Olive 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

Olive Oil is the renfied fixed oil obtained from the ripe fruit of Olea europaea L. (Fam. Oleaceae). 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 〈401〉, Fatty Acid Composition.

Acceptance criteria: Meets the composition proflle of fatty acids in Table 1

B. Identity by Triglyceride Profile

Analysis: Proceed as directed in the test for Identification of Fixed Oils by Thin-Layer Chromatography 〈202〉.

Acceptance criteria: Meets the requirements in the chapter

3 IMPURITIES

Alkaline Impurities

Sample: 10 mL of Olive Oil

Analysis: Mix 10 mL of freshly opened acetone and 0.3 mL of water, and add 0.05 mL of bromophenol blue TS. 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 (Free Fatty Acids) 〈401〉: NMT 0.3. [Note—Petroleum ether with a 100°–120° boiling range can be used to replace ether in the test.]

Fats and Fixed Oils, Peroxide Value 〈401〉: NMT 10.0

Fats and Fixed Oils, Fatty Acid Composition 〈401〉: Olive Oil exhibits the composition proflle of fatty acids shown in Table 1, as determined in the chapter.

Table 1

Absence of Sesame Oil

Sample: 10 mL of Olive Oil

Analysis: Mix the Sample with a mixture of 0.5 mL of a 0.35% (v/v) solution of furfural in acetic anhydride and 4.5 mL of acetic anhydride, and shake the mixture for about 1 min. Pass through a filter paper previously wetted with acetic anhydride. Add 0.2 mL of sulfuric acid to the filtrate.

Acceptance criteria: No bluish-green color develops.

Fats and Fixed Oils, Unsaponifiable Matter 〈401〉: NMT 1.5%. [Note—Petroleum ether with a 40°–60° boiling range can be used to replace ether in the test.]

Ultraviolet Absorbance

Sample solution: Dissolve 1.0 g of Olive Oil in cyclohexane, and dilute with cyclohexane to 100 mL.

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Mode: UV-Vis

Wavelength: 270 nm

Path length of the cell: 1 cm

Analysis: Determine the UV-Vis absorbance using the Instrumental conditions described above.

Acceptance criteria: The absorbance is NMT 1.20.

Water Determination, Method Ic 〈921〉: NMT 0.1%

Sterol Composition

2 M Alcoholic potassium hydroxide solution: Dissolve 12 g of potassium hydroxide in 10 mL of water, and dilute with alcohol (Ethanol) to 100mL.

Sample A: Accurately weigh 5 g of Olive Oil into a 150-mL fllask fitted with a re flux condenser. Add 50 mL of 2 M Alcoholic potassium hydroxide solution, and heat on a water bath for 1 h, shaking frequently. Add 50 mL of water through the top of the condenser, shake, and allow to cool. Transfer the contents of the flask to a separating funnel. Rinse the flask with several portions totaling 50 mL of petroleum ether with a 40°–60° boiling range, and add the rinsings to the separating funnel. Shake vigorously for 1 min. Allow to separate, and transfer the aqueous layer to a second separating funnel. If an emulsion forms, add small quantities of alcohol or a concentrated solution of potassium hydroxide. Shake the aqueous layer with two 50-mL quantities of petroleum ether with a 40°–60° boiling range. Combine the petroleum ether layers in a third separating funnel and wash with three 50-mL quantities of 50% alcohol. Transfer the petroleum ether layer to a tared 250-mL flask. Rinse the separating funnel with small quantities of petroleum ether with a 40°- 60° boiling range, and add to the flask. Evaporate the petroleum ether on a water bath and dry the residue at 100°–105° for 15 min, keeping the flask horizontal. Allow to cool in a desiccator and weigh.

Reference A: Accurately weigh 5 g of sun flower oil into a 150-mL flask fitted with a reflux condenser. Proceed as directed for Sample A, beginning with “Add 50 mL of 2 M Alcoholic potassium hydroxide solution”.

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 petroleum ether with a 40°–60° boiling range to a 15-mL test tube.

[Note - Ether can be used to replace petroleum ether if Sample A is not well soluble in petroleum ether.] 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 suficient 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 the 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. The chromatogram from Sample solution A may have one principal peak.

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 and 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 content of each sterol in the sterol fraction of Olive 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: Olive Oil exhibits the composition profiles of sterols shown in Table 4.

Table 4

The content of stigmasterol is NMT that of campesterol.

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant, well-filled containers, and prevent exposure to excessive heat.

Labeling: Label it to indicate the name and quantity of any suitable antioxidants.