Schizochytrium Oil Capsules
DEFINITION
Schizochytrium Oil Capsules are prepared from Schizochytrium Oil and contain NLT 95.0% and NMT 105.0% of the labeled amount of docosahexaenoic acid (DHA; C22H32O2) (C22:6 n–3).
IDENTIFICATION
•  Long Chain Unsaturated Fatty Acid Profile: Proceed as directed under Strength, Content of DHA.
Analysis
Samples:  Standard Solution 2 and Test Solution 1
Calculate the area percentage for each fatty acid as methyl ester in Test Solution 1:
Result = (rU/rT) × 100
 rU = = peak response of each individual fatty acid as methyl ester rT = = sum of the responses of all the peaks, except the solvent and butylated hydroxytoluene peaks
Acceptance criteria:  The retention times of the peaks of the docosahexaenoic acid methyl ester and the eicosapentanoic acid methyl ester of Test Solution 1 correspond to those of Standard Solution 2, as obtained in the test for Content of EPA and DHA. The area percentage for the methyl esters of the fatty acids from the chromatogram of Test Solution 1 in the test for Content of EPA and DHA meet the requirements for each fatty acid shown in the table below.
Fatty
Acid
Relative
Retention
Time
Shorthand
Notation
Lower
Limit
(Area %)
Upper
Limit
(Area %)
Dihomo-
gammalinolenic
acid
0.71 20:3 n–6 1.7 2.8
Arachidonic acid 0.73 20:4 n–6 0.6 1.3
Eicosapentaenoic acid (EPA) 0.79 20:5 n–3 1.3 3.9
Docosapentaenoic
acid (DPA
n–6)
0.94 22:5 n–6 10.5 16.5
Docosahexaenoic acid
(DHA)
1.00 22:6 n–3 30.0 40.0
STRENGTH
•  Content of DHA
Test Solution 1 and Test Solution 2:  Weigh NLT 10 Capsules in a tared weighing bottle. With a sharp blade or other appropriate means, carefully open the Capsules, without loss of the 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 isooctane. Discard the washings, and allow the empty Capsules to dry in a current of dry air until the isooctane is completely evaporated. Weigh the empty Capsules in the original tared weighing bottle, and calculate the average fill weight (AFW) of schizochytrium oil per Capsule. Proceed with the content of Capsules as directed in the Analysis.
Analysis:
Calculate the percentage of the labeled amount of docosahexaenoic acid (DHA) in the Capsules taken:
Result = R × AFW/L
 R = = percentage of DHA in the portion of oil taken from the Capsules AFW = = average fill weight of the Capsules taken (mg) L = = the labeled amount of DHA (mg/Capsule)
Acceptance criteria:  NLT 95.0% and NMT 105.0% of the labeled amount of DHA
PERFORMANCE TESTS
Meet the requirements of the Rupture Test for Soft Shell Capsules
Meet the requirements
IMPURITIES
Inorganic Impurities
•  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 before use. 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 into 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 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.
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 schizochytrium oil from Capsules, 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 Capsules taken:
Result = (C/W) × 25
 C = = concentration as obtained above W = = weight of Capsule content taken to prepare the Sample solution (g)
Acceptance criteria:  NMT 0.1 µg/g
[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. ]
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 in 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 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.
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 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 Capsules taken:
Result = (C/W) × 25
 C = = concentration, as obtained above W = = weight of Capsule content taken to prepare the Sample solution (g)
Acceptance criteria:  NMT 0.1 µg/g
[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. ]
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 makeup 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 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 Capsules taken:
Result = (C/W) × 25
 C = = concentration, as obtained above W = = weight of Capsule content taken to prepare the Sample solution (g)
Acceptance criteria:  NMT 0.1 µg/g
•  Limit of Mercury
Proceed as directed for Mercury 261, Method IIa and Method IIb, 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
SPECIFIC TESTS
NMT 20.0, determined on the contents of the Capsules
The free fatty acids in 10 g require for neutralization NMT 1.42 mL of 0.1 N sodium hydroxide.
NMT 5.0, determined on the contents of the Capsules
NMT 26 (determined on the contents of the Capsules), calculated as:
Result = (2 × PV) + AV
 PV = = peroxide value AV = = anisidine value
NMT 4.5%, determined on the contents of the Capsules
•  Packaging and Storage: Preserve in tight, light-resistant containers, and avoid exposure to excessive heat.
•  Labeling: The label states the content of docosahexaenoic acid in mg/Capsule. It also states the name and concentration of any added antioxidant.
USP Docosahexaenoic Acid Ethyl Ester RS
USP Eicosapentaenoic Acid Ethyl Ester RS
USP Methyl Tricosanoate RS
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Pharmacopeial Forum: Volume No. 35(5) Page 1192