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.
• Content of Phospholipids
Solution A: 1341.6 g of n-hexane, 334.1 g of 2-propanol, 39.4 g of 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 acetic acid, and 0.58 g of triethylamine
Solvent: n-Hexane, 2-propanol, and water (23:23:4). [NoteTo 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
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.
Detector: evaporative light-scattering detector
Column: 4-mm × 125-mm; 5-µm packing L20
Column temperature: 55
Injection size: 20 µL
Sample: Standard solutions
[NoteThe relative retention times for phosphatidylcholine, phosphatidylethanolamine, and lysophosphatidylcholine are 1.00, 0.85, and 1.25, respectively. ]
Relative standard deviation: NMT 5.0%
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 coefficient 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
Acceptance criteria: NMT 3.0% of lysophosphatidylcholine
• Heavy Metals, Method II 231: NMT 10 ppm
• 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
(See Chromatography 621, Column Chromatography.)
Chromatographic column:Transfer 1000 g of silica gel having a particle size of 0.050.2 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.
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, roundbottom, 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
Acceptance criteria: NMT 7.0%
• Fats and Fixed Oils, Acid Value 401: NMT 20.0
• 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%
• Packaging and Storage: Preserve under nitrogen in a sealed container, and store at a temperature of 10 or below.
• USP Reference Standards 11
USP Endotoxin RS
USP Phosphatidylcholine RS
USP Phosphatidylethanolamine RS
USP Lysophosphatidylcholine RS
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USP35NF30 Page 1789Pharmacopeial Forum: Volume No. 33(4) Page 703