Omega-3-Acid Ethyl Esters Capsules

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 

Omega-3-Acid Ethyl Esters Capsules contain Omega-3-Acid Ethyl Esters, with NLT 95.0% and NMT 105.0% of the labeled sum of eicosapentaenoic acid ethyl ester (EPAee) and docosahexaenoic acid ethyl ester (DHAee) and NLT 95% of the labeled amount of total omega-3-acid ethyl esters, as the sum of alpha-linolenic acid ethyl ester (C18:3 n−3, EE), moroctic acid ethyl ester (C18:4 n−3, EE), eicosatetraenoic acid ethyl ester (C20:4 n−3, EE), eicosapentaenoic acid ethyl ester (EPAee) (C20:5 n−3, EE), heneicosapentaenoic acid ethyl ester (C21:5 n−3, EE), Docosapentaenoic acid ethyl ester (C22:5 n−3, EE), and docosahexaenoic acid ethyl ester (DHAee) (C22:6 n−3, EE). Tocopherol may be added as an antioxidant. 

2 IDENTIFICATION 

A. The retention times of the peaks for eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester of the Sample solution correspond to those of the Standard solution, as obtained in the Assay for Content of EPAee, DHAee, and Total Omega-3-Acid Ethyl Esters.

B. It complies with the Acceptance criteria in the test for Concentration of Omega-3-Acid Ethyl Esters in Specific Tests. 

3 ASSAY 

Content of EPAee, DHAee, and Total Omega-3-Acid Ethyl Esters 

[Note—Carry out the procedure as rapidly as possible, avoiding exposure to actinic light, oxidizing agents, oxidation catalysts (e.g., copper and iron), and air.] 

Antioxidant solution: 50 mg/L of butylated hydroxytoluene in isooctane 

Retention time identification solution: Prepare a mixture containing suitable concentrations of alpha-linolenic acid ethyl ester (C18:3 n−3, EE), moroctic acid ethyl ester (C18:4 n−3, EE), eicosatetraenoic acid ethyl ester (C20:4 n−3, EE), heneicosapentaenoic acid ethyl ester (C21:5 n−3, EE), and docosapentaenoic acid ethyl ester (C22:5 n−3, EE) in Antioxidant solution.¹ 

Internal standard solution: 7.0 mg/mL of USP Methyl Tricosanoate RS in Antioxidant solution 

System suitability solution: 5.5 mg/mL of docosahexaenoic acid methyl ester and 0.5 mg/mL of tetracos-15-enoic acid methyl ester in Antioxidant solution 

Standard solution: Dissolve 60.0 mg of USP Docosahexaenoic Acid Ethyl Ester RS and 90.0 mg of USP Eicosapentaenoic Acid Ethyl Ester RS in 10.0 mL of Internal standard solution. 

Sample solution: Weigh NLT 10 Capsules in a tared weighing bottle. With a sharp blade, carefully open the Capsules, without loss of shell material, and transfer the combined Capsule contents to a 100-mL beaker. Remove any adhering substance from the emptied Capsules by washing with several small portions of diethyl ether. Discard the washings, and allow the empty Capsules to air-dry over a period of NMT 30 min, taking precautions to avoid uptake or loss of moisture. Weigh the empty Capsules in the original tared weighing bottle, and calculate the average ll weight per Capsule (W ). Transfer an amount of the combined Capsule contents equivalent to 225 mg of the labeled 

amount of total omega-3-acid ethyl esters to a suitable flask, and dissolve with 10.0 mL of Internal standard solution. Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: GC 

Detector: Flame ionization 

Column: 0.25-mm × 25–50-m fused silica capillary; coated with a 0.25-µm lm of phase G16 

Temperatures 

Injection port: 250° 

Detector: 270° 

Column: See Table 1. 

Table 1 

Carrier gas: Hydrogen or helium 

Linear velocity: Adjust to obtain a retention time for docosahexaenoic acid ethyl ester of 26 ± 3 min. 

Injection volume: 1 µL 

Injection type: Split; split ratio, 1:220 

System suitability 

Samples: System suitability solution and Standard solution 

Suitability requirements 

Resolution: NLT 1.2 between the docosahexaenoic acid methyl ester and tetracos-15-enoic acid methyl ester peaks, System suitability solution 

Relative standard deviation: NMT 2.0% for the ratios of the peak responses of DHAee and EPAee relative to the internal standard, Standard solution 

Analysis 

Samples: Retention time identification solution, Standard solution, and Sample solution 

Identify the retention times of the relevant fatty acid ethyl esters by comparing the peaks from the Sample solution with those from the Retention time identification solution. 

Calculate the content, in mg/g, of EPAee and DHAee in the portion of Capsules taken: 

Result = (R_U/R_S) × (C_S/C_U) 

R_U = peak area ratio of the EPAee or DHAee peak to the internal standard peak from the Sample solution  

R_S = peak area ratio of the EPAee or DHAee peak to the internal standard peak from the Standard solution 

C_S = concentration of USP Eicosapentaenoic Acid Ethyl Ester RS or USP Docosahexaenoic Acid Ethyl Ester RS in the Standard solution (mg/mL) 

C_U = Capsule ll content of the Sample solution (g/mL) 

Calculate the percentage of the labeled sum of EPAee and DHAee in the portion of Capsules taken: 

Result = (EPAee + DHAee) × W_AF × (100/L)  

EPAee = content of EPAee in the portion of Capsules taken (mg/g) 

DHAee = content of DHAee in the portion of Capsules taken (mg/g) 

W_AF = average ll weight of the Capsules taken (g) 

L = sum of the labeled content of EPAee and DHAee (mg/Capsule) 

Calculate the percentage of the labeled amount of total omega-3-acid ethyl esters in the portion of Capsules taken: 

Result = {r_FAn–3ee × [(EPAee + DHAee)/(r_EPAee + r_DHAee)] + EPAee + DHAee} × W_AF × (100/L) 

r_FAn–3ee = sum of the peak areas of alpha-linolenic acid ethyl ester (C18:3 n−3, EE), moroctic acid ethyl ester (C18:4 n−3, EE),  eicosatetraenoic acid ethyl ester (C20:4 n−3, EE), heneicosapentaenoic acid ethyl ester (C21:5 n−3, EE), and 3ee  docosapentaenoic acid ethyl ester (C22:5 n−3, EE) from the Sample solution 

EPAee= content of EPAee (mg/g) 

DHAee= content of DHAee (mg/g) 

r_EPAee = peak area of EPAee from the Sample solution 

r_DHAee = peak area of DHAee from the Sample solution 

W_AF = average ll weight of the Capsules taken (g) 

L = label claim of total omega-3-acids ethyl esters (mg/Capsule) 

Acceptance criteria: 95.0%–105.0% of the labeled sum of EPAee and DHAee and NLT 95.0% of the labeled amount of total omega-3-acid ethyl esters per Capsule 

4 PERFORMANCE TESTS 

Uniformity of Dosage Units 〈905〉, Weight Variation: Meet the requirements 

Disintegration 〈701〉 

Medium, tier 1: Water 

Medium, tier 2: Simulated gastric uid TS 

Time: 30 min 

Analysis: Perform the test with water as Medium, tier 1. Repeat the test with simulated gastric uid TS as Medium, tier 2, if the disintegration time is more than 30 min in Medium, tier 1. 

Acceptance criteria: Meet the requirements 

5 IMPURITIES 

Oligomers 

Mobile phase: Tetrahydrofuran 

System suitability solution: Monodocosahexaenoin, didocosahexaenoin, and tridocosahexaenoin in Mobile phase, with concentrations of about 0.5, 0.3, and 0.2 mg/mL, respectively. [Note—Suitable grades of monodocosahexaenoin, didocosahexaenoin, and tridocosahexaenoin may be obtained from Nu-Chek Prep.] 

Sample solution 1: 5.0 mg/mL of the Capsule contents in tetrahydrofuran 

Sample solution 2: [Note—Use Sample solution 2 where the results of this test using Sample solution 1 exceed the Acceptance criteria due to the presence of monoglycerides.] Weigh 50 mg of the Capsule contents into a quartz tube, add 1.5 mL of a 20-g/L solution of sodium hydroxide in methanol, cover with nitrogen, cap tightly with a polytef-lined cap, mix, and heat on a water bath for 7 min. Allow to cool. Add 2.0 mL of boron trichloride–methanol solution, cover with nitrogen, cap tightly, mix, and heat on a water bath for 30 min. Cool to 40°–50°, add 1 mL of isooctane, cap, and shake vigorously for NLT 30 s. Immediately add 5 mL of saturated sodium chloride solution, cover with nitrogen, cap, and shake thoroughly for NLT 15 s. Transfer the upper layer to a separate tube. Shake the methanol layer with 1 mL of isooctane. Wash the combined isooctane extracts with 2 quantities, each of 1 mL of water. Carefully evaporate the solvent under a stream of nitrogen, then add 10.0 mL of tetrahydrofuran to the residue. Add a small amount of anhydrous sodium sulfate, and filter. Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: Differential refractometer 

Columns: Three concatenated, 7.8-mm × 30-cm; 7-µm packing L21, with pore sizes in the range of 5–50 nm, arranged with decreasing pore size from the injector to the detector to fulfill the system suitability requirements 

Flow rate: 0.8 mL/min 

Injection volume: 40 µL 

System suitability 

Sample: System suitability solution 

Suitability requirements 

Elution order: Tridocosahexaenoin, didocosahexaenoin, and monodocosahexaenoin 

Resolution: NLT 2.0 between monodocosahexaenoin and didocosahexaenoin; NLT 1.0 between didocosahexaenoin and tridocosahexaenoin 

Analysis 

Samples: Sample solution 1 and Sample solution 2 

Measure the areas of the major peaks. 

Calculate the percentage of oligomers in the portion of omega-3-acid ethyl esters taken to prepare Sample solution 1: 

Result = (r_I/r_T) × 100 

r_I = sum of the peak areas with retention times less than that of the ethyl esters peak from Sample solution 1 

r_T = sum of the areas of all peaks from Sample solution 1 

Calculate the percentage of oligomers in the portion of the Capsules contents taken to prepare Sample solution 2: 

Result = (r_I/r_T) × 100 

r_I = sum of the peak areas with retention times less than that of the methyl esters peak from Sample solution 2 

r_T = sum of the areas of all peaks from Sample solution 2  

Acceptance criteria: NMT 2% of oligomers 

6 SPECIFIC TESTS 

Concentration of Omega-3-Acid Ethyl Esters 

Antioxidant solution, Retention time identification solution, Internal standard solution, System suitability solution, Standard solution, Sample solution, Chromatographic system, System suitability, and Analysis: Proceed as directed in the Assay for Content of EPAee, DHAee, and Total Omega-3-Acid Ethyl Esters. 

Calculate the concentration, in mg/g, of EPAee and DHAee in the portion of Capsules taken: 

Result = (R_U/R_S) × (C_S/C_U) 

R_U = peak area ratio of the EPAee or DHAee peak to the internal standard peak from the Sample solution 

R_S = peak area ratio of the EPAee or DHAee peak to the internal standard peak from the Standard solution 

C_S = concentration of USP Eicosapentaenoic Acid Ethyl Ester RS or USP Docosahexaenoic Acid Ethyl Ester RS in the Standard solution (mg/mL) 

C_U = Capsule ll content of the Sample solution (g/mL) 

Calculate the concentration, in mg/g, of total omega-3-acids ethyl esters in the portion of Capsules taken: 

Result = r_FAn–3ee × [(EPAee + DHAee)/(r_EPAee + r_DHAe  )] + EPAee + DHAee 

r_FAn–3ee = sum of the peak areas of alpha-linolenic acid ethyl ester (C18:3 n−3, EE), moroctic acid ethyl ester (C18:4 n−3, EE), eicosatetraenoic acid ethyl ester (C20:4 n−3, EE), heneicosapentaenoic acid ethyl ester (C21:5 n−3, EE), and docosapentaenoic acid ethyl ester (C22:5 n−3, EE) from the Sample solution 

EPAee = content of EPAee (mg/g) 

DHAee = content of DHAee (mg/g) 

r_EPAee = peak area of EPAee from the Sample solution 

r_DHAe  = peak area of DHAee from the Sample solution 

Acceptance criteria: It meets the requirements in Table 2. Capsules labeled as containing Omega-3-Acid Ethyl Esters type A meet Acceptance Criteria II. 

Table 2 

^aSum of alpha-linolenic acid ethyl ester (C18:3 n−3, EE), moroctic acid ethyl ester (C18:4 n−3, EE), eicosatetraenoic acid ethyl ester (C20:4 n−3, EE), eicosapentaenoic acid ethyl ester (EPAee) (C20:5 n−3, EE), heneicosapentaenoic acid ethyl ester (C21:5 n−3, EE), docosapentaenoic acid ethyl ester (C22:5 n−3, EE), and docosahexaenoic acid ethyl ester (DHAee) (C22:6 n−3, EE). Fats and Fixed Oils 〈401〉, Procedures, Acid Value 

Sample solution: Dissolve about 5.0 g of the oil, accurately weighed, in 100 mL of a mixture of equal volumes of alcohol and ether (which has been neutralized to phenolphthalein with 0.1 M potassium hydroxide) contained in a flask. 

Acceptance criteria: NMT 2.0 

Fats and Fixed Oils 〈401〉, Procedures, Anisidine Value: NMT 25 

Fats and Fixed Oils 〈401〉, Procedures, Peroxide Value: NMT 10 mEq/kg 

Absorbance 

Sample solution: Transfer 300 mg, accurately weighed, to a 50-mL volumetric flask. Dissolve in and dilute immediately with isooctane to volume. Pipet 2.0 mL into a 50-mL volumetric flask, and dilute with isooctane to volume. 

Acceptance criteria: NMT 0.60, determined at 233 nm in a 1-cm cell, with isooctane being used as the blank Microbial Enumeration Tests 〈61〉: NMT 103 cfu/g for the total aerobic microbial count, and NMT 102 cfu/g for the total combined yeasts and molds count. 

Tests for Specified Microorganisms 〈62〉: Meet the requirements for absence of Escherichia coli in 1 g and for absence of Salmonella species in 10 g 

7 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight containers, and store at controlled room temperature. Do not freeze. Protect from light. Labeling: The label states the amount of docosahexaenoic acid (DHA) ethyl ester and eicosapentaenoic acid (EPA) ethyl ester, and the minimum amount of total content of omega-3-acid ethyl esters in mg/Capsule. Capsules which meet Acceptance Criteria II of the test for Concentration of Omega-3-Acid Ethyl Esters are labeled as containing Omega-3-Acid Ethyl Esters type A. It also states the name and content of any added antioxidant. 

USP Reference Standards 〈11〉 

USP Docosahexaenoic Acid Ethyl Ester RS 

All cis-4,7,10,13,16,19-docosahexaenoic ethyl ester. 

C₂₄H₃₆O₂ 356.55 

USP Eicosapentaenoic Acid Ethyl Ester RS 

All cis-5,8,11,14,17-eicosapentaenoic ethyl ester. 

C₂₂H₃₄O₂ 330.51 

USP Methyl Tricosanoate RS 

Tricosanoic acid methyl ester. 

C₂₄H₄₈O₂ 368.64 

¹ The relevant fatty acid ethyl esters are available from Nu-Chek Prep, Inc. (www.nu-chekprep.com); Cayman Chemical (www.caymanchem.com); and Carbosynth (www.carbosynth.com).