Fish Oil Containing Omega-3 Acids
DEFINITION
Fish Oil Containing Omega-3 Acids is the purified, winterized, and deodorized fatty oil obtained from fish of the families Engraulidae, Carangidae, Clupeidae, Osmeridae, Scombroidae, and Ammodytidae. The omega-3 acids are defined as the following: alpha-linolenic acid (C18:3 n
3), moroctic acid (C18:4 n3), eicosatetraenoic acid (C20:4 n3), eicosapentaenoic acid (EPA) (C20:5 n3), heneicosapentaenoic acid (C21:5 n3), docosapentaenoic acid (C22:5 n3), and docosahexaenoic acid (DHA) (C22:6 n3). It contains NLT 28.0% (w/w) of total omega-3 acids, expressed as free acids, consisting of NLT 13.0% of EPA and NLT 9.0% of DHA. Suitable antioxidants in appropriate concentrations may be added.
3), moroctic acid (C18:4 n3), eicosatetraenoic acid (C20:4 n3), eicosapentaenoic acid (EPA) (C20:5 n3), heneicosapentaenoic acid (C21:5 n3), docosapentaenoic acid (C22:5 n3), and docosahexaenoic acid (DHA) (C22:6 n3). It contains NLT 28.0% (w/w) of total omega-3 acids, expressed as free acids, consisting of NLT 13.0% of EPA and NLT 9.0% of DHA. Suitable antioxidants in appropriate concentrations may be added.IDENTIFICATION
• The retention times of the peaks for eicosapentaenoic acid methyl ester and docosahexaenoic acid methyl ester of Test Solution 2 in the test for Content of EPA and DHA correspond to those for the respective compounds of Standard Solution 1. The sum of the area for EPA and DHA methyl esters is NLT 22% of the total detected area for the methyl esters, and no other peak has an area higher than 20% of the total detected area for the methyl esters. The chromatogram of Test Solution 2 exhibits at least 15 additional peaks at the retention times of the methyl esters of unsaturated fatty acids exhibited in Standard Solution 2.
COMPOSITION
• Content of EPA and DHA
Analysis
Samples:
Standard Solution 1, Standard Solution 2, Test Solution 1, and Test Solution 2
Identify the retention times of the relevant fatty acid methyl esters peaks by comparing the chromatogram of Standard Solution 2 with the Reference Chromatogram supplied with the USP Fish Oil RS. Identify the retention time for the internal standard peak in the chromatogram of Test Solution 2 by comparing with that of Test Solution 1.
Calculate the percentage of EPA or DHA in the portion of Fish Oil Containing Omega-3 Acids taken:
Result = (RU/RS) × (WS/WU) × F × 100
| RS | = | = ratio of peak responses of either EPA or DHA relative to the internal standard in the chromatogram of Standard Solution 2 |
| WS | = | = weight of either USP Docosahexaenoic Acid Ethyl Ester RS or USP Eicosapentaenoic Acid Ethyl Ester RS used to prepare Standard Solution 1 (mg) |
| WU | = | = weight of the Fish Oil Containing Omega-3 Acids taken to prepare Test Solution 2 (mg) |
| F | = | = factor to express the content of DHA (0.921) and EPA (0.915) as free fatty acids |
| RU | = | = ratio of peak response of either EPA or DHA to the corrected peak response of the internal standard in the chromatogram of Test Solution 2 calculated as follows: |
RU = 1/[(rU2/rT2) (rU1/rT1)]
(rU1/rT1)]| rU2 | = | = peak response of the peak at the locus of the internal standard in the chromatogram of Test Solution 2 |
| rT2 | = | = peak response of EPA or DHA in the chromatogram of Test Solution 2 |
| rU1 | = | = peak response of any peak at the locus of the internal standard in the chromatogram of Test Solution 1 |
| rT1 | = | = peak response of EPA or DHA in the chromatogram of Test Solution 1. [Note—If no peak is found at the locus of the internal standard in the chromatogram of Test Solution 1, RU = rT2/rU2. ] |
Acceptance criteria:
NLT 13.0% (w/w) of EPA and NLT 9.0% (w/w) of DHA
• Content of Total Omega-3 Acids
Analysis:
Proceed as directed in the test for Content of EPA and DHA, except to calculate the content of the total omega-3 acids:
Result = EPA + DHA + [An3(EPA + DHA)]/(AEPA + ADHA)
3(EPA + DHA)]/(AEPA + ADHA)| EPA | = | = content of EPA from the test for Content of EPA and DHA [% (w/w)] |
| DHA | = | = content of DHA from the test for Content of EPA and DHA [% (w/w)] |
| An3 | = | = sum of the areas of the peaks corresponding to C18:3 n3, C18:4 n3, C20:4 n3, C21:5 n3, and C22:5 n3 methyl esters in the chromatogram of Test Solution 2 |
| AEPA | = | = area of the peak corresponding to the EPA methyl ester in the chromatogram of Test Solution 2 |
| ADHA | = | = area of the peak corresponding to the DHA methyl ester in the chromatogram of Test Solution 2 |
Acceptance criteria:
NLT 28.0% (w/w) of total omega-3 acids, expressed as free acids
CONTAMINANTS
• Limit of Arsenic
[Note—For the preparation of all aqueous solutions and for the rinsing of glass, polytef, and plastic vessels before use, use water that has been passed first through a strong-acid, strong-base, mixed-bed ion-exchange resin. Select all reagents to have as low a content of arsenic as practicable, and store all reagent solutions in containers of borosilicate glass. Cleanse glass, polytef, and plastic vessels before use by soaking in warm 8 N nitric acid for 30 min and by rinsing with deionized water. ]
1% Palladium stock solution:
Transfer 1 g of ultrapure palladium metal into a Teflon beaker. Add 20 mL of water and 10 mL of nitric acid, and warm on a hot plate to dissolve. Allow the solution to cool to room temperature, transfer it into a 100-mL volumetric flask, and dilute with deionized water to volume.
1% Magnesium nitrate stock solution:
Transfer 1 g of ultrapure magnesium nitrate into a Teflon beaker. Add 40 mL of water and 1 mL of nitric acid, and warm on a hot plate to dissolve the solids. Allow the solution to cool to room temperature, transfer it into a 100-mL volumetric flask, and dilute with deionized water to volume.
Modifier working solution:
1% Palladium stock solution, 1% Magnesium nitrate stock solution, and 2% nitric acid (3:2:5). A volume of 5 µL provides 0.015 mg of palladium and 0.01 mg of magnesium nitrate.
Blank:
Nitric acid and water (5 in 100)
Standard stock solution:
Transfer 10.0 mL of Standard Arsenic Solution, prepared as directed in the test for Arsenic 
211
, to a 100-mL volumetric flask. Add 40 mL of water and 5 mL of nitric acid, and dilute with water to volume. This solution contains 0.10 µg/mL of arsenic.
211, to a 100-mL volumetric flask. Add 40 mL of water and 5 mL of nitric acid, and dilute with water to volume. This solution contains 0.10 µg/mL of arsenic.
Standard solutions:
Dilute the Standard stock solution with the Blank to obtain concentrations of 0.002, 0.005, 0.010, 0.025, and 0.050 µg/mL of arsenic.
Sample solution:
For preparation of the Sample solution, use a microwave oven with a magnetron frequency of 2455 MHz and a selectable output power of 0–950 watts in 1% increments, equipped with advanced composite vessels with 100-mL polytef liners. Use rupture membranes to vent vessels should the pressure exceed 125 psi. The vessels fit into a turntable, and each vessel can be vented into an overflow container. Equip the microwave oven with an exhaust tube to ventilate fumes. [Caution—Wear proper eye protection and protective clothing and gloves.
] Transfer approximately 500 mg of Fish Oil Containing Omega-3 Acids, weighed to the nearest 0.1 mg, into a Teflon digestion vessel liner. Prepare samples in duplicate. Add 15 mL of nitric acid, and swirl gently. Cover the vessels with lids, leaving the vent fitting off. Predigest overnight under a hood. Place the rupture membrane in the vent fitting, and tighten the lid. Place all vessels on the microwave oven turntable. Connect the vent tubes to the vent trap, and connect the pressure-sensing line to the appropriate vessel. Initiate a two-stage digestion procedure by heating the microwave at 15% power for 15 min, followed by 25% power for 45 min. Remove the turntable of vessels from the oven, and allow the vessels to cool to room temperature. [Note—A cool water bath may be used to speed the cooling process. ] Vent the vessels when they reach room temperature. Remove the lids, and slowly add 2 mL of 30% hydrogen peroxide to each. Allow the reactions to subside, and seal the vessels. Return the vessels on the turntable to the microwave oven, and heat for an additional 15 min at 30% power. Remove the vessels from the oven, and allow them to cool to room temperature. Transfer the cooled digests into 25-mL volumetric flasks, and dilute with water to volume.
Analysis:
Program the graphite furnace as follows. Dry at 115
using a 1-s ramp, a 65-s hold, and an argon flow of 300 mL/min. Char the sample at 1000 using a 1-s ramp, a 20-s hold, and an airflow of 300 mL/min. Cool down and purge the air from the furnace for 10 s using a 20 set temperature and an argon flow of 300 mL/min. Atomize at 2400 using a 0-s ramp and a 5-s hold with the argon flow stopped. Clean out at 2600 using a 1-s ramp and a 5-s hold. Separately inject equal volumes (20 µL) of the Standard solutions, the Sample solution, and the Blank, followed by a 5-µL injection of the Modifier working solution for each of the samples, into the graphite tube of a suitable graphite furnace atomic absorption spectrometer equipped with a hollow-cathode lamp for arsenic. Determine the peak area at the arsenic emission line at 193.7 nm, corrected for background absorption. Plot the corrected peak areas of the Standard solutions versus their contents of arsenic, in µg/mL, and calculate the regression line best fitting the points. Determine the concentration, C, in µg/mL, of arsenic in each mL of the Sample solution by interpolation from the regression line.
using a 1-s ramp, a 65-s hold, and an argon flow of 300 mL/min. Char the sample at 1000 using a 1-s ramp, a 20-s hold, and an airflow of 300 mL/min. Cool down and purge the air from the furnace for 10 s using a 20 set temperature and an argon flow of 300 mL/min. Atomize at 2400 using a 0-s ramp and a 5-s hold with the argon flow stopped. Clean out at 2600 using a 1-s ramp and a 5-s hold. Separately inject equal volumes (20 µL) of the Standard solutions, the Sample solution, and the Blank, followed by a 5-µL injection of the Modifier working solution for each of the samples, into the graphite tube of a suitable graphite furnace atomic absorption spectrometer equipped with a hollow-cathode lamp for arsenic. Determine the peak area at the arsenic emission line at 193.7 nm, corrected for background absorption. Plot the corrected peak areas of the Standard solutions versus their contents of arsenic, in µg/mL, and calculate the regression line best fitting the points. Determine the concentration, C, in µg/mL, of arsenic in each mL of the Sample solution by interpolation from the regression line.
Calculate the content of arsenic in the portion of Fish Oil Containing Omega-3 Acids taken:
Result = (C/W) × 25
| W | = | = weight of Fish Oil Containing Omega-3 Acids taken to prepare the Sample solution (g) |
Acceptance criteria:
NMT 0.1 µg/g
• Limit of Lead
[Note—For the preparation of all aqueous solutions and for the rinsing of glass, polytef, and plastic vessels before use, use water that has been passed through a strong-acid, strong-base, mixed-bed ion-exchange resin before use. Select all reagents to have as low a content of lead as practicable, and store all reagent solutions in containers of borosilicate glass. Cleanse glass, polytef, and plastic vessels before use by soaking in warm 8 N nitric acid for 30 min and by rinsing with deionized water. ]
10% Monobasic ammonium phosphate solution:
10 g of ultrapure monobasic ammonium phosphate in 1 mL of nitric acid and 40 mL of water to dissolve the phosphate. Dilute with deionized water to 100 mL.
1% Magnesium nitrate solution:
Transfer 1 g of ultrapure magnesium nitrate to a Teflon beaker. Add 40 mL of water and 1 mL of nitric acid, and warm on a hot plate to dissolve the solids. Allow the solution to cool to room temperature, transfer it to a 100-mL volumetric flask, and dilute with deionized water to volume.
Modifier working solution:
10% Monobasic ammonium phosphate solution, 1% Magnesium nitrate solution, and 2% nitric acid (2:1:2). A volume of 5 µL provides 0.2 mg of phosphate plus 0.01 mg of magnesium nitrate.
Blank:
Nitric acid and water (5 in 100)
Standard stock solution:
Transfer 10.0 mL of Lead Nitrate Stock Solution, prepared as directed in the test for Heavy Metals 231, to a 100-mL volumetric flask. Add 40 mL of water and 5 mL of nitric acid, and dilute with water to volume. Transfer 1.0 mL of this solution to a second 100-mL volumetric flask. Add 50 mL of water and 1 mL of nitric acid, and dilute with water to volume. This solution contains 0.10 µg/mL of lead.
231, to a 100-mL volumetric flask. Add 40 mL of water and 5 mL of nitric acid, and dilute with water to volume. Transfer 1.0 mL of this solution to a second 100-mL volumetric flask. Add 50 mL of water and 1 mL of nitric acid, and dilute with water to volume. This solution contains 0.10 µg/mL of lead.
Standard solutions:
Dilute the Standard stock solution with the Blank to obtain concentrations of 0.002, 0.005, 0.010, 0.025, and 0.050 µg/mL of lead.
Sample solution:
Prepare as directed in the test for Limit of Arsenic.
Analysis:
Program the graphite furnace as follows. Dry at 120 using a 1-s ramp, a 55-s hold, and an argon flow of 300 mL/min. Char the sample at 850 using a 1-s ramp, a 30-s hold, and an airflow of 300 mL/min. Cool down and purge the air from the furnace for 10 s using a 20 set temperature and an argon flow of 300 mL/min. Atomize at 2100 using a 0-s ramp and a 5-s hold with the argon flow stopped. Clean out at 2600 using a 1-s ramp and a 5-s hold. Separately inject equal volumes (20 µL) of the Standard solutions, the Sample solution, and the Blank, followed by a 5-µL injection of Modifier working solution for each of the samples, into the graphite tube of a suitable graphite furnace atomic absorption spectrometer equipped with a hollow-cathode lamp for lead. Determine the peak area at the lead emission line at 283.3 nm, corrected for background absorption. Plot the corrected peak areas of the Standard solutions versus their contents of lead, in µg/mL, and calculate the regression line best fitting the points. Determine the concentration, C, in µg/mL, of lead in each mL of the Sample solution by interpolation from the regression line.
using a 1-s ramp, a 55-s hold, and an argon flow of 300 mL/min. Char the sample at 850 using a 1-s ramp, a 30-s hold, and an airflow of 300 mL/min. Cool down and purge the air from the furnace for 10 s using a 20 set temperature and an argon flow of 300 mL/min. Atomize at 2100 using a 0-s ramp and a 5-s hold with the argon flow stopped. Clean out at 2600 using a 1-s ramp and a 5-s hold. Separately inject equal volumes (20 µL) of the Standard solutions, the Sample solution, and the Blank, followed by a 5-µL injection of Modifier working solution for each of the samples, into the graphite tube of a suitable graphite furnace atomic absorption spectrometer equipped with a hollow-cathode lamp for lead. Determine the peak area at the lead emission line at 283.3 nm, corrected for background absorption. Plot the corrected peak areas of the Standard solutions versus their contents of lead, in µg/mL, and calculate the regression line best fitting the points. Determine the concentration, C, in µg/mL, of lead in each mL of the Sample solution by interpolation from the regression line.
Calculate the content of lead in the portion of Fish Oil Containing Omega-3 Acids taken:
Result = (C/W) × 25
| W | = | = weight of Fish Oil Containing Omega-3 Acids taken to prepare the Sample solution (g) |
Acceptance criteria:
NMT 0.1 µg/g
• Limit for Cadmium
[Note—For the preparation of all aqueous solutions and for the rinsing of glass, polytef, and plastic vessels before use, use water that has been passed through a strong-acid, strong-base, mixed-bed ion-exchange resin before use. Select all reagents to have as low a content of cadmium as practicable, and store all reagent solutions in containers of borosilicate glass. Cleanse glass, polytef, and plastic vessels before use by soaking in warm 8 N nitric acid for 30 min and by rinsing with deionized water. ]
10% Monobasic ammonium phosphate solution:
10 g of ultrapure monobasic ammonium phosphate in 40 mL of water and 1 mL of nitric acid to dissolve the phosphate. Dilute with deionized water to 100 mL.
1% Magnesium nitrate solution:
Transfer 1 g of ultrapure magnesium nitrate to a Teflon beaker. Add 40 mL of water and 1 mL of nitric acid, and warm on a hot plate to dissolve the solids. Allow the solution to cool to room temperature, transfer it to a 100-mL volumetric flask, and dilute with deionized water to volume.
Modifier working solution:
10% Monobasic ammonium phosphate solution, 1% Magnesium nitrate solution, and 2% nitric acid to volume (2:1:2). A volume of 5 µL provides 0.2 mg of phosphate and 0.01 mg of magnesium nitrate.
Blank:
Nitric acid and water (5 in 100)
Standard stock solution A:
0.1372 mg/mL of cadmium nitrate in water
Standard stock solution B:
Standard stock solution A, nitric acid, and water (2:1:97). This solution contains 0.10 µg/mL of cadmium. [Note—Before make up to final volume dissolve in a portion of water and nitric acid. ]
Standard solutions:
Dilute Standard stock solution B with the Blank to obtain concentrations of 0.002, 0.005, 0.010, 0.025, and 0.050 µg/mL of cadmium.
Sample solution:
Prepare as directed in the test for Limit of Arsenic.
Analysis:
Program the graphite furnace as follows. Dry at 120 using a 1-s ramp, a 55-s hold, and an argon flow of 300 mL/min. Char the sample at 850 using a 1-s ramp, a 30-s hold, and an airflow of 300 mL/min. Cool down and purge the air from the furnace for 10 s using a 20 set temperature and an argon flow of 300 mL/min. Atomize at 2400 using a 0-s ramp and a 5-s hold with the argon flow stopped. Clean out at 2600 using a 1-s ramp and a 5-s hold. Separately inject equal volumes (20 µL) of the Standard solutions, the Sample solution, and the Blank, followed by a 5-µL injection of Modifier working solution for each of the samples, into the graphite tube of a suitable graphite furnace atomic absorption spectrometer equipped with a hollow-cathode lamp for cadmium. Determine the peak area at the cadmium emission line at 228.8 nm, corrected for background absorption. Plot the corrected peak areas of the Standard solutions versus their contents of cadmium, in µg/mL, and calculate the regression line best fitting the points. Determine the concentration, C, in µg/mL, of cadmium in each mL of the Sample solution by interpolation from the regression line. Calculate the content of cadmium in the Fish Oil Containing Omega-3 Acids taken:
using a 1-s ramp, a 55-s hold, and an argon flow of 300 mL/min. Char the sample at 850 using a 1-s ramp, a 30-s hold, and an airflow of 300 mL/min. Cool down and purge the air from the furnace for 10 s using a 20 set temperature and an argon flow of 300 mL/min. Atomize at 2400 using a 0-s ramp and a 5-s hold with the argon flow stopped. Clean out at 2600 using a 1-s ramp and a 5-s hold. Separately inject equal volumes (20 µL) of the Standard solutions, the Sample solution, and the Blank, followed by a 5-µL injection of Modifier working solution for each of the samples, into the graphite tube of a suitable graphite furnace atomic absorption spectrometer equipped with a hollow-cathode lamp for cadmium. Determine the peak area at the cadmium emission line at 228.8 nm, corrected for background absorption. Plot the corrected peak areas of the Standard solutions versus their contents of cadmium, in µg/mL, and calculate the regression line best fitting the points. Determine the concentration, C, in µg/mL, of cadmium in each mL of the Sample solution by interpolation from the regression line. Calculate the content of cadmium in the Fish Oil Containing Omega-3 Acids taken:
Result = (C/W) × 25
| W | = | = weight of Fish Oil Containing Omega-3 Acids taken to prepare the Sample solution (g) |
Acceptance criteria:
NMT 0.1 µg/g
• Limit for Mercury:
Proceed as directed for Mercury 261, Method IIa, except use a Standard Mercury Solution having the equivalent of 0.1 µg/mL of mercury.
261, Method IIa, except use a Standard Mercury Solution having the equivalent of 0.1 µg/mL of mercury.
Sample solution:
Prepare as directed for the Sample solution in the test for Limit of Arsenic combining the 2 duplicate cooled digests into 1.0 mL of Potassium Permanganate Solution.
Acceptance criteria:
NMT 0.1 µg/g
• Limit of Dioxins, Furans, and Polychlorinated Biphenyls
Analysis:
Determine the content of polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) by method No. 1613 revision B of the Environmental Protection Agency. Determine the content of polychlorinated biphenyls (PCBs) by method No. 1668 revision A of the Environmental Protection Agency.
Acceptance criteria:
The sum of PCDDs and PCDFs is NMT 2.0 pg/g of WHO toxic equivalents. The sum of PCDDs, PCDFs, and dioxin-like PCBs (polychlorinated biphenyls, non-ortho IUPAC congeners PCB-77, PCB-81, PCB-126, and PCB-169, and mono-ortho IUPAC congeners PCB-105, PCB-114, PCB-118, PCB-123, PCB-156, PCB-157, PCB-167, and PCB-189) is NMT 10.0 pg/g of WHO toxic equivalents.
SPECIFIC TESTS
• Fats and Fixed Oils, Acid Value 401:
NMT 3
401:
NMT 3
• Fats and Fixed Oils, Anisidine Value 401:
NMT 20.0
401:
NMT 20.0
• Fats and Fixed Oils, Peroxide Value 401:
NMT 5.0
401:
NMT 5.0
• Fats and Fixed Oils, Total Oxidation Value (TOTOX) 401:
NMT 26, calculated:
401:
NMT 26, calculated:
Result = (2 × PV) + AV
| PV | = | = peroxide value |
| AV | = | = anisidine value |
• Fats and Fixed Oils, Unsaponifiable Matter 401:
NMT 1.5%
401:
NMT 1.5%
• Stearin:
10 mL remains clear after cooling at 0 for 3 h
for 3 h
• Absorbance
Sample solution:
0.24 mg/mL in isooctane
Acceptance criteria:
The absorbance is NMT 0.70, determined at 233 nm.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers, and store at controlled room temperature. It may be bottled or otherwise packaged in containers from which air has been expelled by production of a vacuum or by an inert gas.
• Labeling:
The label states the average content of DHA and EPA in mg/g. It also states the name and concentration of any added antioxidant.
• USP Reference Standards 11
11
USP Docosahexaenoic Acid Ethyl Ester RS 
all cis-4,7,10,13,16,19-Docosahexaenoic ethyl ester.
C24H36O2 356.55
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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
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all cis-5,8,11,14,17-Eicosapentaenoic ethyl ester.
C22H34O2 330.51
USP Fish Oil RS
USP Methyl Tricosanoate RS
Tricosanoic acid methyl ester.
C24H48O2 368.64
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Tricosanoic acid methyl ester.
C24H48O2 368.64
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Huy T. Dinh, M.S.
Scientific Liaison 1-301-816-8594 |
(DS2010) Monographs - Dietary Supplements |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1291
Pharmacopeial Forum: Volume No. 34(5) Page 1207