Omega-3-Acid Ethyl Esters

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 is a mixture of ethyl esters, principally the ethyl esters of eicosapentaenoic acid (EPAee) (C20:5 n−3, EE) and docosahexaenoic acid (DHAee) (C22:6 n−3, EE). It may also contain ethyl esters of alpha-linolenic acid (C18:3 n−3, EE), moroctic acid (C18:4 n−3, EE), eicosatetraenoic acid (C20:4 n−3, EE), heneicosapentaenoic acid (C21:5 n−3, EE), and Docosapentaenoic acid (C22:5 n−3, EE). Tocopherol may be added as an antioxidant. 

2 IDENTIFICATION 

A. The retention times of the principal peaks in Test solution 4 correspond to those of eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester in Standard solution 1b and Standard solution 1a, as obtained in the Assay. 

B. It meets the acceptance criteria in Table 1 of the Assay. 

3 ASSAY 

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

(See Fats and Fixed Oils 〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole.) 

Test solution 3, Test solution 4, Standard solution 1a, Standard solution 1b, System suitability solution 1, Chromatographic system, and System suitability: Proceed as directed in the chapter. 

Analysis 

Samples: Test solution 3, Test solution 4, Standard solution 1a, and Standard solution 1b 

Calculate the content of EPAee and DHAee in the portion of Omega-3-Acid Ethyl Esters taken: 

Result = (R_U/R_S) × (W_S/W_U) 

R_U = peak area ratio of EPAee or DHAee to the internal standard from Test solution 3 

R_S = peak area ratio of the EPAee to the internal standard from Standard solution 1b or DHAee to the internal standard from Standard solution 1a 

W_S = weight of DHAee taken for preparing Standard solution 1a or weight of EPAee taken for preparing Standard solution 1b (mg) S 

W_U = weight of sample taken for preparing Test solution 3 (g) 

Calculate the content of total omega-3-acid ethyl esters in the portion of Omega-3-Acid Ethyl Esters 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) in Test solution 4

EPAee = content of EPAee (mg/g) 

DHAee = content of DHAee (mg/g) 

r_EPAee = peak area of EPAee in Test solution 4

r_DHAe  = peak area of DHAee in Test solution 4

Acceptance criteria: It conforms to the acceptance criteria in Table 1. Articles labeled as Omega-3-Acid Ethyl Esters type A meet Acceptance Criteria II. 

Table 1 

^a Alpha-linolenic acid ethyl ester. 

^b Moroctic acid ethyl ester. 

^c Eicosatetraenoic acid ethyl ester. 

^d Eicosapentaenoic acid ethyl ester. 

^e Heneicosapentaenoic acid ethyl ester. 

^f Docosapentaenoic acid ethyl ester (clupanodonic acid ethyl ester). 

^g Docosahexaenoic acid ethyl ester. 

4 IMPURITIES 

Fats and Fixed Oils 〈401〉, Procedures, Trace Metals: NMT 0.1 ppm each of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) Cholesterol 

Internal standard stock solution: 3 mg/mL of 5α-cholestane in n-heptane. [Note—Prepare fresh before use.] 

Internal standard solution: 0.3 mg/mL of 5α-cholestane in n-heptane. [Note—Prepare fresh before use.] 

Standard stock solution: 3.0 mg/mL of cholesterol in n-heptane. [Note—This solution is stable for 6 months stored in a freezer.] Transfer 1.0 mL of this solution to a 10.0-mL volumetric flask. Dilute with n-heptane to volume. [Note—Prepare this solution fresh daily.] 

Standard solution: Transfer 1.0 mL each of the Standard stock solution and the Internal standard solution to a 15-mL centrifuge tube. Evaporate to dryness at about 50° with a gentle stream of nitrogen. Add 0.5 mL of 50% potassium hydroxide and 3 mL of alcohol, ll the tube with nitrogen, and cap. Heat the sample at 100° for 60 min, using a heating block. Cool for about 10 min. Add 6 mL of water to the tube, and shake for 1 min. Extract the solution four times with 2.5-mL portions of ethyl ether, using a vortex mixer or suitable shaker for 1 min for each extraction. Transfer and combine the extracts into a large centrifuge tube, and wash with 5 mL of water, mixing completely with gentle inversion. Remove the water phase, and add 5 mL of 0.5 M potassium hydroxide to the ether phase, mixing carefully to avoid an emulsion. Remove the potassium hydroxide, and add another 5 mL of water, mixing carefully. Transfer the ether phase to a small centrifuge tube. [Note—If an emulsion has occurred, a small amount of sodium chloride may be added to obtain a separation of the phases.] Evaporate the ether phase to dryness under a stream of nitrogen with careful heating. Dissolve the sample in 600 µL of ethyl acetate, and mix well. Transfer 200 µL of this solution to a sample vial, and dilute with ethyl acetate to about 2 mL. 

Alpha tocopherol stock solution: 1.5–2.0 mg/mL of USP Alpha Tocopherol RS in n-heptane. [Note—This solution is stable for 12 months stored in a freezer.] 

System suitability solution: Mix 1.0 mL of the Standard stock solution, 1.0 mL of the Internal standard stock solution, and 2.0 mL of the Alpha tocopherol stock solution in a 50-mL volumetric flask. Evaporate to dryness with the aid of heat, and dilute with ethyl acetate to volume. Dilute 1.0 mL of this solution with ethyl acetate to 10.0 mL. [Note—This solution is stable for 6 months stored in a freezer.] 

Sample solution: Transfer 100 mg of Omega-3-Acid Ethyl Esters to a 15-mL centrifuge tube. Add 1.0 mL of the Internal standard solution. Prepare as directed in the Standard solution beginning with “Evaporate to dryness”. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: GC 

Detector: Flame ionization 

Column: 0.25-mm × 30-m capillary; coated with a 0.25-µm lm of G27 phase 

Temperatures 

Injection port: 320° Detector: 300° 

Column: See Table 2. 

Table 2 

Carrier gas: Helium Flow rate: 1.3 mL/min Injection volume: 1 µL 

Injection type: Splitless injection system 

System suitability 

Sample: System suitability solution 

Suitability requirements 

Resolution: NLT 1.2 between alpha tocopherol and cholesterol 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the content of total cholesterol in the portion of Omega-3-Acid Ethyl Esters taken: 

Result = (R_U/R_S) × (W_S/W_U) 

R_U = peak area ratio of the cholesterol peak to the internal standard from the Sample solution 

R_S = peak area ratio of the cholesterol peak to the internal standard from the Standard solution  

W_S = weight of cholesterol in the Standard solution (mg) 

W_U = weight of Omega-3-Acid Ethyl Esters in the Sample solution (g) 

Acceptance criteria: NMT 3.0 mg/g 

Oligomers 

Mobile phase: Tetrahydrofuran 

System suitability solution: Monodocosahexaenoin, didocosahexaenoin, and tridocosahexaenoin1in Mobile phase, with concentrations of about 0.5, 0.3, and 0.2 mg/mL, respectively. 

Sample solution 1: 5.0 mg/mL of Omega-3-Acid Ethyl Esters 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 Omega-3-Acid Ethyl Esters 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 two quantities, each of 1 mL of water. Carefully evaporate the solvent under a stream of nitrogen, and 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 in concatenated series, 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 injection port 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 

[Note—The elution order is tridocosahexaenoin, didocosahexaenoin, and monodocosahexaenoin.] 

Suitability requirements 

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 peaks

r_T = sum of all the peak areas

Calculate the percentage of oligomers in the portion of Omega-3-Acid Ethyl Esters 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 ethyl esters peaks

r_T = sum of all the peak areas

Acceptance criteria: NMT 1.0% of oligomers 

Change to read: 

Limit of Dioxins, Furans, and Polychlorinated Biphenyls (PCBs): Determine the content of polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) using the current revision of (USP 1-Dec-2022) method No. 1613  (USP 1-Dec-2022) of the Environmental Protection Agency. Determine the content of polychlorinated biphenyls (PCBs) using the current revision of (USP 1-Dec-2022) method No. 1668  (USP 1-Dec-2022) of the Environmental Protection Agency. 

Acceptance criteria: The sum of PCDDs and PCDFs is NMT 1 pg/g of WHO toxic equivalents. The sum of PCBs (IUPAC congeners PCB-28, PCB-52, PCB-101, PCB-118, PCB-138, PCB-153, and PCB-180) is NMT 0.5 ppm. 

Limit of Total Unidentified Fatty Acid Ethyl Esters 

[Note—This test is not required for articles labeled as Omega-3-Acid Ethyl Esters type A.] 

From the chromatogram obtained with Test solution 4 in the Assay for Content of EPAee, DHAee, and Total Omega-3-Acid Ethyl Esters, determine the peak area of the largest single unidentified peak with a relative retention time different from those in Table 3. 

Table 3 

Calculate the content of unidentified fatty acid ethyl esters in area percentage: 

Result = 100 − (100 × Σ Aiee/r_T) 

Aiee = peak area of each identified ethyl ester in Table 3 

r_T = sum of all the peak areas except solvents and butylated hydroxytoluene 

Acceptance criteria: The area of the largest single unidentified peak is NMT 0.5% of the total area. The total area of unidentified peaks as calculated above is NMT 2%. 

Limit of Non-Omega-3-Acid Ethyl Esters 

[Note—This test is only required for articles labeled as Omega-3-Acid Ethyl Esters type A.] 

From the chromatogram obtained with Test solution 4 in the Assay for Content of EPAee, DHAee, and Total Omega-3-Acid Ethyl Esters, calculate the amounts of C18:1 n−9 ethyl ester and C20:4 n−6 ethyl ester in the portion of Omega-3-Acid Ethyl Esters taken: 

Result = (Aiee/r_T) × 100 

Aiee = peak area of C18:1 n−9 ethyl ester or C20:4 n−6 ethyl ester 

r_T = sum of all the peak areas except solvents and BHT 

Acceptance criteria 

C18:1 n−9 ethyl ester: NMT 6.0% 

C20:4 n−6 ethyl ester: NMT 4.0% 

5 SPECIFIC TESTS 

Fats and Fixed Oils 〈401〉, Procedures, Acid Value: NMT 2.0 

Fats and Fixed Oils 〈401〉, Procedures, Anisidine Value: NMT 15 

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

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 0.55, determined at 233 nm, with isooctane being used as the blank 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight, light-resistant containers under a nitrogen atmosphere. Store at controlled room temperature. Labeling: The label states the content of DHA ethyl ester and EPA ethyl ester in mg/g, the sum of the EPA and DHA ethyl esters contents in mg/g, and the content of the total omega-3-acid ethyl esters in weight percentage (w/w). It also states the name of any added antioxidant. Articles which meet Acceptance Criteria II of the Assay and the Limit of Non-Omega-3-Acid Ethyl Esters are labeled as Omega-3-Acid Ethyl Esters type A. 

USP Reference Standards 〈11〉 

USP Alpha Tocopherol RS 

¹ Suitable grades of monodocosahexaenoin, didocosahexaenoin, and tridocosahexaenoin may be obtained from Nu-Chek Prep.