Crypthecodinium cohnii Oil Capsules
DEFINITION
Crypthecodinium cohnii Oil Capsules are prepared from Crypthecodinium cohnii Oil and contain NLT 95.0% and NMT 105.0% of the labeled amount of docosahexaenoic acid (DHA, C22H32O2) (C22:6 n
3).
3).IDENTIFICATION
• Long-Chain Unsaturated Fatty Acid Profile:
Proceed as directed in 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 all the peak responses, except the solvent and butylated hydroxytoluene peaks |
Acceptance criteria:
The retention time of the peaks of the docosahexaenoic acid methyl ester and the eicosapentaenoic acid methyl ester from Test solution 1 corresponds to that from Standard Solution 2, as obtained in the test for Content of EPA and DHA. The area percent for the methyl esters of the fatty acids from Test solution 1 in the test for Content of EPA and DHA meet the requirements for each fatty acid indicated in Table 1.
Table 1
| Fatty Acid |
Relative Retention Time |
Shorthand Notation |
Lower Limit (Area, %) |
Upper Limit (Area, %) |
|---|---|---|---|---|
| Linoleic acid | 0.52 | 18:2 n6 |
0 | 1.0 |
| Eicosapentanoic acid | 0.79 | 20:5 n3 |
0 | 0.1 |
| Docosapentaenoic acid | 0.94 | 22:5 n6 |
0 | 0.1 |
| Docosahexaenoic acid | 1.00 | 22:6 n3 |
35.0 | 47.0 |
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 Crypthecodinium cohnni oil/Capsule. Proceed with the content of capsules as directed in the Analysis.
Analysis:
Proceed as directed in Fats and Fixed Oils 
401
, Omega-3 Fatty Acids Determination and Profile, Content of EPA and DHA.
401, Omega-3 Fatty Acids Determination and Profile, Content of EPA and DHA.
Calculate the percentage of the labeled amount of docosahexaenoic acid (DHA) in the Capsules taken:
Result = R × AFW/L
| R | = | = determined percentage of DHA in the portion of oil taken from the Capsules (%) |
| AFW | = | = average fill weight of the Capsules taken (mg) |
| L | = | = labeled amount of DHA (mg/Capsule) |
Acceptance criteria:
NLT 95.0% and NMT 105.0% of the labeled amount of DHA
PERFORMANCE TESTS
• Disintegration and Dissolution 2040:
Meet the requirements for Rupture Test for Soft Shell Capsules
2040:
Meet the requirements for Rupture Test for Soft Shell Capsules
• Weight Variation 2091:
Meet the requirements
2091:
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.
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 Crypthecondinium cohnii 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.
, 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 Capsules content 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. ]
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:
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 (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:
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.
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.
, 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 Capsules taken:
Result = (C/W) × 25
| C | = | = concentration as obtained above |
| W | = | = weight of Capsules content 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 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 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.
, 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 Capsules taken:
Result = (C/W) × 25
| C | = | = concentration as obtained above |
| W | = | = weight of Capsules content 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.
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
SPECIFIC TESTS
• Fats and Fixed Oils, Anisidine Value 401:
NMT 20.0, determined on the contents of the Capsules
401:
NMT 20.0, determined on the contents of the Capsules
• Fats and Fixed Oils, Free Fatty Acids 401:
The free fatty acids in 10 g require for neutralization NMT 1.42 mL of 0.1 N sodium hydroxide.
401:
The free fatty acids in 10 g require for neutralization NMT 1.42 mL of 0.1 N sodium hydroxide.
• Fats and Fixed Oils, Peroxide Value 401:
NMT 5.0, determined on the contents of the Capsules
401:
NMT 5.0, determined on the contents of the Capsules
• Fats and Fixed Oils, Total Oxidation Value (TOTOX) 401:
NMT 26 (determined on the contents of the Capsules), calculated as
401:
NMT 26 (determined on the contents of the Capsules), calculated as
Result = (2 × PV) + AV
| PV | = | = peroxide value |
| AV | = | = anisidine value |
• Fats and Fixed Oils, Unsaponifiable Matter 401:
NMT 3.5%, determined on the contents of the Capsules
401:
NMT 3.5%, determined on the contents of the Capsules
• Specific Gravity 841:
0.91–0.93, determined on the contents of the Capsules
841:
0.91–0.93, determined on the contents of the Capsules
ADDITIONAL REQUIREMENTS
• 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 Reference Standards 11
11
USP Docosahexaenoic Acid Ethyl Ester RS 
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USP Eicosapentaenoic Acid Ethyl Ester RS
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USP Methyl Tricosanoate RS
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Natalia Davydova
Scientific Liaison 1-301-816-8328 |
(DS2010) Monographs - Dietary Supplements |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1258
Pharmacopeial Forum: Volume No. 35(5) Page 1187