Polysorbate 80

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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Sorbitan, mono-9-octadecenoate, poly(oxy-1,2-ethanediyl) derivs., (Z)-;

Polyoxyethylene 20 sorbitan monooleate

CAS RN^®: 9005-65-6.

1 DEFINITION

Polysorbate 80 is a mixture of partial esters of fatty acids, mainly oleic acid, with Sorbitol and its anhydrides ethoxylated with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides.

2 IDENTIFICATION

A. It meets the requirements of the test for Composition of Fatty Acids.

Change to read:

B. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197F _{▲(CN 1-MAY-2020)}

3 ASSAY

COMPOSITION OF FATTY ACIDS

Diluent: 20 g/L of sodium hydroxide in methanol

Boron trifluoride-methanol solution: 140 g/L of boron trifluoride in methanol

Saturated sodium chloride solution: Sodium chloride and water (1:2). Before use, decant the solution from any undissolved substance and filter, if necessary.

Reference solution A: Prepare 0.50 g of the mixture of calibrating substances with the composition described in Table 1. Dissolve in heptane, and dilute with heptane to 50.0 mL.

Reference solution B: Reference solution A in heptane (1 in 10)

Reference solution C: Prepare 0.50 g of a mixture of fatty acid methyl esters, which corresponds to the composition of the substance to be examined. Dissolve in heptane, and dilute with heptane to 50.0 mL. [NOTE-Commercially available mixtures of fatty acid methyl esters may also be used.]

Sample solution: Dissolve 0.10 g of Polysorbate 80 in 2 mL of Diluent in a 25-mL conical flask, and boil under a reflux condenser for 30 min. Add 2.0 mL of Boron trifluoride-methanol solution through the condenser, and boil for 30 min. Add 4 mL of heptane through the condenser, and boil for 5 min. Cool, add 10.0 mL of Saturated sodium chloride solution, shake for about 15 s, and add a quantity of Saturated sodium chloride solution such that the upper phase is brought into the neck of the flask. Collect 2 mL of the upper phase, wash with three quantities, each of 2 mL, of water, and dry over anhydrous sodium sulfate.

Table 1

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 0.32 - mm x 30 - m G16 on fused silica; film thickness 0.5 µm

Temperatures

Injection port: 250°

Detector: 250°

Column: See Table 2.

Table 2

Carrier gas: Helium

Linear velocity: 50 cm/s

Injection volume: 1 µL

Injection type: Split ratio, 50:1

System suitability

Samples: Reference solution A and Reference solution B

Suitability requirements

Resolution: NLT 1.8 between the peaks due to methyl oleate and methyl stearate, Reference solution A

Theoretical plates: NLT 30,000 calculated for the peak of methyl stearate, Reference solution A

Signal-to-noise ratio: NLT 5 for the peak of methyl myristate, Reference solution B OFFICIA

Analysis

Sample: Sample solution

Identify the peaks from Reference solution C.

Calculate the percentage of each component in the Sample solution:

Result = (A_C/A_T) x 100

A_C = peak area for the component of interest

A_T = total area of all peaks related to fatty acids

Acceptance criteria: See Table 3.

Table 3

4 IMPURITIES

4.1 RESIDUE ON IGNITION

Sample: 2.00 g

Analysis: Heat a silica or platinum crucible to redness for 30 min, allow to cool in a desiccator, and weigh. Evenly distribute the Sample in the crucible. Dry at 100-105 for 1 h and ignite to constant mass in a muffle furnace at 600 ± 25°, allowing the crucible to cool in a desiccator after each ignition. Flames should not be produced at any time during the procedure. If after prolonged ignition the ash still contains black particles, take up with hot water, pass through an ashless filter paper, and ignite the residue and the filter paper. Combine the filtrate with the ash, carefully evaporate to dryness, and ignite to constant mass.

Acceptance criteria: NMT 0.25%

4.2 ETHYLENE OXIDE AND DIOXANE

Ethylene oxide standard solution: Dilute 0.5 mL of a commercially available solution of ethylene oxide in methylene chloride (50 mg/mL) with water to 50.0 mL. [NOTE-The solution is stable for 3 months, if stored in vials with a Teflon-coated, silicon membrane and crimped caps at -20°.] Allow to reach room temperature. Dilute 1.0 mL of this solution with water to 250.0 mL.

Dioxane standard solution: Dioxane in water (v/v) 1 in 20,000

Acetaldehyde standard solution: 0.01 mg/mL of acetaldehyde in water

Standard solution: Dilute 6.0 mL of Ethylene oxide standard solution and 2.5 mL of Dioxane standard solution with water to 25.0 mL.

Sample solution A: Transfer 1.0 g of Polysorbate 80 to a 10-ml headspace vial. Add 2.0 ml of water, and seal the vial immediately with a Teflon-coated, silicon membrane and an aluminum cap.

Sample solution B: Transfer 1.0 g of Polysorbate 80 to a 10-ml headspace vial. Add 2.0 ml. of the Standard solution, and seal the vial immediately with a Teflon-coated, silicon membrane and an aluminum cap.

Reference solution: Transfer 2.0 mL of Acetaldehyde standard solution and 2.0 mL of Ethylene oxide standard solution to a 10-mL headspace vial, and seal the vial immediately with a Teflon-coated, silicon membrane and an aluminum cap. 

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: Headspace GC

Detector: Flame ionization

Column:¹ 0.53 - mm x 50 - 1 m G27 on fused silica; film thickness 5 µm

Temperatures

Injection port: 85°

Detector: 250°

Column: See Table 4.

Table 4

Split ratio: 3.5:1

Carrier gas: Helium

Flow rate: 4.0 mL/min

Injection volume: 1 mL

System suitability

Sample: Reference solution

[NOTE-The relative retention times for ethylene oxide, acetaldehyde, and dioxane are 1.0, 0.9, and 1.9, respectively. The retention time for ethylene oxide is about 6.5 min.]

Suitability requirements

Resolution: NLT 2.0 between the peaks due to acetaldehyde and ethylene oxide

Analysis

Samples: Sample solution A and Sample solution B

Calculate the content, in ppm, of ethylene oxide:

Result = (2 × C_ED × A_A)/(A_B - A_A)

C_ED = concentration of ethylene oxide in Sample solution B (µg/mL)

A_A = peak area of ethylene oxide from Sample solution A

A_B = peak area of ethylene oxide from Sample solution B

Calculate the content, in ppm, of dioxane:

Result = (2 × D × C_D × A_A) × 1000/(A_B - A_A)

D = density of dioxane, 1.03 g/mL

 C_D = concentration of dioxane in Sample solution B (µL/mL) 

A_A = peak area of dioxane from Sample solution A

A_B = peak area of dioxane from Sample solution B

Acceptance criteria: NMT 1 ppm for ethylene oxide; NMT 10 ppm for dioxane

5 SPECIFIC TESTS

5.1 SPECIFIC GRAVITY (841)

1.06-1.09,

5.2 VISCOSITY-CAPILLARY METHODS (911) or VISCOSITY ROTATIONAL METHODS (912)

300-500 centistokes at 25",

5.3 FATS AND FIXED OILS, Acid Value (401)

Light petroleum: It has the following properties: a clear, colorless, flammable liquid without fluorescence; practically insoluble in water, miscible with alcohol; density at 20° about 0.720; distillation range 100-120"; water content NMT 0.03%.²

Sample solution: Dissolve 5.0 g in 50 mL of a mixture of equal volumes of alcohol and Light petroleum (previously neutralized with 0.1 N potassium hydroxide or 0.1 N sodium hydroxide), using 0.5 mL of phenolphthalein TS as the indicator. If necessary, heat to about 90° to dissolve the substance to be examined.

Analysis: Titrate the Sample solution with 0.1 N potassium hydroxide VS or 0.1 N sodium hydroxide VS until the pink color persists for at least 15 s. When heating has been applied to aid dissolution, maintain the temperature at about 90° during the titration.

Acceptance criteria: NMT 2.0

5.4 FATS AND FIXED OILS, Hydroxyl Value (401)

Sample: 2.0 g

Analysis: Transfer the Sample into a 150-mL acetylation flask fitted with an air condenser. Add 5.0 mL of Pyridine-Acetic Anhydride Reagent, and attach the air condenser. Heat the flask in a water bath for 1 h keeping the level of the water about 2.5 cm above the level of the liquid in the flask. Withdraw the flask, and allow to cool. Add 5 mL of water through the upper end of the condenser. If a cloudiness appears, add sufficient pyridine to clear it, noting the volume added. Shake the flask, and replace in the water bath for 10 min. Withdraw the flask, and allow to cool. Rinse the condenser and the walls of the flask with 5 mL of alcohol, previously neutralized with phenolphthalein TS. Titrate with 0.5 N alcoholic potassium hydroxide VS using 0.2 mL of phenolphthalein TS as the indicator. Carry out a blank test under the same conditions.

Acceptance criteria: 65-80

5.5 FATS AND FIXED OILS, Peroxide Value (401)

Sample: 10.0 g

Saturated potassium iodide solution: Prepare a saturated solution of potassium iodide in carbon dioxide-free water. Make sure the solution remains saturated as indicated by the presence of undissolved crystals.

Analysis: Transfer the Sample into a 100-mL beaker, and dissolve with 20 mL of glacial acetic acid. Add 1 mL of Saturated potassium iodide solution, and allow to stand for 1 min. Add 50 mL of carbon dioxide-free water and a magnetic stirring bar. Titrate with 0.01 M sodium thiosulfate VS, determining the endpoint potentiometrically. Carry out a blank titration.

Acceptance criteria: NMT 10

5.6 FATS AND FIXED OILS, Saponification Value (401)

Sample: 4.0 g

Analysis: Transfer the Sample into a 250-mL borosilicate glass flask fitted with a reflux condenser. Add 30.0 mL of 0.5 N alcoholic potassium hydroxide VS and a few glass beads. Attach the condenser, and heat under reflux for 60 min. Add 1 mL of phenolphthalein TS and 50 mL of dehydrated alcohol, and titrate immediately with 0.5 N hydrochloric acid VS. Carry out a blank test under the same conditions.

Acceptance criteria: 45-55

5.7 WATER DETERMINATION, Method / (921)

NMT 3.0%, determined on 1.0 g

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Store in an airtight container, protected from light.

USP REFERENCE STANDARDS (11)

USP Polysorbate 80 RS

¹ CP-Sil 8 CB is suitable.

² Petroleum ether, boiling range 100°-140°; [CAS 64742-49-0] from Fisher Scientific; catalog number AC23302-0025 is suitable.