Egg Phospholipids

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

Egg Phospholipids is a mixture of naturally occurring phospholipids obtained from the yolk of hens’ eggs that is suitable for use as an emulsifying agent in injectable emulsions. The content of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, and other related phospholipids is to be reported in the certificate of analysis. It may also contain a suitable stabilizer.

2 ASSAY

2.1 Content of Phospholipids

Solution A: 1341.6 g of n-hexane, 334.1 g of 2-propanol, 39.4 g of glacial acetic acid, and 1.45 g of triethylamine (or 2.0 mL triethylamine)

Solution B: 663.5 g of 2-propanol, 140.0 g of water, 15.8 g of glacial acetic acid, and 0.58 g of triethylamine

Solvent: n-Hexane, 2-propanol, and water (23:23:4). [Note—To avoid the formation of two phases, mix the 2-propanol and water first, and then add the n-hexane.]

Mobile phase: See the gradient table below.

Standard solutions: Transfer USP Phosphatidylcholine RS, USP Phosphatidylethanolamine RS, and USP Lysophosphatidylcholine RS to separate flasks, dissolve each in Solvent, and dilute. Standard solutions of five different concentrations are prepared on the basis of the expected content of phosphatidylcholine, phosphatidylethanolamine, and lysophosphatidylcholine in the sample. The Standard solutions should cover a range of 60% to 140%. Calculate the concentrations of the Standards:

Result = WP/V

W = weight of the Standard (mg)

P = purity of the designated Reference Standard

V = volume of each of the Standard solutions (mL)

Sample solution: 100 mg of Egg Phospholipids in a 25-mL volumetric flask. Dissolve in Solvent, and dilute. Calculate the concentration, in mg/mL: this value is used as the sample amount.

2.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: evaporative light-scattering detector

Column: 4-mm × 125-mm; 5-μm packing L20

Column temperature: 55°

Injection size: 20 μL

2.3 System suitability

Sample: Standard solutions

[Note-The relative retention times for phosphatidylcholine, phosphatidylethanolamine, and lysophosphatidylcholine are 1.00, 0.85, and 1.25, respectively.]

2.4 Suitability requirements

Relative standard deviation: NMT 5.0%

2.5 Analysis

Samples: Each of the Standard solutions and Sample solution

Identify the peaks of the relevant analytes in the chromatogram of the Sample solution by comparison with the chromatograms obtained from the Standard solutions. Measure the areas of the analyte peaks. Plot the logarithms of the relevant responses versus the logarithms of the concentrations, in mg/mL, of each analyte obtained from the Standard solutions, and determine the linear regression line using a least-squares analysis. The correlation coeficient for the linear regression line is NLT 0.995. From the graphs so obtained, determine the concentration, C, in mg/mL, of the relevant analyte in the Sample solution.

Separately calculate the percentages of phosphatidylethanolamine, phosphatidylcholine, and lysophosphatidylcholine in the portion of

Egg Phospholipids taken:

Result = (CV/W) × 100

C = concentration of the relevant analyte in the Sample solution (mg/mL)

V = volume of the relevant analyte in the Sample solution (mL)

W = weight of Egg Phospholipids in the Sample solution (mg)

Acceptance criteria: NMT 3.0% of lysophosphatidylcholine

3 IMPURITIES

Organic Impurities

Procedure: Limit of Nonphosphatidyl Lipids

Solvent: Diethyl ether

Sample solution: 500 mg of Egg Phospholipids, dissolved in 15 mL of Solvent, in a 50-mL conical flask

Chromatographic system

(See Chromatography 〈621〉, Column Chromatography.)

Mode: Column

Chromatographic column:

Transfer 1000 g of silica gel having a particle size of 0.05–0.2 mm into a container with well-closing screw caps. Add 150 g of water, shake well, and allow to stand for 24 h. Suspend 15 g of prepared adsorbent in 50 mL of Solvent, and introduce into a 1- to 2-cm chromatographic column. Drain the Solvent through the column to a level of about 1 cm above the silica gel bed.

Analysis

Sample: Sample solution

Transfer the Sample solution to the Chromatographic column. Rinse the column containing the Sample solution with two 15-mL portions of Solvent, allowing each rinse to pass through the column before adding the next. After rinsing, elute with 105 mL of Solvent.

Evaporate the eluate (150 mL) in a tared, round–bottom, 250-mL conical flask to dryness, using a suitable rotary evaporator. The volatiles are blown out with a stream of nitrogen, and the residue is dried at 105° for 20 min. The weight of the residue gives the oil fraction, determined as nonpolar lipids, in Egg Phospholipids.

Calculate the percentage of the nonphosphatidyl lipids taken:

Result = A/W × 100

A = weight of the residue (mg)

W = weight of Egg Phospholipids taken in the Sample solution (mg)

Acceptance criteria: NMT 7.0%

4 SPECIFIC TESTS

Fats and Fixed Oils, Acid Value 〈401〉: NMT 20.0

Fats and Fixed Oils, Peroxide Value 〈401〉: NMT 3

Bacterial Endotoxins Test 〈85〉: NMT 6 USP Endotoxin Units/g

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total microbial count does not exceed 100 cfu/g. It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.

Water Determination, Method I 〈921〉

Sample: 2 g in 50 mL of anhydrous methyl alcohol

Acceptance criteria: NMT 6.0%

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve under nitrogen in a sealed container, and store at a temperature of −10° or below.

USP Reference Standards 〈11〉

USP Phosphatidylcholine RS

USP Phosphatidylethanolamine RS

USP Lysophosphatidylcholine RS