8/12/2019
`Printed on: Mon Aug 12 2019, 11:36:29 am
`Printed by: Nicole Chang
`Currently Ocial as of: 12-Aug-2019
`Ocial as of 1-May-2019
`DocId: GUID-B2C1B38E-606D-4103-857F-4DBF2B9F18EC_2_en-US
`Printed from: https://online.uspnf.com/uspnf/document/GUID-B2C1B38E-606D-4103-857F-4DBF2B9F18EC_2_en-US?highlight=KrillOil
`© 2019 USPC
`
`USP-NF
`
`Krill Oil Capsules
`
`DEFINITION
`Change to read:
`▲Krill Oil Capsules contain NLT 95.0% of the labeled amount of Krill Oil calculated through the content of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) ,
`and total phospholipids.▲ (USP 1-May-2019)
`
`IDENTIFICATION
`Change to read:
`• A. FATTY ACID PROFILE
`
`Antioxidant solution, System suitability solution 1, and Chromatographic system: Proceed as directed in Fats and Fixed Oils 〈401〉, Procedures, Omega-3 Fatty
`Standard solution: Prepare as directed in Fats and Fixed Oils 〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole, Test solution 1, except use 250 mg of
`Sample solution: Using the portion of oil from NLT 10 Capsules, prepare as directed in Fats and Fixed Oils 〈401〉, Procedures, Omega-3 Fatty Acids Determination and
`
`Acids Determination and Prole.
`
`USP Krill Oil RS.
`
`Prole, Test solution 1.
`System suitability
`Samples: System suitability solution 1 and Standard solution
`Suitability requirements
`Chromatogram similarity: The chromatogram from the Standard solution is similar to the reference chromatogram provided with the lot of USP Krill Oil RS being
`used.
`Resolution: NLT 1.0 between the methyl oleate and methyl cis-vaccinate peaks, Standard solution
`Theoretical area percentages: Meet the requirements for System suitability solution 1
`Analysis
`Sample: Standard solution and Sample solution
`Identify the retention times of the peaks corresponding to the relevant fatty acid methyl esters by comparing the chromatogram of the Standard solution to the
`reference chromatogram provided with the lot of USP Krill Oil RS used.
`Calculate the area percentage for each fatty acid as methyl esters in the portion of oil taken from the Capsules:
`
`= peak area of each individual fatty acid from the Sample solution
`
`= total area of all peaks, except the solvent and butylated hydroxytoluene peaks, from the Sample solution
`
`Result = (r /r ) × 100
`A B
`
`r
`
`r
`
`A B
`
`Acceptance criteria: See Table 1.
`
`Table 1
`
`Fatty Acid
`
`Short-Hand Notation
`
`Lower Limit (Area %)
`
`Upper Limit (Area %)
`
`Saturated fatty acids
`
`Myristic acid
`
`Palmitic acid
`
`14:0
`
`16:0
`
`Palmitic acid:myristic acid ratio
`
`16:0/14:0
`
`Monounsaturated fatty acids
`
`Palmitoleic acid
`
`cis-Vaccenic acid
`
`Oleic acid
`
`Eicosenic acid
`
`16:1 n-7
`
`18:1 n-7
`
`18:1 n-9
`
`20:1 n-9
`
`▲5.0▲ (USP 1-May-2019)
`
`17.0
`
`1.6
`
`2.5
`
`4.7
`
`▲6.0▲ (USP 1-May-2019)
`
`0.0
`
`13.0
`
`24.6
`
`▲3.6▲ (USP 1-May-2019)
`
`▲12.0▲ (USP 1-May-2019)
`
`8.0
`
`14.5
`
`2.0
`
`Erucic acid
`
`22:1 n-9
`
`0.0
`
`1.5
`
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`USP-NF
`
`Fatty Acid
`
`Short-Hand Notation
`
`Lower Limit (Area %)
`
`Upper Limit (Area %)
`
`Polyunsaturated fatty acids
`
`Linoleic acid
`
`Eicosapentaenoic acid
`
`Docosapentaenoic acid
`
`Docosahexaenoic acid
`
`18:2 n-6
`
`20:5 n-3
`
`22:5 n-3
`
`22:6 n-3
`
`0.0
`
`14.0
`
`0.0
`
`7.1
`
`3.0
`
`▲28.0▲ (USP 1-May-2019)
`
`0.7
`
`15.7
`
`Change to read:
`• B. PHOSPHOLIPID PROFILE
`Solution A, ▲▲ (USP 1-May-2019) Internal standard, ▲Sample stock solution,▲ (USP 1-May-2019) Sample solution, Standard solution, Instrumental conditions, System
`suitability, and Analysis: Proceed as directed in the test for Content of Total Phospholipids in Strength.
`Acceptance criteria: ▲The Sample solution shows P nuclear magnetic resonance (NMR) spectra similar to those obtained with USP Krill Oil RS. The main signal in
`31
`the P NMR is due to phosphatidylcholine (PC); a signal due to phosphatidylcholine ether has an intensity of about 10% of that due to PC; and the second signal in
`31
`intensity is due to 2-lysophosphatidylcholine (2-LPC). Minor signals due to phosphatidylethanolamine (PE), N-acylphosphatidylethanolamine (NAPE),
`lysophosphatidylethanolamine (LPE), and 1-lysophosphatidylcholine (1-LPC) among others are also observed.▲ (USP 1-May-2019)
`
`STRENGTH
`Delete the following:
`▲• CONTENT OF KRILL OIL
`Analysis: Weigh NLT 10 Capsules in a tared weighing bottle, and carefully open the Capsules, without loss of shell material. 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 2,2,4-trimethylpentane. Discard the
`washings, and allow the empty Capsules to dry in a current of dry air until the 2,2,4-trimethylpentane is completely evaporated. Weigh the empty Capsules in the
`original tared weighing bottle, and calculate the average net weight per Capsule.
`Acceptance criteria: 95.0%–105.0%▲ (USP 1-May-2019)
`Change to read:
`• CONTENT OF EPA AND DHA
`
`(See Fats and Fixed Oils 〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole).
`
`Standard solution 1a, Standard solution 1b, Standard solution 2a, Standard solution 2b, System suitability solution 1, and Chromatographic system: Proceed as
`directed in the chapter.
`Test solution 1: ▲Weigh NLT 10 Capsules in a tared weighing bottle, and carefully open the Capsules without loss of shell material. Transfer the combined contents
`to a 100-mL beaker. Remove any adhering substance from the emptied Capsules by washing with several small portions of acetone. Discard the washings, and
`allow the empty Capsules to dry in a current of air until the acetone is completely evaporated. Weigh the empty Capsules in the original tared weighing bottle, and
`
`calculate the average ll weight per Capsule. Take 250 mg of the combined Capsule contents and▲ (USP 1-May-2019) proceed as directed in Fats and Fixed Oils 〈401〉,
`Test solution 2: ▲▲ (USP 1-May-2019) Prepare as directed in Fats and Fixed Oils 〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole, Test solution 2▲using
`Analysis: Proceed as directed in Fats and Fixed Oils 〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole, Analysis (for triglycerides).
`
`Procedures, Omega-3 Fatty Acids Determination and Prole, Test solution 1.
`
`250 mg of the combined Capsule contents.▲ (USP 1-May-2019)
`
`▲Calculate the percentage of the labeled amounts of EPA and DHA in the portion of Capsules taken:
`
`Result = A × W × 100/L
`Av
`
`A
`
`= content of EPA or DHA in the portion of Capsules taken (mg/mg)
`
`W
`Av
`
`L
`
`= average ll weight (mg/Capsule)
`
`= label claim of EPA or DHA (mg/Capsule)▲ (USP 1-May-2019)
`
`Acceptance criteria: ▲NLT 95.0% of the labeled amounts of EPA and DHA▲ (USP 1-May-2019)
`Change to read:
`• CONTENT OF TOTAL PHOSPHOLIPIDS
`
`(See Nuclear Magnetic Resonance Spectroscopy 〈761〉, Qualitative and Quantitative NMR Analysis.)
`
`[NOTE—All deuterated solvents used in this method should be NLT 99.8 atom % D. ▲▲ (USP 1-May-2019)]
`Solution A: 0.2 M ethylenediaminetetraacetic acid (EDTA) adjusted with a 1 M cesium carbonate solution to a pH of 7.2–7.5. ▲▲ (USP 1-May-2019)[NOTE—Use cesium
`carbonate of a sucient grade for trace metals analysis.]
`▲▲ (USP 1-May-2019)
`Internal standard: Use a triphenyl phosphate nuclear magnetic resonance (NMR) reference standard with NLT 99% purity.
`▲Sample stock solution: Transfer a number of Capsules (NLT 5) to a conical ask with stopper. Add 2 mL/Capsule of water and melt the gelatin shells at 50°–60°.
`Add 10 mL/Capsule of chloroform and methanol (2:1), insert the stopper and shake intensively for 30 min. Transfer to a separation funnel tted with glass wool.
`Collect the organic phase. Wash the conical ask with chloroform and methanol (2:1) and transfer to a separation funnel. Collect the organic phase and combine
`with previous extract in a suitable volumetric ask. Dilute with chloroform and methanol (2:1) to volume.
`
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`USP-NF
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`Sample solution: Transfer an aliquot of the Sample stock solution equivalent to 300 mg of Krill Oil to a suitable sealable glass vial, and add about 25 mg of the
`Internal standard accurately weighed. Evaporate to dryness. Add 2 mL of deuterated chloroform (chloroform-d) containing 0.05% tetramethylsilane (TMS), 2 mL of
`deuterated methanol (methanol-d4), and 2 mL of Solution A. Seal the vial and vortex intensively for 30–60 s, shake for an additional 30 min on a shaking device,
`and centrifuge the contents of the vial. Pass the entire amount of the lower organic phase through a glass ber lter, and collect the ltrate in the appropriate NMR
`tube.▲ (USP 1-May-2019)
`Standard solution: ▲Transfer 300 mg of USP Krill Oil RS to a suitable sealable glass vial, and add about 25 mg of the Internal standard accurately weighed. Add 2 mL
`of methanol-d4, 2 mL of chloroform-d containing 0.05% TMS, and 2 mL of Solution A. Seal the vial and vortex intensively for 30–60 s, shake for an additional 30
`min on a shaking device, and centrifuge the contents of the vial. Pass the entire amount of the lower organic phase through a glass ber lter, and collect the
`ltrate in the appropriate NMR tube.▲ (USP 1-May-2019)
`Instrumental conditions
`Magnetic eld strength: ▲NLT 7.05 Tesla (resonance frequencies of 121 MHz for P or 300 MHz for H)▲ (USP 1-May-2019)
`31
`1
`Probe: Direct observe probe capable of tuning to the resonance frequency of P (dependent on the specic magnetic eld strength used) ▲at a temperature of
`31
`35°▲ (USP 1-May-2019)
`Data collection: Use the parameters specied in Table 2. Use 90° pulses, and calibrate pulses before use according to the recommendations supplied by the
`instrument manufacturer.
`
`Table 2
`
`Parameter
`
`P NMR Quantitative Measurement
`31
`
`Pulse program
`
`Spectral width
`
`H-decoupled P (inverse gated)
`1
`31
`
`50 ppm (25 to –25 ppm)
`
`Transmitter offset
`
`Center of spectral width, 0 ppm
`
`Relaxation delay
`
`Acquisition time
`
`Size of data set
`
`5–15 s
`
`1–6 s
`
`NLT 64k (32k with zero-lling)
`
`▲ ▲ (USP 1-May-2019)
`
`▲ ▲ (USP 1-May-2019)
`
`▲ ▲ (USP 1-May-2019)
`
`▲ ▲ (USP 1-May-2019)
`
`▲ ▲ (USP 1-May-2019)
`
`▲ ▲ (USP 1-May-2019)
`
`▲ ▲ (USP 1-May-2019)
`
`[NOTE—The acquisition time is dependent upon the dwell time and the number of data points collected. The number of scans acquired using a ▲7.05 Tesla
`magnet▲ (USP 1-May-2019) must be NLT 512.]
`System suitability: ▲The Standard solution shows the P NMR signal for triphenyl phosphate at –17.80 ppm, and the signal for phosphatidylcholine at –0.89 ppm.
`31
`The signal-to-noise ratio for the phosphatidylcholine signal in the P spectrum of the Sample solution obtained in the Analysis is NLT 2000. Using the baseline as a
`31
`reference, determine the height of the phosphatidylcholine ether peak and draw a line parallel to the baseline at 30% of that total peak height (intensity). The valley
`between the peaks of phosphatidylcholine ether and phosphatidylcholine is below the line drawn.▲ (USP 1-May-2019)
`Analysis: ▲Obtain the quantitative P spectrum of the Sample solution and the Standard solution as directed in Data collection. Record the resulting spectra, and
`31
`integrate the complete set of phospholipid peaks as identied by a comparison with the reference spectrum provided with USP Krill Oil RS. The integration region
`for each signal must extend ±0.05 ppm on either side of the P signal.▲ (USP 1-May-2019)
`31
`Calculations: Use the following equations and molecular weights listed in Table 3 to ▲calculate the content of the phospholipid of interest in the Capsules taken:▲
`(USP 1-May-2019)
`
` = (W × C )/(MW × 100)
`mmol
`IS
`IS
`IS
`IS
`
`= millimoles of the Internal standard in the Sample solution (mmol)
`
`= weight of the Internal standard added to the Sample solution (mg)
`
`= purity value of the Internal standard, based on quantitative P NMR analysis (% by weight)
`31
`
`= molecular weight of the Internal standard, 326.28 g/mol (for triphenyl phosphate)
`
`= millimoles of the phospholipid of interest in the Sample solution (mmol)
`
` × A )
`)/(I
` × A × mmol
` = (I
`mmol
`PL
`PL
`IS
`IS
`IS
`PL
`
`= integrated area under the phospholipid signal of interest obtained from the spectrum of the Sample solution
`
`= number of phosphorus atoms per molecule expected from the Internal standard, 1 (for triphenyl phosphate)
`
`= millimoles of the Internal standard in the Sample solution
`
`mmol
`IS
`
`W
`IS
`
`C
`IS
`
`MW
`IS
`
`mmol
`PL
`
`I
`PL
`
`A
`IS
`
`mmol
`IS
`
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`I
`IS
`
`A
`PL
`
`C
`PL
`
`n
`
`MW
`PL
`
`mmol
`PL
`
`V
`
`a
`
`USP-NF
`= integrated area under the Internal standard obtained from the spectrum of the Sample solution
`
`= number of phosphorus atoms per molecule expected from the phospholipid of interest, 1 (for any phospholipid listed in Table 3)
`
`▲C = 1/n × MW × mmol
` × V/a
`PL
`PL
`PL
`
`= content of the phospholipid of interest in the Capsules taken (mg/Capsule)
`
`= number of Capsules used to prepare the Sample stock solution
`
`= molecular weight of the phospholipid of interest (mg/mmol, from Table 3)
`
`= millimoles of the phospholipid of interest in the Sample solution (mmol)
`
`= volume of volumetric ask used to prepare the Sample stock solution (mL)
`
`= volume of the aliquot of Sample stock solution used to prepare the Sample solution (mL)▲ (USP 1-May-2019)
`
`Table 3
`
`Component
`
`Approximate Chemical Shift (ppm) in
`Reference to Triphenyl Phosphate
`
`Molecular Weight (g/mol)
`
`Triphenyl phosphate (Internal standard)
`
`Phosphatidylcholine, including ether (PC)
`
`1-Lysophosphatidylcholine (1-LPC)
`a
`
`2-Lysophosphatidylcholine (2-LPC)
`a
`
`Phosphatidylethanolamine (PE)
`
`N-Acylphosphatidylethanolamine (NAPE)
`
`Lysophosphatidylethanolamine (LPE)
`
`Other
`
`–17.8
`
`–0.89
`
`–0.48
`
`–0.4
`
`–0.24
`
`0
`
`0.25
`
`—
`
`—
`
`791
`
`534.5
`
`534.5
`
`770
`
`1032
`
`492.5
`
`800
`
`  Ability to resolve the signals of 1-LPC and 2-LPC will depend upon the applied magnetic eld strength of the NMR spectrometer used for the test procedure.
`a
`
`▲Calculate the percentage of the labeled amount of total phospholipids per Capsule taken:
`
`Result = ΣC × 100/L
`
`PL
`
`C
`PL
`
`L
`
`= sum of the individual amounts of phospholipids of interest in the Capsules taken (mg/Capsule)
`
`= label claim of total phospholipids (mg/Capsule)▲ (USP 1-May-2019)
`
`Acceptance criteria: ▲NLT 95.0% of the labeled amount of total phospholipids▲ (USP 1-May-2019)
`Change to read:
`• CONTENT OF ASTAXANTHIN
`[NOTE—Perform this analysis in subdued light using low-actinic glassware.]
`Sample solution: 0.005 g/mL of Krill Oil in chloroform using the portion of oil from NLT 10 Capsules. [NOTE—If the solution is not clear, centrifuge it with an
`appropriate centrifuge to obtain a clear supernatant.]
`Instrumental conditions
`
`(See Ultraviolet-Visible Spectroscopy 〈857〉.)
`
`Analytical wavelength: 486 nm
`Cell: 1 cm
`Blank: Chloroform
`Analysis
`Sample: Sample solution
`Calculate the percentage of astaxanthin in the portion of Krill Oil taken from the Capsules:
`
`Result = A/(F × C)
`
`A
`
`F
`
`C
`
`= absorbance of the Sample solution
`
`= coecient of extinction (E ) of pure astaxanthin in chloroform (100 mL · g  · cm ), 1692
`1%
`– 1
`– 1
`
`= concentration of the Sample solution (g/mL)
`
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`Acceptance criteria: NLT ▲0.005%▲ (USP 1-May-2019)
`
`PERFORMANCE TESTS
`
`• DISINTEGRATION AND DISSOLUTION 〈2040〉, Rupture Test for Soft Shell Capsules: Meet the requirements
`• WEIGHT VARIATION 〈2091〉: Meet the requirements
`
`USP-NF
`
`CONTAMINANTS
`• LIMIT OF DIOXINS, FURANS, AND POLYCHLORINATED BIPHENYLS
`Analysis: Determine the content of polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) by Method 1613, Revision B of the
`Environmental Protection Agency (EPA). Determine the content of polychlorinated biphenyls (PCBs) by Method 1668, Revision A of the EPA.
`Acceptance criteria: The sum of PCDDs and PCDFs is NMT 2.0 pg/g of World Health Organization (WHO) toxic equivalents. The sum of PCDDs, PCDFs, and dioxin-
`like PCBs [non-ortho International Union of Pure and Applied Chemistry (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.
`3
`
`2
`
`• MICROBIAL ENUMERATION TESTS 〈2021〉: The total aerobic microbial count does not exceed 10 cfu/g, and the combined molds and yeasts count does not exceed 10
`• ABSENCE OF SPECIFIED MICROORGANISMS 〈2022〉, Test Procedures, Test for Absence of Salmonella Species and Test for Absence of Escherichia coli: Meet the requirements
`
`cfu/g.
`
`SPECIFIC TESTS
`• ASTAXANTHIN ESTERIFICATION
`Standard solution A: 10 mg/mL of USP Astaxanthin Esters from Haematococcus pluvialis RS in acetone
`Standard solution B: 10 mg/mL of USP Astaxanthin (Synthetic) RS in acetone
`Sample solution: Using the portion of oil from NLT 10 Capsules, prepare a solution of 250 mg/mL in acetone.
`Chromatographic system
`
`(See Chromatography 〈621〉, General Procedures, Thin-Layer Chromatography.)
`
`Mode: TLC
`Adsorbent: 0.25-mm layer of chromatographic silica gel. [NOTE—Dry silica gel at 110° for 1 h before use.]
`Application volume: 5 µL
`Developing solvent system: Hexane and acetone (70:30)
`Analysis
`Samples: Standard solution A, Standard solution B, and Sample solution
`Develop the chromatogram in the Developing solvent system until the solvent front has moved about 15 cm of the length of the plate. Remove the plate from the
`chamber, and allow to dry.
`Acceptance criteria: The principal spot from Standard solution B, located in the bottom half of the plate, is free astaxanthin. The Sample solution may exhibit a light,
`minor spot in the same location. The principal spots from Standard solution A are from monoesters (primary spot, located slightly above the middle of the plate)
`and diesters (secondary spot, located in the top third of the plate). The principal spot from the Sample solution should correspond in color and R value to the
`F
`diester spot from Standard solution A. The secondary spot from the Sample solution should correspond in color and approximately the same R value to the
`F
`monoester spot from Standard solution A. [NOTE—Slight differences in R values within monoester spots and within diester spots may exist because of different
`F
`intensities.]
`
`• FATS AND FIXED OILS 〈401〉, Procedures, Peroxide Value: NMT 5.0 mEq peroxide/kg
`
`ADDITIONAL REQUIREMENTS
`• PACKAGING AND STORAGE: Preserve in tight containers, and store at room temperature. Protect from light.
`Change to read:
`• LABELING: The label states the amount of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and total phospholipids ▲in mg/Capsule.▲ (USP 1-May-2019)
`Change to read:
`
`• USP REFERENCE STANDARDS 〈11〉
`
`USP Astaxanthin (Synthetic) RS
`USP Astaxanthin Esters from Haematococcus pluvialis RS
`▲
`▲ (USP 1-May-2019)
`USP Krill Oil RS
`▲
`▲ (USP 1-May-2019)
`
`Auxiliary Information- Please check for your question in the FAQs before contacting USP.
`
`Topic/Question
`
`KRILL OIL CAPSULES
`
`Contact
`
`Natalia Davydova
`Scientic Liaison
`+1 (301) 816-8328
`
`Expert Committee
`
`NBDS2015 Non-botanical Dietary Supplements
`2015
`
`Chromatographic Columns Information: Chromatographic Columns
`
`Most Recently Appeared In:
`Pharmacopeial Forum: Volume No. 44(1)
`
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`Page Information:
`
`USP42-NF37 - 5046
`USP41-NF36 - 4721
`USP40-NF35 1S - 8452
`
`Current DocID: GUID-B2C1B38E-606D-4103-857F-4DBF2B9F18EC_2_en-US
`
`USP-NF
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