8/12/2019
`Printed on: Mon Aug 12 2019, 11:33:39 am
`Printed by: Nicole Chang
`Currently Ocial as of: 12-Aug-2019
`Ocial as of 1-Mar-2019
`DocId: GUID-6F51ACE9-9EB4-4779-A35A-E3E084C99C73_5_en-US
`Printed from: https://online.uspnf.com/uspnf/document/GUID-6F51ACE9-9EB4-4779-A35A-E3E084C99C73_5_en-US?highlight=Krill%20Oil
`© 2019 USPC
`
`USP-NF
`
`Krill Oil
`
`Change to read:
`
`DEFINITION
`Krill Oil is the xed oil extracted from Antarctic krill (Euphausia superba Dana) biomass using appropriate food-grade organic solvents. Krill Oil contains NLT 30% (w/w)
`and NMT 59% (w/w) of total phospholipids, of which 60%–96% are phosphatidylcholine, ▲1-lysophosphatidylcholine, and 2-lysophosphatidylcholine.▲2S (USP41) It
`contains NLT 10% (w/w) of eicosapentaenoic acid (EPA) and NLT 5.0% (w/w) of docosahexaenoic acid (DHA) mostly in the form of phospholipids. It also contains
`NLT ▲0.005%▲2S (USP41) of astaxanthin.
`
`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 Acids
`Standard solution: Prepare as directed in Fats and Fixed Oils〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole, Content of EPA and DHA, Test Solution
`
`Determination and Prole.
`
`1, except use 250 mg of USP Krill Oil RS.
`Sample solution: Prepare as directed in the Standard solution, except replace USP Krill Oil RS with Krill Oil.
`System suitability
`Samples: System suitability solution 1 and Standard solution
`Suitability requirements
`Resolution: NLT 1.0 between the methyl oleate and methyl cis-vaccinate peaks, Standard solution
`Theoretical area percentages: Meets the requirements for System suitability solution 1
`Chromatogram similarity: The chromatogram obtained from the Standard solution is similar to the reference chromatogram provided with the lot of USP Krill Oil
`RS being used.
`Analysis
`Sample: Sample solution
`Identify the retention times of the relevant fatty acid methyl esters in the Sample solution by comparing the chromatogram of the Sample solution with that of
`the Standard solution and the USP reference chromatogram.
`Calculate the area percentage for each fatty acid as methyl esters in the portion of Krill Oil taken:
`
`= 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: The fatty acids obtained from the Sample solution meet the limit requirements in Table 1.
`
`Table 1
`
`Fatty Acid
`
`Shorthand 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
`
`Eicosenoic acid
`
`16:1 n−7
`
`18:1 n−7
`
`18:1 n−9
`
`20:1 n−9
`
`▲5.0▲2S (USP41)
`
`17.0
`
`1.6
`
`2.5
`
`4.7
`
`▲6.0▲2S (USP41)
`
`0.0
`
`13.0
`
`24.6
`
`▲3.6▲2S (USP41)
`
`▲12.0▲2S (USP41)
`
`8.0
`
`14.5
`
`2.0
`
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`Fatty Acid
`
`Shorthand Notation
`
`Lower Limit (Area %)
`
`Upper Limit (Area %)
`
`Erucic acid
`
`Polyunsaturated fatty acids
`
`Linoleic acid
`
`Eicosapentaenoic acid
`
`Docosapentaenoic acid
`
`Docosahexaenoic acid
`
`22:1 n−9
`
`18:2 n−6
`
`20:5 n−3
`
`22:5 n−3
`
`22:6 n−3
`
`0.0
`
`0.0
`
`14.0
`
`0.0
`
`7.1
`
`1.5
`
`3.0
`
`▲28.0▲2S (USP41)
`
`0.7
`
`15.7
`
`Change to read:
`• B. PHOSPHOLIPID PROFILE
`Solution A, Line shape standard ( H), Sensitivity standard ( H), Sensitivity standard ( P), Internal standard, Sample solution, Standard solution, Instrumental
`1
`1
`31
`conditions, System suitability, and Analysis: Proceed as directed in the test for Content of Total Phospholipids.
`Acceptance criteria: The Sample solution contains all of the following phospholipids: phosphatidylcholine, ▲lysophosphatidylcholine, and
`phosphatidylethanolamine. The sum of phosphatidylcholine, 1-lysophosphatidylcholine, and 2-lysophosphatidylcholine is 60%–96%.▲2S (USP41)
`
`COMPOSITION
`• CONTENT OF EPA AND DHA
`Standard solution 1a, Standard solution 1b, Standard solution 2a, Standard solution 2b, System suitability solution 1, and Chromatographic system: Proceed as
`
`directed in Fats and Fixed Oils〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole.
`Test solution 1: Prepare as directed in Fats and Fixed Oils〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole, Content of EPA and DHA, Test Solution 1,
`Test solution 2: Prepare as directed in Fats and Fixed Oils〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole, Content of EPA and DHA, Test Solution 2,
`Analysis: Proceed as directed in Fats and Fixed Oils〈401〉, Procedures, Omega-3 Fatty Acids Determination and Prole, Content of EPA and DHA, Analysis (for
`(See Nuclear Magnetic Resonance Spectroscopy 〈761〉, Qualitative and Quantitative NMR Analysis.)
`
`except use 250 mg of Krill Oil.
`
`except use 250 mg of Krill Oil.
`
`triglycerides).
`Acceptance criteria: NLT 10.0% (w/w) of EPA and NLT 5.0% (w/w) of DHA
`Change to read:
`• CONTENT OF TOTAL PHOSPHOLIPIDS
`
`[NOTE—All deuterated solvents used in this method should be NLT 99.8 atom % D. Whenever water is used in this method, it should be of sucient quality to ensure that
`no trace metals or other contaminants that may affect the analysis are present.]
`Solution A: 0.2 M EDTA, adjusted with a 1 M cesium carbonate solution to a pH of 7.2–7.5. Document the nal pH and the amount of 1 M cesium carbonate solution
`necessary to attain the desired pH. [NOTE—Use cesium carbonate of a sucient grade for trace metals analysis.]
`Line shape standard ( H): 1% chloroform in acetone-d
`1
`6
`Sensitivity standard ( H): 0.1% ethylbenzene in chloroform-d
`Sensitivity standard ( P): 0.0485 M triphenyl phosphate in acetone-d
`1
`6
`Internal standard: Use a suitable triphenyl phosphate NMR analytical standard with purity of NLT 99.0%.
`Sample solution: [NOTE—NMR solvents containing tetramethylsilane (TMS) are readily available. If the solvents used do not contain TMS, it must be added to the
`Sample solution at an approximate concentration of 0.05% (v/v) for use as a chemical shift scale reference.] Transfer 300–350 mg of Krill Oil to a 5-mL sealable
`glass vial. Add 25.0 mg of the Internal standard to the vial. Add 1 mL each of deuterated chloroform (chloroform-d) and deuterated methanol (methanol-d ) of a
`4
`grade suitable for NMR analysis to the vial to dissolve the sample. Once dissolution is complete, add 1 mL of Solution A, seal the vial, and shake the solution for
`10–20 min, then centrifuge the contents of the vial. Transfer the lower organic phase to an appropriate NMR tube. It is critical to collect the entire organic phase
`and transfer it to the NMR tube. It may be unavoidable to also transfer small amounts of the aqueous phase when collecting the organic phase in the NMR tube.
`This is acceptable practice, so long as the aqueous phase remains completely separated and atop the organic phase in the NMR tube. The entire amount of
`aqueous phase must be above the probe's radio frequency (RF) coil (outside the analysis area of the tube). Should the organic phase contain undissolved
`materials, they must remain suspended at the aqueous-organic interface and be outside the analysis area of the tube as well. The organic phase must be free of
`bubbles and suspended materials that may interfere with NMR data acquisition.
`Standard solution: Prepare as directed in the Sample solution, using 300–350 mg of USP Krill Oil RS in place of the sample.
`Instrumental conditions
`
`(See Nuclear Magnetic Resonance Spectroscopy 〈761〉.)
`
`1 3
`
`Magnetic eld strength: NLT 300 MHz for H frequency
`1
`Probe: Direct observe probe capable of tuning to the resonance frequency of P (dependent on the specic magnetic eld strength used)
`31
`Instrument performance qualication
`[NOTE—Testing for sensitivity and line shape should be performed on the interval specied by the manufacturer of the instrument used. Performing these tests on a
`minimum of a monthly basis is required for this method, but may be done more often, as required. Resolution testing is to be performed during each analysis and
`documented as a part of the analytical results.]
`H Line shape test: Using the Line shape standard ( H) and the protocol recommended by the instrument manufacturer, the instrument must achieve the line
`1
`1
`shape specications for the probe in use, as required by the instrument manufacturer. [NOTE—A different standard solution may be required or recommended
`by the manufacturer of the instrument; 1% chloroform in acetone-d is most commonly used.]
`6
`H Sensitivity test: Using the Sensitivity standard ( H) and the protocol recommended by the instrument manufacturer, the instrument must achieve the
`1
`1
`sensitivity specications required by the instrument manufacturer. [NOTE—A different standard solution may be required or recommended by the manufacturer
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`of the instrument; 0.1% ethylbenzene in chloroform-d is most commonly used.]
`P Sensitivity test: Using the Sensitivity standard ( P) and the protocol recommended by the instrument manufacturer, the instrument must achieve the
`31
`31
`sensitivity specications required by the instrument manufacturer. [NOTE—A different standard solution may be required or recommended by the manufacturer
`of the instrument; 0.0485 M triphenyl phosphate in acetone-d is most commonly used.]
`H Resolution test: The resolution is demonstrated by the ability to detect both of the Si satellite signals of TMS. The satellites must be resolved from the TMS
`29
`1
`signal in the spectrum with a line broadening factor of NMT 0.5 ppm.
`P Resolution test: The resolution is demonstrated using the phosphatidylcholine ether peak and the phosphatidylcholine peak. The separation of these peaks
`31
`(with an applied line broadening factor of 1.0) must be demonstrated, as follows. Using the baseline as a reference, determine the total peak height of the
`phosphatidylcholine ether peak , and draw a line at 30% of that total peak height (intensity). [NOTE—The phosphatidylcholine ether signal appears just
`downeld from the phosphatidylcholine signal.] The phosphatidylcholine ether peak and the neighboring phosphatidylcholine peak must be fully resolved at a
`point that is NMT 30% of the peak height of the phosphatidylcholine ether peak.
`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
`
`H NMR Qualitative Measurement
`1
`
`Pulse program
`
`Spectral width
`
`Transmitter offset
`
`Relaxation delay
`
`Acquisition time
`
`Size of data set
`
`H-decoupled P (inverse gated)
`1
`31
`
`Single pulse H
`1
`
`50 ppm (25 ppm to −25 ppm)
`
`20 ppm (−3 ppm to 17 ppm)
`
`Center of spectral width, 0 ppm
`
`Center of spectral width, 7 ppm
`
`5–15 s
`
`1–6 s
`
`2–5 s
`
`1–6 s
`
`NLT 64k (32k with zero-lling)
`
`NLT 64k (32k with zero-lling)
`
`[NOTE—The acquisition time is dependent upon the dwell time and the number of data points collected. The number of scans acquired using a 300 MHz instrument
`must be NLT 512.]
`System suitability: Under the conditions outlined in Data collection, the P NMR signal of triphenyl phosphate should be observed at −17.80 ppm, and the H NMR
`31
`1
`spectrum should be referenced to the H signal of TMS (0 ppm) for all spectra acquired in the Analysis. For quantitative analysis, a sucient number of scans
`1
`should be acquired such that the signal-to-noise ratio for the phosphatidylcholine signal in the P spectrum of the Sample solution acquired in the Analysis is NLT
`31
`2000.
`Analysis: Acquire the data outlined in Data collection. Minimally acquire the H spectrum (ngerprint) of the Sample solution and the Standard solution as well as the
`1
`quantitative P spectrum of the Sample solution and the Standard solution. Record the resulting spectra, and perform integration by hand or automated means on
`31
`the quantitative P NMR spectrum of the Sample solution. Integration of the peaks contained in the spectrum of the Sample solution must be performed such that
`31
`the complete set of phospholipid peaks (as identied by comparison to the spectrum of the Standard solution and its reference spectrum) is included in the
`integration. The integration region for each signal must extend ±0.05 ppm on either side of the P signal. Quantify the total phospholipids present, the
`31
`phosphatidylcholine ether content, and the phosphatidylcholine content in the Sample solution using comparison to the concentration of the Internal standard.
`Compare the H spectrum of the Sample solution to that of the Standard solution to determine the similarity of ngerprints according to which phospholipids
`1
`identied in the reference spectrum of the Standard solution are present in the spectrum of the Sample solution.
`Calculations: Use the following equations and molecular weights listed in Table 3 to determine the phospholipids content in the sample taken:
`
` = (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)
`
`= 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)
`
`mmol
`IS
`
`W
`IS
`
`C
`IS
`
`MW
`IS
`
`mmol
`PL
`
`I
`PL
`
`A
`IS
`
`mmol
`IS
`
`I
`IS
`
`A
`PL
`
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` × 100)/W
`C = (MW × mmol
`PL
`PL
`PL
`S
`
`C
`PL
`
`MW
`PL
`
`mmol
`PL
`
`W
`S
`
`= concentration of the phospholipid of interest in the Sample solution (%, w/w)
`
`= molecular weight of the phospholipid of interest (g/mol, from Table 3) in the Sample solution (mg)
`
`= millimoles of the phospholipid of interest in the Sample solution (mmol)
`
`= weight of the sample present in the Sample solution (mg)
`
`[NOTE—Use the molecular weight specied in Table 3 for the calculations.]
`
`Table 3
`
`Component
`
`Approximate Chemical Shift (ppm) in Reference
`to Triphenyl Phosphate
`
`Molecular Weight (g/mol)
`
`Triphenyl phosphate (Internal standard)
`
`Phosphatidylcholine (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-lysophosphatidylcholine and 2-lysophosphatidylcholine will depend upon the applied magnetic eld strength of the NMR spectrometer used for the test procedure.
`a
`
`Acceptance criteria
`Total phospholipids: 30%–59% (w/w)
`▲Sum of phosphatidylcholine, 1-lysophosphatidylcholine, and 2-lysophosphatidylcholine:▲2S (USP41) 60%–96% (w/w) of the Total phospholipids content
`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. [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:
`
`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)
`
`Acceptance criteria: NLT ▲0.005%▲2S (USP41)
`
`CONTAMINANTS
`• 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 World Health Organization (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 3.0 pg/g of WHO toxic equivalents.
`
`SPECIFIC TESTS
`
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`• 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: 250 mg/mL of Krill Oil in acetone
`Chromatographic system
`
`(See Chromatography 〈621〉, General Procedures, Thin-Layer Chromatography.)
`
`USP-NF
`
`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, 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.
`Change to read:
`
`• USP REFERENCE STANDARDS 〈11〉
`
`USP Astaxanthin Esters from Haematococcus pluvialis RS
`USP Astaxanthin (Synthetic) RS
`▲▲ (CN 1-May-2018)
`USP Krill Oil RS
`▲▲ (CN 1-May-2018)
`
`Auxiliary Information- Please check for your question in the FAQs before contacting USP.
`
`Topic/Question
`
`KRILL OIL
`
`Contact
`
`Expert Committee
`
`Fatkhulla K Tadjimukhamedov
`Associate Scientic Liaison
`+1 (301) 230-3216
`
`NBDS2015 Non-botanical Dietary Supplements
`2015
`
`Chromatographic Columns Information: Chromatographic Columns
`
`Most Recently Appeared In:
`Pharmacopeial Forum: Volume No. 43(4)
`
`Page Information:
`
`USP42-NF37 - 5042
`USP41-NF36 2S - 9015
`USP41-NF36 - 4718
`
`Current DocID: GUID-6F51ACE9-9EB4-4779-A35A-E3E084C99C73_5_en-US
`Previous DocID: GUID-6F51ACE9-9EB4-4779-A35A-E3E084C99C73_2_en-US
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