Soybean Oil

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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1 DEFINITION

Soybean Oil is the refined fixed oil obtained from the seeds of the soya plant Glycine max Merr. (Fam. Fabaceae). It may contain suitable antioxidants.

2 IDENTIFICATION

A. IDENTITY BY FATTY ACID COMPOSITION

Analysis: Proceed as directed in the test for Fats and Fixed Oils, Fatty Acid Composition. Acceptance criteria: Meets the composition profile of fatty acids in Table 1

B. IDENTITY BY TRIGLYCERIDE PROFILE

Analysis: Proceed as directed in Identification of Fixed Oils by Thin-Layer Chromatography 〈202〉. Acceptance criteria: Meets the requirements in the chapter

3 IMPURITIES

ALKALINE IMPURITIES

Sample: 10 mL

Analysis: Mix 10 mL of acetone and 0.3 mL of water, and add 0.05 mL of bromophenol blue TS. If necessary, neutralize the solution to a green color with 0.01 N hydrochloric acid or 0.01 N sodium hydroxide. Add the Sample, shake, and allow to stand. Titrate with 0.01 N hydrochloric acid VS to change the color of the upper layer to yellow.

Acceptance criteria: NMT 0.1 mL of 0.01 N hydrochloric acid is required.

4 SPECIFIC TESTS

FATS AND FIXED OILS, Acid Value〈401〉: NMT 0.3

FATS AND FIXED OILS, Peroxide Value〈401〉

Solution A: Glacial acetic acid and chloroform (60:40)

Solution B: Prepare a saturated solution of potassium iodide in freshly boiled and cooled water, and store it protected from light. Discard it if it gives a color on addition of Solution A and starch TS.

Sample: 10 g Titrimetric system (See Titrimetry 〈541〉.)

Mode: Direct titration

Titrant: 0.01 N sodium thiosulfate VS Endpoint detection: Visual

Analysis: Transfer the Sample to a conical flask. Add 30 mL of Solution A, and swirl to dissolve. Add 0.5 mL of Solution B, swirl the flask for 1 min, accurately timed, and add 30 mL of water. Titrate with Titrant, with vigorous agitation, to a light yellow color. Add 0.5 mL of starch TS, and continue the titration until the blue color has disappeared. Perform a blank test, and make any necessary correction.

Calculate the peroxide value:

Result = [(V × N)/W] × F

V = volume of sodium thiosulfate (mL)

N = normality of the sodium thiosulfate solution (mEq/mL)

W = weight of Soybean Oil taken (g)

F = conversion factor, 1000 g/kg

Acceptance criteria: NMT 10.0

FATS AND FIXED OILS, Fatty Acid Composition〈401〉: Soybean Oil exhibits the composition profile of fatty acids shown in Table 1, as determined in the chapter.

Table 1

FATS AND FIXED OILS, Unsaponifiable Matter〈401〉: NMT 1.5%

STEROL COMPOSITION

Sample A: Transfer 5 g of Soybean Oil to a 250-mL conical flask, add 50 mL of an alcoholic potassium hydroxide solution prepared by dissolving 12 g of potassium hydroxide in 10 mL of water and diluting with alcohol to 100 mL, and heat the flask on a steam bath under a suitable condenser to maintain reflux for 1 h, swirling frequently. Cool to a temperature below 25°, and transfer the contents of the flask to a separator with a polytetrafluoroethylene stopcock, rinsing the flask with two 50-mL portions of water that are added to the separator (do not use grease on the stopcock). Extract with three 100-mL portions of ether, combining the ether extracts in another separator containing 40 mL of water. Gently rotate or shake the separator for a few minutes. [NOTE—Violent agitation may result in the formation of a difficult-to- separate emulsion.] Allow the mixture to separate, and discard the lower aqueous phase. Wash the ether extract with two additional 40-mL portions of water, and discard the lower aqueous phase. Wash the ether extract successively with a 40-mL portion of potassium hydroxide solution (3 in 100) and a 40-mL portion of water. Repeat this potassium hydroxide solution–water wash sequence three times. Wash the ether extract with 40-mL portions of water until the last washing is not reddened by the addition of 2 drops of phenolphthalein TS. Transfer the ether extract to a tared flask, and rinse the separator with 10 mL of ether, adding the rinsings to the flask. Evaporate the ether on a steam bath, and add 6 mL of acetone to the residue. Remove the acetone in a current of air, and dry the residue at 105°.

Reference A: Transfer 5 g of sunflower oil to a 250-mL conical flask, and proceed as directed in Sample A, beginning with “add 50 mL of an alcoholic potassium hydroxide solution prepared by dissolving 12 g of potassium hydroxide in 10 mL of water and diluting with alcohol to 100 mL”.

Separation of the sterol fraction by LC

Mobile phase: Isopropyl alcohol and n-hexane (1:99)

Sample solution A: Transfer Sample A with three 4-mL quantities of ether to a 15-mL test tube. Evaporate to dryness under a stream of nitrogen. Dissolve Sample A in Mobile phase to obtain a solution with an approximate concentration of 40 mg/mL. Add a few drops of isopropyl alcohol to improve the solubility. [NOTE—3 drops are normally sufficient to ensure complete solubilization.] Pass through a membrane filter (nominal 0.45-µm pore size).

Reference solution A: Prepare as directed for Sample solution A except use Reference A instead of Sample A.

Chromatographic system

(See Chromatography 〈621〉.)

Mode: LC

Detector: UV 210 nm Columns

Guard: 4.6-mm × 0.5-cm (or 4.6-mm × 1.0-cm); 5-µm packing L3 with a 6-nm pore size Analytical: 4.6-mm × 25-cm; 5-µm packing L3 with a 6-nm pore size

Flow rate: 1.0 mL/min

Injection volume: 50 µL

Identification of peaks due to sterols

Samples: Sample solution A and Reference solution A

Sterol identification: The sterol fraction elutes at the end of the chromatogram. Locate the fraction to be collected by using the chromatogram from Reference solution A. The chromatogram from Reference solution A shows two or three principal peaks, which elute at approximately 21–35 min depending on the column used.

Sterol collection: Collect the fraction at the detector outlet in a 15-mL tube with a screw cap. Evaporate the solvent under a stream of nitrogen. [NOTE—If necessary, to increase the sample amount for later analysis, make the second injection of 50 µL on the HPLC column and collect the fraction at the detector outlet in the same 15-mL test tube with a screw cap. Evaporate the solvent under a stream of nitrogen.]

Determination of sterols by GC

Sample solution B: Dissolve the residue of the sterol fraction obtained from Sample solution A in the previous LC step in 0.2 mL of anhydrous pyridine and 0.2 mL of a mixture of 1 volume of chlorotrimethylsilane and 99 volumes of bis(trimethylsilyl)trifluoroacetamide. Insert the stopper into the test tube tightly and heat at 80° for 20 min. Allow to cool and use the liquid phase.

Reference solution B: Dissolve 9 parts of the residue of the sterol fraction obtained from Reference solution A in the previous LC step, 1 part of cholesterol in 0.2 mL of anhydrous pyridine, and 0.2 mL of a mixture of 1 volume of chlorotrimethylsilane and 99 volumes of bis(trimethylsilyl)trifluoroacetamide. Insert the stopper into the test tube tightly, and heat at 80° for 20 min. Allow to cool and use the liquid phase.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 0.25-mm × 30-m fused-silica capillary; 0.25-µm layer of phase G27

Temperatures

Injection port: 290°

Detector: 290°

Column: See Table 2.

Table 2

Carrier gas: Helium Flow rate: 2.6 mL/min

Injection volume: 1–3 µL (depending on the expected amount of sterols in the test sample). Injection type: Split injection; split ratio is 25:1

System suitability

Sample: Reference solution B

The chromatogram from Reference solution B shows five principal peaks corresponding to cholesterol, campesterol, stigmasterol, β- sitosterol, and Δ7-stigmastenol. 

[NOTE—The retention times of the sterols with reference to β-sitosterol are given in Table 3.]

Table 3

Suitability requirements

Resolution: NLT 3.0 between the campesterol and stigmasterol peaks

Analysis

Samples: Sample solution B and Reference solution B

Use the chromatogram from Reference solution B to identify the peaks due to cholesterol, campesterol, stigmasterol, β-sitosterol and Δ7-stigmastenol. Identify the peaks due to the sterols in the chromatogram from Sample solution B using the chromatograms from Reference solution B and the relative retention times with reference to β-sitosterol (main peak) given in Table 3.

Calculate the percentage of the content of each sterol in the sterol fraction of Soybean Oil taken:

Result = (r_U/r_T) × 100

r_U = area of the peak due to the sterol component to be determined

r_T = sum of the areas of the peaks due to the components indicated in Table 3

Acceptance criteria: NMT 0.3% of brassicasterol

WATER DETERMINATION, Method Ic〈921〉: NMT 0.1%

5 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers, and avoid exposure to excessive heat.

LABELING: Label it to indicate the name and quantity of any added antioxidant. Where Soybean Oil is intended for use in the manufacture of injectable dosage forms, it is so labeled.

OTHER REQUIREMENTS: For Soybean Oil intended for use in injectable dosage forms, which is specified in the labeling, the requirements for Unsaponifiable Matter, Acid Value, Peroxide Value, and Water, Method Ic in Injections and Implanted Drug Products 〈1〉, Specific Tests, Vehicles and added substances, Nonaqueous vehicles must be met.