, Quillay bark extract, Bois de Panama, Panama bark extract, Quillai extract; INS No. 999



DEFINITION

Quillaia extract (Type 1) is obtained by aqueous extraction of the milled inner bark or of the wood of pruned stems and branches of Quillaja saponaria Molina (family Rosaceae). It contains triterpenoid saponins (quillaia saponins, QS) consisting predominantly of glycosides of quillaic acid. Polyphenols and tannins are major components and some sugars and calcium oxalate will be present. Quillaia extract (Type 1) is available commercially as liquid product or as spray-dried powder that may contain carriers such as lactose, maltitol or maltodextrin. The liquid product is usually preserved with sodium benzoate or ethanol.



C.A.S. number

68990-67-0



Formula weight

Monomeric saponins range from ca. 1800 to ca. 2300, consistent with a triterpene with 8-10 monosaccharide residues



ASSAY

Saponin content: not less than 20 % and not more than 26 % on the dried basis



DESCRIPTION

Red-brownish liquid or light brown powder with a pink tinge



FUNCTIONAL USES

Emulsifier, foaming agent



CHARACTERISTICS




IDENTIFICATION




Solubility (FNP 5)

Very soluble in water, insoluble in ethanol, acetone, methanol and butanol



Foam

Dissolve 0.5 g of powder extract in 9.5 g of water or 1 ml of liquid extract in 9 ml of water. Add 1 ml of this mixture to 350 ml of water in a 1000-ml graduated cylinder. Cover the cylinder, vigorously shake it 30 times, and allow settling. Record the foam level (ml) after 30 min. Typical values are 150 ml of foam



Chromatography

Determine as in METHOD OF ASSAY. The retention time of major peak of the sample corresponds to the major saponin peak (QS-18) of the standard.



Colour and turbidity

Powder form only: Dissolve 0.5 g in 9.5 g of water. The solution is not turbid. Determine the absorbance of the solution against water at 520 nm. The absorbance is less than 1.2.



PURITY




Water (FNP 5)

Powder form: not more than 6% (Karl Fischer Method)



Loss on drying (FNP 5)

Liquid form: 50 to 80% (2 g, 105°, 5h)



pH (FNP 5)

3.7 - 5.5 (4 % solution)



Ash (FNP 5) Tannins

Not more than 14% on a dried basis (use 1.0 g for powder samples; for liquid samples, use the residue from loss on drying) Not more than 8% on a dried basis See description under TESTS



Lead (FNP 5)

Not more 2 mg/kg.
Determine using an atomic absorption technique appropriate to the specified level. The selection of the sample size and method of sample preparation may be based on the principles of the method described in FNP 5, "Instrumental Methods".



TESTS




PURITY TESTS




Tannins

Weigh either 3.0 g of the powder form or an equivalent amount of liquid sample, accounting for solids content determined from loss on drying. Dissolve in 250 ml of water. Adjust the pH to 3.5 with acetic acid. Dry 25 ml of this solution at 105° for 5 h and determine the weight of the dried solid, in g (Wi). Mix 50 ml of the solution with 360 mg of polyvinyl polypyrrolidone. Stir the solution for 30 min at room temperature; then centrifuge at 800 ×g. Recover the supernatant and dry this solution at 105o (5h).Weigh the recovered solid (Wf, ing). The percentage of tannins in the sample is:

% tannins (dried basis) = 100 × (Wi - Wf/2)/Wi



METHOD OF ASSAY

Principle
The saponins QS-7, QS-17, QS-18 and QS-21 are separated by reversed phase HPLC and their quantitation is used as an indicator for total saponins levels in Quillaia extract (Type 1).

Sample preparation
Powders: Weigh 0.5 g of sample and dissolve in 9.5 g of water. Filter through a0.2 µm filter.
Aqueous extracts (~ 550 mg solids/ml): Weigh 1 g of sample and dilute with 9 g of water. Filter through a 0.2 µm filter.
In either case, the sample volume is ca. 10 ml.

Standard preparation
Weigh 1.5 g of purified saponins (SuperSap, Natural Response, Chile; Quil-A, Superfos, Denmark or similar, containing a known saponin content) and dissolve in 100 ml of water. Filter through a 0.2 µm filter.

High performance liquid chromatography (HPLC)
HPLC conditions




Column:

Vydac 214TP54 (4.6 x 250 mm length, 5 µm pore) or equivalent


Column temperature:

room temperature


Pump:

gradient


Solvent A:

0.15% trifluoroacetic acid in HPLC-grade water.


Solvent B:

0.15% trifluoroacetic acid in HPLC-grade acetonitrile.


Gradient:

Time(min) 0

% solvent A 70

% solvent B 30



40

55

45



45

70

30


Flow rate:

1 ml/min


Detection wavelength:

220 nm


Injection volume:

20 µl




Calculation
The concentration of saponins, Csap, in mg/ml, in the solution prepared as directed under sample preparation is:

Csap= (Asample/Astandard) x CStandard

where CStandard (mg/ml) is the saponins concentration of the standard injected (e.g., CStandard = 13.5 mg/ml if the saponin content of 1.5 g of standard sample is 90 %) and Asample and Astandard are the sums of the peak areas attributed to the four principle saponins in the sample preparation and in the standard preparation, respectively, as noted in the figure. (Tannins and Polyphenols will elute before the saponins. The peaks due to the saponins will appear after the major peak due to the polyphenols - see figure.)

The percentage of saponins in the test sample is:

% Saponins = 100 × Csap/(0.1Wsample)

where Wsample is the weight of the sample (mg) taken for the sample preparation and 0.1 is the inverse of the sample volume, 10 ml.

Appendix

Chromatogram of Standard (15 mg solids/ml equivalent to 13.5 mg saponins/ml).

Chromatogram of Quillaia extract (Type 1) (55 mg solids/ml)

Quillaia extract (Type 2)


Revised specifications prepared at the 65th JECFA and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 61st JECFA (2003) and published in FNP 52 Add 11 (2003). A group ADI of 0.1 mg quillaia saponins per kg bw was established at the 65th JECFA (2005) for Quillaia Extract (Type 1) and Quillaia Extract (Type 2).



SYNONYMS

Quillaja extract, Soapbark extract, Quillay bark extract, Bois de Panama, Panama bark extract, Quillai extract



DEFINITION

Quillaia extract (Type 2) is obtained either by chromatographic separation or ultrafiltration of the aqueous extraction of the milled inner bark or of the wood of pruned stems and branches of Quillaja saponariaMolina (family Rosaceae). It contains triterpenoid saponins (quillaia saponins, QS) consisting predominantly of glycosides of quillaic acid. Polyphenols and tannins are minor components. Some sugars and calcium oxalate will also be present.
Quillaia extract (Type 2) is available commercially as a liquid product or as a spray-dried powder that may contain carriers such as lactose, maltitol or maltodextrin. The liquid product is usually preserved with sodium benzoate or ethanol.



C.A.S. number

68990-67-0



Formula weight

Monomeric saponins range from ca. 1800 to ca. 2300, consistent with a triterpene with 8-10 monosaccharide residues



Assay

Saponin content: not less than 65% and not more than 90% on the dried basis



DESCRIPTION

Light red-brownish liquid or powder



FUNCTIONAL USES

Emulsifier, foaming agent



CHARACTERISTICS




IDENTIFICATION




Solubility (FNP 5)

Very soluble in water, insoluble in ethanol, acetone, methanol, and butanol



Foam

Dissolve 0.5 g of the powder form in 9.5 ml of water or 1 ml of the liquid form in 9 ml of water. Add 1 ml of this solution to 350 ml of water in a 1000-ml graduated cylinder. Cover the cylinder, vigorously shake it 30 times, and allow settling. Record the foam volume (ml) after 30 min. Typical volumes are about 260 ml.



Chromatography

Determine as in METHOD OF ASSAY. The retention time of major sample peak corresponds to the major saponin peak (QS-18) of the standard.



Colour and turbidity

Powder form only: Dissolve 0.5 g in 9.5 ml of water. The solution shall not turbid Determine the absorbance of the solution against water at 520 nm. The absorbance shall be less than 0.7.



PURITY




Water (FNP 5)

Powder form: not more than 6% (Karl Fischer Method)



Loss on drying (FNP 5)

Liquid form: 50 to 80% (2 g, 105°, 5h)



pH (FNP 5)

3.7 - 5.5 (4 % solution)



Ash (FNP 5)

Not more than 5% on a dried basis (use 1.0 g for powder samples; for liquid samples, use the residue from Loss on drying)



Tannins

Not more than 8% on a dried basis
See description under TESTS



Lead (FNP 5)

Not more 2 mg/kg.
Determine using an atomic absorption technique appropriate to the specified level. The selection of the sample size and method of sample preparation may be based on the principles of the method described in FNP 5, "Instrumental Methods".



TESTS




PURITY TESTS




Tannins

Weigh either 3.0 g of the powder form or an equivalent amount of liquid sample, accounting for solids content determined from loss on drying. Dissolve in 250 ml of water. Adjust the pH to 3.5 with acetic acid. Dry 25 ml of this solution at 105° for 5 h and determine the weight of the dried solid, in g (Wi). Mix 50 ml of the solution with 360 mg of polyvinyl polypyrrolidone. Stir the solution for 30 min at room temperature; then centrifuge at 800 ×g. Recover the supernatant and dry this solution at 105o(5h).Weigh the recovered solid (Wf, ing). The percentage of tannins in the sample is:

% tannins (dried basis) = 100 × (Wi - Wf/2)/Wi



METHOD OF ASSAY

Principle:
The saponins QS-7, QS-17, QS-18 and QS-21 are separated by reversed phase HPLC and their quantitation is used as an indicator for total saponins levels in Quillaia extract (Type 2).

Sample preparation:
Powders:Weigh 0.5 g of sample and dissolve in 9.5 ml of water. Filter through a0.2 µm filter.

Aqueous extracts (~ 550 mg solids/ml):Weigh 1 g of sample and dilute with 9 ml of water. Filter through a 0.2 µm filter.
In each case, the sample volume is ca. 10 ml.

Standard preparation:
Weigh 1.5 g of purified saponins (SuperSap, Natural Response, Chile; Quil-A, Superfos, Denmark or similar, containing a known saponin content) and dissolve in 100 ml of water. Filter through a 0.2 µm filter.

High performance liquid chromatography (HPLC):
HPLC conditions:




Column:

Vydac 214TP54 (4.6 x 250 mm length, 5 µm particle size) or equivalent


Column temperature:

Room temperature


Pump:

Gradient


Solvent A:

0.15% trifluoroacetic acid in HPLC-grade water.


Solvent B:

0.15% trifluoroacetic acid in HPLC-grade acetonitrile.


Gradient:

Time(min)

% solvent A

% solvent B



0

70

30



40

55

45



45

70

30


Flow rate:

1 ml/min


Detection wavelength:

220 nm


Injection volume:

20 µl




Calculation:
The concentration of saponins, Csap, in mg/ml, in the solution prepared as directed under sample preparation is:

Csap= (Asample/Astandard)CStandard

where CStandard (mg/ml) is the saponins concentration of the standard injected (e.g., CStandard = 13.5 mg/ml if the saponin content of 1.5 g of standard sample is 90 %) and Asample and Astandard are the sums of the peak areas attributed to the four principle saponins in the sample preparation and in the standard preparation, respectively, as noted in the figure. (Tannins and polyphenols will elute before the saponins. The peaks corresponding to the saponins will appear after the major peak corresponding to the polyphenols)

The percentage of saponins in the test sample is:

% Saponins = 100 × Csap/(0.1Wsample)

where Wsample is the weight of the sample (mg) taken for the sample preparation and 0.1 is the inverse of the sample volume, 10 ml.

Appendix

Chromatogram of standard (15 mg solids/ml equivalent to 13.5 mg saponins/ml).

Sucrose esters of fatty acids


(Tentative)

Information required on

  • method of analysis for the determination of free sucrose using capillary GC or HPLC

  • an alternative and less toxic solvent than pyridine for preparing the standard and sample solutions for the determinations of free sucrose and propylene glycol

  • method of analysis for the determination of dimethyl sulfoxide that does not require a packed column

Note: The tentative specifications will be withdrawn unless the requested information is received before the end of the year 2006.

Tentative specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding the specifications prepared at the 61st JECFA (2003) and published in FNP 52 Add 11 (2003). An ADI of 0-30 mg/kg bw for this substance together with sucroglycerides was established at the 49th JECFA (1997).



SYNONYMS

Sucrose fatty acid esters, INS No. 473



DEFINITION

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



Assay

Not less than 80% of sucrose esters



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



Fatty acids

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 has no odour of isobutyric acid, and which dissolves when 3 ml of diethyl ether are added. Use the aqueous layer separated from the diethyl ether in the Test for sugars.



Sugars

To 2 ml of the aqueous layer separated from the diethyl ether in the test for fatty acids, carefully add 1 ml of anthrone TS down the inside of a test tube; the boundary surface of the two layers turns 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



Dimethylformamide

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
Determine using an atomic absorption 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 FNP 5, "Instrumental methods".



TESTS




PURITY TESTS




Free sucrose

Determine by gas liquid chromatography (FNP 5).

Standard solutions
Prepare a stock solution containing 5.0 mg/ml of sucrose in N,N- dimethylformamide. Prepare a range of standard solutions containing 0.5, 1.25 and2.5 mg/ml of sucrose by dilutions of the stock solution with pyridine.

Internal standard solution
Weigh accurately 0.25 g of octacosane into a 50-ml volumetric flask, add 25 ml of tetrahydrofuran to dissolve the octacosane, and add pyridine to the mark.

Chromatography conditions


Column:

2% Dexsil 300GC on Uniport HP 80/100 mesh (slightly polar, 2.1 m x 3.2 mmi.d.) or equivalent


Carrier gas:

Nitrogen


Flow rate:

40 ml/min


Detector:

FID


Temperatures:

- injection:

280°



- column:

Hold for 1 min at 160°, then 160-300° at15°/min, hold for 60 min at 300°



- detector:

320°


The retention times of free sucrose and octacosane measured under the above conditions are approx. 8.2 and 9.8 min, respectively.

Procedure
Weigh accurately 20-50 mg of the sample into a centrifugation tube, add 1 ml internal standard solution, 1 ml pyridine, 0.4 ml of N,O-bis(trimethylsilyl)acetamide(BSA) and 0.2 ml trimethylchlorosilane (TMCS). After sealing the tube, shake and let stand for 5 min at room temperature. Inject 1 µl into the gas liquid chromatograph.

Standard curve
Prepare silylate standard solutions following the same procedure using 1 ml each of the standard solutions in place of the sample and pyridine. Draw a standard curve by plotting amount of sucrose (mg) in 1 ml of the standard solution (X-axis) vs. ratio of peak area of sucrose/internal standard (Y-axis).

Measure the peak areas for sucrose and internal standard. Calculate the ratio of their peak areas, and obtain the amount of sucrose in sample from the standard curve.

Calculate the percentage of free sucrose from:



Dimethylformamide

Determine by gas liquid chromatography (FNP 5).

Standard solutions
Prepare a stock solution containing 1.00 mg/ml of dimethylformamide in tetrahydrofuran. Prepare a range of standard solutions containing 0.05, 0.1 and 0.2 µg/ml of dimethylformamide by diluting the stock solution with tetrahydrofuran.

Chromatography conditions


Column:

Polyethylene glycol (30 m x 0.32 mmi.d. with a 0.5 µm film)


Carrier gas:

Helium


Pressure:

150 kPa (constant pressure)


Detector:

Nitrogen phosphorus detector (NPD) (synonym: Flame thermoionic detector (FTD))


Temperatures:

- injection:

180°



- column:

Hold for 2 min at 40°, then 40-160° at 20°/min, hold for 2 min at 160°



- detector:

325°


Injection method:

Splitless injection of 1.0 µl with auto-injector, followed by start of purge after 1.0 min.




The retention time of dimethylformamide measured under the above conditions is approx. 6.4 min.

Procedure
Weigh accurately 2 g of sample into a 20-ml volumetric flask, add 10 ml of tetrahydrofuran to dissolve the sample, add tetrahydrofuran to the mark, and mix the solution well. Inject 1.0 µl of the sample solution into the chromatograph.

Standard curve
Prepare daily by injecting 1.0 µl of each of the standard solutions into the chromatograph.

Calculate the concentration CDFA of dimethylformamide from:

CDFA (mg/kg) = [C (µg/ml) x 20 (ml)]/W (g)

where

C= dimethylformamide concentration detected (µg/ml)
W= weight of sample (g)

Note: The nitrogen phosphorus detector is insensitive to components that do not contain nitrogen or phosphorus. As a consequence, the capillary column can become obstructed with compounds of low volatility, although the baseline of the chromatogram is stable. Accordingly, the column must be reconditioned frequently. Overnight reconditioning (flow carrier gas in the reverse direction at 180°) is required after about every 15 samples.



Dimethyl sulfoxide

Determine by gas liquid chromatography (FNP 5).

Standard solutions
Prepare a 0.25 mg/ml stock solution of dimethyl sulfoxide in tetrahydrofuran. Prepare a range of solutions containing 0.5, 1 and 5 µg/ml of dimethyl sulfoxide by dilutions of the stock solution with tetrahydrofuran.

Chromatography conditions


Column:

10% PEG 20M and 3% KOH on Gas Chrom Z (2 m x 3 mmi.d.) or equivalent. Raise the oven temperature to 180° at a rate of10º/min and let stabilize for 24 to 48 h with 30 to 40 ml/min of nitrogen for conditioning


Carrier gas:

Nitrogen


Flow rate:

50 ml/min


Detector:

Flame photometric detector (using 394 nm sulfur filter)


Temperatures

- injection:

210°



- column:

160°




The retention time of dimethyl sulfoxide measured under the above conditions is approx. 3.4 min.

Procedure
Weigh accurately 5 g of the sample into a 25-ml volumetric flask, add 10 ml of tetrahydrofuran to dissolve the sample, add tetrahydrofuran to the mark, and mix the solution well. Inject 3 µl of the sample solution into the chromatograph.

Standard curve
Prepare daily by injecting 3 µl of each of the standard solutions into the chromatograph.

Calculate the concentration CDMSO of dimethyl sulfoxide in mg/kg from:

CDMSO (mg/kg) = [C (µg/ml) x 25 (ml)]/W (g)

where

C = dimethyl sulfoxide concentration determined (µg/ml)
W = weight of sample (g)



Propylene glycol

Determine by gas liquid chromatography (FNP 5).

Internal standard solution
Prepare a 500 µg/ml solution of ethylene glycol in pyridine.

Standard solution
Prepare a 50 µg/ml solution of propylene glycol in pyridine.

Chromatography conditions




Column:

Polydimethylsiloxane (30 m x 0.32 mmi.d. with 0.25 µm film)


Carrier gas:

Helium


Flow rate:

1.5 ml/min (Constant flow)


Detector:

FID


Temperatures

- injection:

230°



- column:

Hold for 5 min at 60°, then 60-250° at 20°/min, hold for 5 min at 250°



- detector:

250°


The retention times of ethylene glycol and propylene glycol derivatives are approx. 7.7 min and 7.9 min, respectively.




Procedure
Weigh accurately 1 g of the sample in a 10-ml volumetric flask, and add 100 µl ofthe internal standard solution. Dissolve and make to volume with pyridine. Take 0.5ml of sample solution in a centrifugation tube, and add 0.25 ml of 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and 0.1 ml of trimethylchlorosilane (TMCS). After sealing the tube, shake it vigorously, let stand for 30 min at room temperature, then centrifuge. Inject 1.0 µl of this centrifugal supernatant into the chromatograph. Standard curve
Prepare following the same procedure using 0.05, 0.2, 0.5 and 1 ml of the standard solution in place of the sample.

Calculate the concentration CPG of propylene glycol in mg/kg from:

where

CPG (mg/kg) = [C (µg/ml) x 10 (ml)]/W (g)
C = polyethylene glycol concentration determined(µg/ml)
W = weight of sample (g)

Note: It will be necessary to clean the injection port and to recondition column at300° after about every 20 samples, because of contamination of the column.



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

Determined by gas chromatography with a head space sampler.

Standard solutions
Prepare standard solution A containing 4000 mg/l each of methanol, isopropanol, isobutanol, ethyl acetate and methyl ethyl ketone by weighing accurately 0.2 g of each solvent into a 50-ml volumetric flask containing approx. 20 ml of water, then adding water to volume. By dilutions of this solution, prepare solutions containing2000 mg/l (standard solution B) and 1000 mg/l (standard solution C).

Procedure
Weigh accurately 1 g of the sample in each of four sample vials. To one vial add 5 µl of water, to the second, third and fourth, add, respectively, standard solutions A, B and C, and seal them quickly with a septum. (The concentrations of each solvent after adding 5 µl of standard solutions A, B and C to 1 g of the sample are equal to20, 10 and 5 mg/kg of sample, respectively). Place the sample vials in a head space sampler and analyse using the following conditions:


Column:

100% Polydimethylsiloxane (30 m x 0.53 mmi.d. with 1.5 µm film, for example DB-1 manufactured by J&W Co. Ltd.)


Carrier gas:

Nitrogen


Flow rate:

3.5 ml/min


Detector:

FID


Temperatures

- injection:

110°



- column:

40°



- detector:

110°


Head space sampler:




- sample heat insulating temperature:

80°



- sample heat insulating period:

40 min



- syringe temperature:

85°



- sample gas injection:

1.0 ml




Calculation
Plot the relationship between the added amount against the peak area for each solvent using the analytical results. The relationship should be linear (R2 > 0.99). Extrapolate and determine the x-intercept, wi, and calculate the solvent concentrations Ci in mg/kg in the sample from:

Ci = wi/W

where

wi = x-intercept of relationship line using the standard addition method (µg)
W = weight of sample (g)



METHOD OF ASSAY

Determine by HPLC using the following conditions:

Procedure
Accurately weigh 250 mg of the sample and transfer to a 50-ml volumetric flask. Dilute to volume with tetrahydrofuran and mix. Filter through a 0.5 µm membrane filter. Inject 100 µl of the sample into the pre-stabilized chromatograph.

Chromatography conditions


Column:

Styrene-divinylbenzene copolymer for gel permeation chromatography (TSK-GEL G2000 (Tosoh) or equivalent)


Mobile phase:

HPLC-grade degassed tetrahydrofuran


Flow rate:

0.7 ml/min


Detector:

RI


Temperatures:

- Column:

38°



- Detector:

38°




Record the chromatogram for about 90 min.

Calculate the percentage of sucrose ester content in the sample from:

% sucrose ester = 100 A/T

where

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

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


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