Omega-3 Acid Triglycerides
DEFINITION
Omega-3 Acid Triglycerides is a mixture of mono-, di-, and triesters of omega-3 acids with glycerol containing mainly triesters and obtained either by esterification of concentrated and purified omega-3 acids with glycerol or by transesterification of the omega-3 acid ethyl esters with glycerol. The omega-3 acids are from the body oil of fish of the families Engraulidae, Carangidae, Clupeidae, Osmeridae, Salmonidae, and Scombridae and are defined as the following: alpha-linolenic acid (C18:3 n3), 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 58.0% of total omega-3 acids expressed as triglycerides and NLT the labeled amount of EPA and DHA, expressed as the free fatty acids. Suitable antioxidants in appropriate concentrations may be added.
IDENTIFICATION
•  A. The retention times of the peaks for eicosapentaenoic acid methyl ester and docosahexaenoic acid methyl ester of the Sample solution in the tests for Content of EPA and DHA and Content of Total Omega-3 Acids correspond to those for the respective compounds of the Standard solutions. If either EPA or DHA is not claimed on the labeling, the peak corresponding to that omega-3 acid does not exceed 15.0% of the total detected area of the Sample solution in the test for Content of EPA and DHA and Content of Total Omega-3 Acids.
•  B. The retention time of the peak corresponding to the triglycerides of the Sample solution corresponds to the triglycerides peak of the System suitability solution in the test for Content of Oligomers and Partial Glycerides.
COMPOSITION
•  Content of EPA and DHA
Acceptance criteria:  NLT the labeled amount, expressed as free acids
•  Content of Total Omega-3 Acids
Acceptance criteria:  NLT 58.0% of total omega-3 acids, expressed as triglycerides
•  Content of Oligomers and Partial Glycerides
Mobile phase:  Use tetrahydrofuran.
Sample solution:  1.00 mg/mL of Omega-3 Acid Triglycerides in Mobile phase
System suitability solution:  0.5 mg/mL of monodocosahexaenoin, 0.3 mg/mL of didocosahexaenoin, and 0.2 mg/mL of tridocosahexaenoin in Mobile phase. [Note—Suitable grades of monodocosahexaenoin, didocosahexaenoin, and tridocosahexaenoin may be obtained from Nu-Chek Prep. ]
Chromatographic system 
Mode:  LC
Detector:  Differential refractometer
Columns:  Three 7.8-mm × 30-cm; connected in series; packing L21, 7 µm. Two columns are 50 nm in pore size, and the other is 10 nm, arranged so that the 50-nm pore size columns are closer to the injector.
Flow rate:  0.8 mL/min
Injection size:  40 µL
System suitability 
Sample:  System suitability solution
Suitability requirements 
Elution order:   Tridocosahexaenoin, didocosahexaenoin, and monodocosahexaenoin
Resolution:  NLT 2.0 between monodocosahexaenoin and didocosahexaenoin and NLT 1.0 between didocosahexaenoin and tridocosahexaenoin
Analysis 
Sample:  Sample solution
Measure the areas of the major peaks.
Calculate the percentage of oligomers in the portion of Omega-3 Acid Triglycerides taken:
Result = (rU/rT) × 100
rU== sum of the areas of the peaks with a retention time less than that of the triglyceride peak
rT== sum of the areas of all peaks in the chromatogram
Calculate the percentage of partial glycerides (mono- and diglycerides) in the portion of Omega-3 Acid Triglycerides taken:
Result = (rU/rT) × 100
rU== sum of the areas of the peaks corresponding to diglycerides and monoglycerides
rT== sum of the areas of all peaks in the chromatogram
Acceptance criteria:  NMT 3.0% of oligomers and NMT 50.0% of partial glycerides
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 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. ]
Solution A:  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.
Solution B:  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 to a 100-mL volumetric flask, and dilute with deionized water to volume.
Solution C:  Solution A, Solution B, 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 (1:19)
Standard stock solution:  Transfer 10.0 mL of Standard Arsenic Solution, prepared as directed in 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 Omega-3 Acid Triglycerides, 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; and clean out at 2600 with 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 an injection of 5 µL of Solution C 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 Omega-3 Acid Triglycerides taken:
Result = (C × V)/W
C== concentration of arsenic, as obtained above, in the Sample solution (µg/mL)
V== final volume of the Sample solution (mL)
W== weight of Omega-3 Acid Triglycerides 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. 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. ]
Solution A:  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.
Solution B:  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.
Solution C:  Solution A, Solution B, 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 (1:19)
Standard stock solution:  Transfer 10.0 mL of Lead Nitrate Stock Solution, prepared as directed in 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.
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 for Sample solution 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; and clean out at 2600 with 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 an injection of 5 µL of the Solution C 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 Omega-3 Acid Triglycerides taken:
Result = (C × V)/W
C== concentration of lead, as obtained above, in the Sample solution (µg/mL)
V== final volume of the Sample solution (mL)
W== weight of Omega-3 Acid Triglycerides taken to prepare the Sample solution (g)
Acceptance criteria:  NMT 0.1 µg/g
•  Limit of 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. 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. ]
Solution A:  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.
Solution B:  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.
Solution C:  Solution A, Solution B, 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 (1:19)
Standard stock solution A:  0.1372 mg/mL of cadmium nitrate
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 for Sample solution 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; and clean out at 2600 with 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 an injection of 5 µL of the Solution C 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 Omega-3 Acid Triglycerides taken:
Result = (C × V)/W
C== concentration of cadmium, as obtained above, in the Sample solution (µg/mL)
V== final volume of the Sample solution (mL)
W== weight of Omega-3 Acid Triglycerides taken to prepare the Sample solution (g)
Acceptance criteria:  NMT 0.1 µg/g
•  Limit of Mercury: Proceed as directed in 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 two 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 (PCBs)
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
•  Absorbance
Sample solution:  0.24 mg/mL in isooctane
Acceptance criteria:  The absorbance is NMT 0.73, 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 as free acids, in mg/g, and the total content of omega-3 acids as free acids, in mg/g. It also states the name and concentration of any added antioxidant.
•  USP Reference Standards 11
USP Docosahexaenoic Acid Ethyl Ester RS Click to View Structure
all cis-4,7,10,13,16,19-Docosahexaenoic ethyl ester.
    C24H36O2    356.55
USP Eicosapentaenoic Acid Ethyl Ester RS Click to View Structure
all cis-5,8,11,14,17-Eicosapentaenoic ethyl ester.
     C22H34O2        330.51
USP Methyl Tricosanoate RS Click to View Structure
Tricosanoic acid methyl ester.
    C24H48O2        368.64
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