IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`
`
`AKER BIOMARINE AS
`Petitioner
`
`v.
`
`NEPTUNE TECHNOLOGIES AND BIORESOURCES INC.
`Patent Owner
`
`
`CASE IPR: Unassigned
`
`Declaration of Dr. Albert C. Lee
`
`
`
`
`
`\\
`
`000001
`
`

`

`I.
`
`INTRODUCTION
`
`1. My name is Dr. Albert C. Lee. I have been retained by counsel for
`
`Petitioners Aker BioMarine AS to testify as an expert in this inter partes review.
`
`2.
`
`I am currently a Senior Scientist at Chemir Analytical Services, a
`
`division of Evans Analytical Group. Chemir is a commercial laboratory,
`
`specializing in, amongst other things, analytical chemistry. Among other services,
`
`Chemir designs and conducts analytical testing of investigational and commercial
`
`products and samples. Chemir has particular expertise in gas chromatography
`
`(GC), high performance liquid chromatography (HPLC), and inductively coupled
`
`plasma mass spectrometry (ICP-MS). These techniques are described in Exhibit
`
`A.
`
`3.
`
`In my capacity as a Senior Scientist, I regularly supervise the
`
`development and performance of known and novel analytical techniques for the
`
`identification and quantification of components in various substances. My duties
`
`include developing and reviewing analytical methods, reviewing and interpreting
`
`data, and preparing reports describing analytical testing and results. Since
`
`obtaining my Ph.D. in Organic Chemistry from Northwestern University, I have
`
`obtained more than 20 years of experience in synthetic and analytical chemistry,
`
`including designing, conducting and interpreting results derived from chemical
`
`analyses, and supervising the design and performance of analytical methods. I am
`
`000002
`
`

`

`an expert in the fields of gas chromatography (GC) high performance liquid
`
`chromatography (HPLC), and inductively coupled plasma mass spectrometry (ICP-
`
`MS). My curriculum vitae is attached as Exhibit B.
`
`4.
`
`In forming my opinions, I have considered and relied upon my
`
`education, background, and experience in analytical chemistry, as well as the
`
`methods and data generated in carrying out the analyses described herein.
`
`II.
`
`SAMPLE ANALYSIS
`
`5.
`
`Chemir received eleven samples for analysis, identified as follows:
`
`Sample Name
`
`Chemir Sample Identifier
`
`VB2 8/8/13 FH
`
`VB8 8/9/13 FHE
`
`SB2 8/19/2013 BEA-P0
`
`SB6 8/19/2013 BEA-P1
`
`SB10 8/19/2013 BEA-P2
`
`SB14 8/19/2013 BEA-S0
`
`SB18 8/19/2013 BEA-S1
`
`SB22 8/19/2013 BEA-S2
`
`SB26 09/082013 BEA-SU0
`
`SB30 09/08/2013 BEA-SU1
`
`S1
`
`S2
`
`S6
`
`S7
`
`S8
`
`S9
`
`S10
`
`S11
`
`S18
`
`S19
`
`000003
`
`

`

`SB34 09/08/2013 BEA-SU2
`
`S20
`
`
`
`6.
`
`Each of these samples was prepared and analyzed for fatty acid
`
`concentration, astaxanthin concentration, and metal concentration, in accordance
`
`with the methods described in Exhibit A. The results of these analyses are also
`
`provided in Exhibit A.
`
`7.
`
`I supervised these analyses, including the method development,
`
`sample preparation, sample analysis and data analysis, and confirm that Chemir
`
`properly implemented and adhered to these methods, and obtained the reported
`
`results. It is my opinion that the results reported herein reliably reflect the
`
`compositions of the above-referenced samples.
`
`III. ADDITIONAL INFORMATION
`
`8.
`
`During the previous four years, I have testified as an expert, either at
`
`trial or by deposition, in the following cases: Albany Molecular Research, Inc. and
`
`Sanofi-Aventis vs. DRL, Ltd. and DRL, Inc. Civil Action No. 09-4638 Deposition
`
`December 2010, US District Court District of New Jersey; Solutec Corp., Inc. v.
`
`Steve Agnew, Terry Ingle and Suzette Ingle., Civil Action No. 95-2-00234-9
`
`Testimony December 2011, Superior Court of Washington for Yakima County;
`
`Cephalon, Inc. and Cephalon France vs. Watson Laboratories, Inc., Sandoz Inc.,
`
`Lupin Ltd., Apotex Corp. and Apotex Inc., MDL Docket No. 1:10-md-2200-GMS
`
`000004
`
`

`

`Deposition January 2012, Testimony July 2012 US District Court District of
`
`Delaware; Kaneka Corporation vs. Zhejiang Medicine Co., Ltd., ZMC-USA, LLC,
`
`Kiamen Kingdomway Group Company, Pacific Rainbow International, Inc.,
`
`Mitsubishi Gas Company, Inc., Maypro Industries, Inc. and Shenzhou Biology &
`
`Technology Co., Ltd., Deposition May 2012, Testimony July 2012; Endo
`
`Pharmaceuticals, Inc. vs. Mylan Pharmaceuticals, Inc. and Mylan, Inc., Civil
`
`Action No. 11-cv-00717-JEI-KW Deposition March 2013, US District Court of
`
`Delaware.
`
`9.
`
`For completing the analyses described in this report and preparing this
`
`report, total fees paid to Chemir has been ca. $215,220based on a standard rate of
`
`$550/h plus a 70% surcharge for expedited work. Chemir will be compensated at
`
`its standard rate of $550/h for consulting and/or testimony that I provide in this
`
`case. I am a salaried employee of Chemir and receive no additional compensation
`
`for working on this case.
`
`10.
`
`I further declare that all statement made herein of my own knowledge
`
`are true and that all statements made on information and belief are believed to be
`
`true; and further that these statements were made with the knowledge that willful
`
`false statements and the like so made are punishable by fine or imprisonment, or
`
`both, under section 1001 of title 18 of the United States Code, and that such willful
`
`000005
`
`

`

`false statements may jeopardize the validity of the application or any patent issued
`
`thereon.
`
`
`
`000006
`
`

`

`
`
`
`
`
`
`
`
`
`EXHIBIT A
`
`EXHIBIT A
`
`000007
`
`000007
`
`

`

`
`
`
`
`Quantitation of Krill Oil Components
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`September 16, 2013
`
`
`
`Chemir Job #V1DJU114
`
`
`
`
`
`
`
`
`
`
`SCIENTIFIC SOLUTIONS
`
`
`RELIABLE RESULTS
`
`
`IDEAS TO INNOVATION
`
`
`EXTEND YOUR R&D
`
`
`WWW.CHEMIR.COM
`
`
`
`000008
`
`

`

`
`
`Executive Summary
`
`September 16, 2013
`
`Study Objective
`V1DJU114
`
`
`
`The objective of this study was to quantitate the amount of fatty acids, omega-3 fatty acids,
`metals and astaxanthin in the submitted krill oil samples.
`
`Analysis Conclusions
`
`
`Quantitation of fatty acids, omega-3 fatty acids, metals and astaxanthin in Krill Oil Samples
`1
`AVERAGE CONCENTRATION
`S20
`S19
`S18
`S11
`S8
`S9
`S10
`S7
`S6
`S2
`S1
`24.56 24.35 13.12 11.73 21.91 29.51 18.05 16.00 18.17 20.51 29.01
`
`ANALYTES
`
`Saturated fatty acids2
`Monounsaturated fatty
`acids2
`Polyunsaturated fatty
`acids2,3
`Total fatty acids2,4
`Total Omega-3 fatty
`acids2
` C20:5n3 (EPA)
` C22:6n3 (DHA)
`Zinc5
`Selenium
`Astaxanthin6
`
`10.85 8.68
`
`8.16
`
`6.56 10.82 12.92 12.34 12.88
`
`8.35
`
`9.06 12.68
`
`22.56 20.97 17.59 15.75 24.77 31.05 34.01 33.37 27.11 28.28 38.91
`
`59.14 55.43 39.49 34.74 58.59 77.62 66.85 64.10 53.64 57.85 80.60
`
`18.62 17.91 15.42 14.32 22.25 24.06 25.43 23.51 22.09 22.89 31.53
`
`8.55
`5.71
`5
`< 1
`26.9
`
`6.87 10.67 7.14
`7.42
`8.43
`5.07
`7.84
`3.88
`5.23
`5.57
`< 1
`3
`< 1
`3
`2
`< 1
`< 1
`< 1
`< 1
`< 1
`23.7 154.5 161.6 96.6
`80.0
`
`6.96
`3.78
`< 1
`< 1
`79.3
`
`7.48 10.97 11.44 15.59
`4.10
`6.37
`6.62
`9.05
`< 1
`2
`1
`4
`< 1
`< 1
`< 1
`< 1
`67.2 33.4
`38.4
`12.3
`
`
`
`Sample Log-In7
`SAMPLE NUMBER
`S1
`S2
`S4
`S5
`S6
`S7
`S8
`
`VB2 8/8/13 FH
`VB8 8/9/13 FHE
`SPLIT OF S1 for ICP-MS
`SPLIT OF S2 for ICP-MS
`SB2 8/19/2013 BEA-P0
`SB6 8/19/2013 BEA-P1
`SB10 8/19/2013 BEA-P2
`
`DESCRIPTION
`
`
`1 Fatty acids and astaxanthin results are an average of duplicate preparations and duplicate analysis of each preparation,
`zinc and selenium at an average of triplicate preparations and a single analysis of each preparation.
`2 Fatty acid concentrations are expressed as gram of analyte per 100 gram of krill oil.
`3 Polyunsaturated fatty acids includes omega-3 fatty acids.
`4 Total fatty acids value includes saturated, monounsaturated, polyunsaturated fatty acids.
`5 Metals are reported as ppm which is equivalent to milligrams of analyte per kilogram of krill oil.
`6 Astaxanthin is reported as milligram of astaxanthin per 100 gram of krill oil and accounting for three forms of astaxanthin.
`7 Samples S1, S2 and S6-S11 were received packaged with dry ice. Samples S18-S20 were received packaged with cold
`packs. Samples were either used immediately upon receipt or stored at -20°C prior to analysis. Subsamples for ICP analysis
`were shipped with cold packs.
`
`V1DJU114
`
`000009
`
`

`

`3 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`SB14 8/19/2013 BEA-S0
`SB18 8/19/2013 BEA-S1
`SB22 8/19/2013 BEA-S2
`SPLIT OF S6 for ICP-MS
`SPLIT OF S7 for ICP-MS
`SPLIT OF S8 for ICP-MS
`SPLIT OF S9 for ICP-MS
`SPLIT OF S10 for ICP-MS
`SPLIT OF S11 for ICP-MS
`SB26 09/08/2013 BEA-SU0
`SB30 09/08/2013 BEA-SU1
`SB34 09/08/2013 BEA-SU2
`
`S9
`S10
`S11
`S12
`S13
`S14
`S15
`S16
`S17
`S18
`S19
`S20
`
`
`
`
`
`
`Prepared by
`
` Reviewed by
`
`
`
`
`Albert C. Lee, Ph.D.
`Senior Manager
`
`
`
`
`
`
`
`
`
`
`
`
`Ila Sharm, M.S., M.B.A.
`Technical Specialist
`
`
`
`
`
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000010
`
`

`

`4 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`
`
`Table of Contents
`Executive Summary .................................................................................................................................................... 2
`Study Objective ...................................................................................................................................................... 2
`Analysis Conclusions .............................................................................................................................................. 2
`Sample Log-In ......................................................................................................................................................... 2
`Analysis of Krill Oil Samples ........................................................................................................................................ 5
`Sample Preparation .................................................................................................................................................... 5
`Results and Technique Descriptions .......................................................................................................................... 5
`FATTY ACID ANALYSIS ................................................................................................................................................. 5
`OMEGA-3 FATTY ACID ANALYSIS................................................................................................................................... 7
`ASTAXANTHIN ANALYSIS .............................................................................................................................................. 8
`Instrument and Chromatographic Conditions ................................................................................................... 9
`Representative Chromatographs: ................................................................................................................... 10
`Qualifications results ....................................................................................................................................... 13
`Analysis results ................................................................................................................................................ 14
`METALS ANALYSIS .................................................................................................................................................... 15
`
`
`Table 1 Fatty acid analysis and Omega-3 fatty acid results ....................................................................................... 8
`Table 2 HPLC Gradient ................................................................................................................................................ 9
`Table 3 Accuracy test of 0.85 ppm astaxanthin solution. ........................................................................................ 10
`Table 4 Spike Recovery Results ................................................................................................................................ 10
`Table 5 Astaxanthin content expressed as mg of astaxanthin per 100 g of sample ................................................ 14
`Table 6 Metal analysis results (results are in ppm which is equivalent to mg/kg) .................................................. 16
`Table 7 Quantitation of fatty acids, omega-3 fatty acids, metals and astaxanthin in Krill Oil Samples .................. 17
`
`Figure 1 Astaxanthin ester “as received” from USP. ................................................................................................ 11
`Figure 2 Astaxanthin ester USP digested with cholesterol esterase. ....................................................................... 11
`Figure 3 Astaxanthin USP, 1 ppm ............................................................................................................................. 11
`Figure 4 Chromatograph of digested astaxanthin ester .......................................................................................... 11
`Figure 5 MS of 5.36 min peak ................................................................................................................................... 12
`Figure 6 MS of 5.77 min peak ................................................................................................................................... 12
`Figure 7 MS of 7.14 min ........................................................................................................................................... 12
`Figure 8 Astaxanthin USP calibration curve prepared from 347 ppm stock solution .............................................. 13
`Figure 9 Astaxanthin USP calibration curve prepared from 352 ppm stock solution .............................................. 13
`
`
`
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000011
`
`

`

`5 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`
`
`Analysis of Krill Oil Samples
`Analytical Results and Discussion
`
`
`
`Sample Preparation
`
`The samples were prepared for quantitation using the following approaches:
` Fatty acid analysis – hydrolysis followed by methylation;
` Omega-3 fatty acid analysis – hydrolysis followed by methylation;
` Metals analysis – acid/microwave digestion;
` Astaxanthin analysis – digestion with cholesterol esterase to cleave astaxanthin esters to give free
`astaxanthin.
`
`Results and Technique Descriptions
`
`FATTY ACID ANALYSIS
`Analysis for fatty acids was performed following procedures described in AOAC 996.06 “Fat (Total,
`Saturated and Unsaturated) in Foods.” The sample was subjected to saponification and the resulting carboxylic
`acids were converted to the corresponding methyl esters. The composition of the methyl ester mixture was
`compared to a commercial standard of methyl esters purchased from Restek called “Food Industry FAME Mix”
`Cat. No. 35077 using gas chromatography.
`Gas Chromatography (GC) is a separation technique used for the analysis of volatile components (i.e.,
`exhibit a vapor pressure of at least 60 torr). The sample can be introduced into the Gas Chromatograph through
`direct liquid injection, static headspace, or dynamic headspace. Upon injection, the components enter the GC
`column that is often an open tubular capillary column with an inner coating of a stationary phase. The column
`technology is precisely designed to control the chemical properties of the stationary phase and minimize active
`sites from the glass. Components within the column migrate by use of a carrier gas (e.g., helium) and
`appropriate thermal conditions while separation occurs by exploiting a difference in the volatility and polarity of
`the components. The resulting chromatograms can be used to confirm identity of a component through
`retention time and the intensity is indicative of the concentration.
`
`Sample Preparation:
`1. 100-200 mg of sample was placed in a vial; the following was added:
`a. ~100 mg pyrogallic acid
`b. 2 mL C11 triglyceride internal standard solution
`c. 2 mL ethanol
`d. 10 mL 8.3M hydrochloric acid
`2. The preparation was heated at 70-80°C for 40 minutes, with magnetic stir bar. The vials were vortexed
`every 10 minutes. The vials were then cooled to room temperature.
`3. ~12.5 mL diethyl ether was added, and the preparations put on the wrist shaker for 5 minutes.
`4. ~12.5 mL petroleum ether was added, and the preparations put on the wrist shaker for an additional 5
`minutes.
`5. The vials were centrifuged for 5 minutes at 600 rpm.
`6. The top ether layer was removed into new vial, and dried under nitrogen.
`7. The extraction was repeated with another ~12.5 mL diethyl ether, and the preparations put on the wrist
`shaker for 5 minutes.
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000012
`
`

`

`6 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`8. ~12.5 mL petroleum ether was added, and the preparations put on the wrist shaker for an additional 5
`minutes.
`9. The vials were centrifuged for 5 minutes at 600 rpm.
`10. The top ether layer was again removed into the new vial, and the pooled ether was dried to residue
`under nitrogen.
`11. The residue was redissolved in 2 mL chloroform and 2 mL diethyl ether, and transferred to a new vial,
`the residue vial was rinsed with an additional 1 mL each chloroform and diethyl ether and the rinse
`pooled in the new vial as well.
`12. To the solvent was evaporated under nitrogen at 40°C, and to the residue the following was added:
`a. 2 mL ampule of BF3 methanol solution.
`b. 1 mL toluene
`13. The vials were heated in an oven at 100ºC for 45 minutes, shaking every 10 minutes. The vials were
`cooled to room temperature.
`14. The following was added:
`a. 5 mL MilliQ H2O
`b. 1 mL hexane,
`c. ~1 gram sodium sulfate
`15. The vials were put on the wrist shaker for 1 minute.
`16. The top layer was transferred to another vial containing 1 gram sodium sulfate.
`17. A portion of step 16 was transferred to an autosampler vial for analysis by gas chromatography.
`
`Standard:
`The standard used was Food Industry Fame Mix, Restek Cat# 35077.
`
`Instrument and Analysis Conditions:
`The analysis was carried out on an Agilent 6890 gas chromatograph with flame-ionization detector, and an
`Agilent 6890 autosampler, under the following conditions:
`Column: Supelco SP-2560, 100 m x 0.25 mm inner diameter, 0.25 m film thickness.
`Inlet Temperature: 225°C
`Injection Volume: 2 L
`Carrier Gas: Helium
`Split Ratio: 200:1
`Column Flow: 1.2 mL/minute
`Temperature Program: 100°C for 4.00 minutes. Ramp to 240°C at 3°C/minute, hold for 20.00 minutes.
`Detector Temperature: 250°C
`
`Run Sequence:
`The samples and standards were analyzed in the following manner.
`
`
`1. Blank
`2. Mixed Standard – duplicate injections
`3. Blank
`4. Sample 1 Prep 1 – duplicate injections
`5. Sample 1 Prep 2 – duplicate injections
`6. Blank
`7. Sample 2 Prep 1 – duplicate injections
`8. Sample 2 Prep 2 – duplicate injections
`9. Blank
`
`The sequence continued in the same manner until all samples were analyzed, finishing with two more injections
`of the mixed standard and a blank.
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000013
`
`

`

`7 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`OMEGA-3 FATTY ACID ANALYSIS
`Analysis for omega-3 fatty acids was performed following procedures described in AOAC 996.06 and AOAC
`991.39 “Fatty Acids in Encapsulated Fish Oils and Fish Oil Methyl and Ethyl Esters.” Sample preparation is similar
`to that described above and gas chromatography is used for analysis. The commercial standard “Food Industry
`FAME Mix” Cat. No. 35077 contains the following omega-3 fatty acid methyl esters:
`1. C18:3n3 (ALA methyl ester)
`2. C20:3n3 (ETE methyl ester)
`3. C20:5n3 (EPA methyl ester)
`4. C22:6n3 (DHA methyl ester)
`Additional standards were obtained from Sigma-Aldrich and Cayman Chemicals:
`1. C22:5n3 (DPA methyl ester)
`2. C18:4n3 (SDA methyl ester)
`3. C21:5n3 (HPA methyl ester)
`Internal Standard Preparation:
`1. 25 mg 23:0 methyl ester was weighed into 25 mL volumetric flask and diluted to volume with isooctane.
`2. 1.0 mL portions were pipetted into 16 mL glass vials and evaporated to residue under nitrogen.
`Sample Preparation:
`1. 25-35 mg of sample was added to a prepared internal standard vial described above.
`2. 1.5 mL methanolic sodium hydroxide was added, the solution blanketed with nitrogen, vortexed, and
`heated 5 min at ~100ºC. The preparation was then cooled to room temperature.
`3. 2 mL ampule of BF3 methanol solution was added, blanketed with nitrogen, heated for 30 min at ~100°C.
`The preparations were cooled to warm.
`4. 1 mL isooctane was added, blanketed with nitrogen, capped, shook for 30 seconds while warm.
`5. 5 mL saturated sodium chloride solution was added, blanketed with nitrogen, and vortexed. The
`preparation was then cooled to room temperature.
`6. The isooctane layer was transferred to a new tared vial, blanketed with nitrogen and capped.
`7. The aqueous layer was extracted with another 1 mL isooctane.
`8. The isooctane was transferred to the extract vial, and concentrated to ~1 mL under nitrogen.
`9. A portion of step 8 was transferred to an autosampler vial for analysis by gas chromatography.
`Standard Preparation:
`1. The following standards were obtained:
`a. Stearidonic acid methyl ester, Cayman Chemical, Item # 10005000, 10 mg/mL in ethanol
`b. Heneicosapentaenoic acid methyl ester, Cayman Chemical, Item # 11622, 10 mg/mL in ethanol
`c. Docosapentaenoic acid methyl ester, Supelco, Cat # 47563, 10 mg/mL in heptane
`2. 100 L of each standard was added to a prepared internal standard vial with 700 L of isooctane to form
`a mixed standard solution.
`Instrument and Analysis Conditions:
`The analysis was carried out on an Agilent 6890 gas chromatograph with flame-ionization detector, and
`an Agilent 6890 autosampler, under the following conditions:
`
`Column: Phenomenex ZB-Wax plus. 30 m length, 0.25 mm inner diameter, 0.25 m film thickness.
`Inlet Temperature: 250°C
`Injection Volume: 1 L
`Carrier Gas: Helium
`Split Ratio: 25:1
`Column Flow: 1.2 mL/minute
`Temperature Program: 170°C, no hold. Ramp to 225°C at 1°C/minute, no hold.
`Detector Temperature: 270°C
`
`Run Sequence:
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000014
`
`

`

`8 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`The samples and standards were analyzed in the following manner.
`
`
`1. Blank
`2. Mixed Standard – duplicate injections
`3. Blank
`4. Sample 1 Prep 1 – duplicate injections
`5. Sample 1 Prep 2 – duplicate injections
`6. Blank
`7. Sample 2 Prep 1 – duplicate injections
`8. Sample 2 Prep 2 – duplicate injections
`9. Blank
`
`The sequence continued in the same manner until all samples were analyzed, finishing with two more injections
`of the mixed standard and a blank.
`
`Analysis results are shown in Table 1.
`
`Table 1 Fatty acid analysis and Omega-3 fatty acid results
`Fatty acid content8 (g/100 g)
`S20
`S19
`S18
`S7
`S8
`S9
`S10
`S11
`S6
`S2
`S1
`24.56 24.35 13.12 11.73 21.91 29.51 18.05 16.00 18.17 20.51 29.01
`
`ANALYTES
`
`Saturated fatty acids9
`Monounsaturated fatty
`acids
`Polyunsaturated fatty
`acids10
`Total fatty acids11
`55.58 52.23 37.32 32.84 55.65 67.50 54.99 55.16 53.64 57.85 80.60
`Total Omega-3 fatty acids 17.46 16.47 14.79 13.62 21.16 19.91 22.98 21.67 22.09 22.89 31.53
` C20:5n3 (EPA)
`8.55
`8.43
`7.42
`6.87
`10.67 7.14
`6.96
`7.48
`10.97 11.44 15.59
` C22:6n3 (DHA)
`5.71
`5.57
`5.23
`5.07
`7.84
`3.88
`3.78
`4.10
`6.37
`6.62
`9.05
`
`
`10.85 8.68
`
`8.16
`
`6.56
`
`10.82 12.92 12.34 12.88 8.35
`
`9.06 12.68
`
`22.56 20.97 17.59 15.75 24.77 31.05 34.01 33.37 27.11 28.28 38.91
`
`ASTAXANTHIN ANALYSIS
`
`
`The content of astaxanthin in krill oil was determined by High Performance Liquid Chromatography
`(HPLC). HPLC is used for separation, identification, and quantitation of a variety of compounds in complex
`matrices. A mixture of solvents or solutions, called the mobile phase, is forced at high pressure through a
`packed column, usually of coated silica particles, called the stationary phase. Components are separated based
`on the difference in their affinities for the stationary phase and the mobile phase, and are detected and
`measured as they elute from the column. The time a chemical component spends in the column from injection
`until detection is known as retention time, and is an indicator of component identity. The measured peak area
`or height is concentration dependent, and is used to quantitate the component.
`Astaxanthin is usually found in nature as astaxanthin ester, both as the monoester and diester. The acid
`portion of the ester typically consists of fatty acids with variable carbon chain lengths and degrees of
`unsaturation. Krill oil samples were treated with cholesterol esterase to cleave the astaxanthin esters to
`astaxanthin and HPLC was used to quantify astaxanthin.
`
`
`8 Average of duplicate preparations krill oil samples and duplicate injections of each preparation.
`9 Fatty acid concentrations are expressed as gram of analyte per 100 gram of krill oil.
`10 Polyunsaturated fatty acids includes omega-3 fatty acids.
`11 Total fatty acids value includes saturated, monounsaturated, polyunsaturated fatty acids.
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000015
`
`

`

`9 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`
`
`
`Instrument and Chromatographic Conditions:
`Instrument: Agilent 1100 Series HPLC system which is composed of:
`
`
`Agilent 1100 Quaternary Pump
`
`
`Agilent 1100 Variable Wavelength Detector
`
`
`Agilent 1100 Column Thermostat
`
`
`Agilent 1100 Thermostatted Autosampler
`Column: YMC-Carotenoid S5 5 250 x 4.6 mm
`Detection: 474 nm
`Flow rate: 1.00 mL/min
`Gradient:
`
`Table 2 HPLC Gradient
`Time
`1% aq H3PO4
`0.00
`4.0
`15.00
`4.0
`23.00
`4.0
`27.10
`4.0
`35.00
`4.0
`
`MeOH
`81.0
`66.0
`16.0
`81.0
`81.0
`
`MTBE
`15.0
`30.0
`80.0
`15.0
`15.0
`
`Flow rate (mL/min)
`1.000
`1.000
`1.000
`1.000
`1.000
`
`
`Sample Preparation:
`1. Cholesterol esterase solution was prepared at ca. 4 unit/mL in 50 mM Tris-HCl at pH 7.0
`dissolving 300 units of cholesterol esterase into 75 mL of 50 mM Tris-HCl at pH 7.0.
`2. Stock sample solutions were prepared by weighing ca. 100-250 mg of sample into 500 mL
`volumetric flask and diluting with acetone.
`3. Transferred ca. 3.0 mL of sample solution and ca. 3.0 mL Cholesterol esterase solution to a vial.
`4. Heated to 37 °C for 45 min in a shaking incubator.
`5. Added 1 g of sodium sulfate decahydrate and 2 mL petroleum ether, vortex for 30 sec and
`centrifuge for 3 min at ca. 1,500 rpm.
`6. Transfer the petroleum ether layer to a 14 mL glass vial and evaporated under nitrogen.
`7. Added 0.5 g acetone and recorded mass.
`8. Duplicate preparations were made from the stock sample solutions.
`9. Additional samples were prepared in the same manner.
`Standards Preparation:
`1. A 347 ppm stock solution of Astaxanthin was prepared by weighing 3.1 mg Astaxanthin into an
`amber vial, diluting with acetone to a total mass of 8.94 g.
`2. The stock solution was used to prepare concentrations of ca. 0.019, 0.039, 0.081, 0.138, 0.277,
`0.559, 1.12, 2.12, 5.57 and 10.53 ppm.
`3. A 352 ppm stock solution of Astaxanthin was prepared by weighing 3.2 mg Astaxanthin into an
`amber vial, diluting with acetone to a total mass of 9.10 g.
`4. The stock solution was used to prepare concentrations of ca. 0.020, 0.043, 0.078, 0.127, 0.285,
`0.584, 1.06, 2.50, 4.89 and 10.48 ppm.
`Controls:
`1. System suitability was determined by six replicate injections of the 0.138 ppm standard during
`the first round of analyses.
`2. The standard at 0.138 ppm was used as a control during the HPLC analyses of samples S1, S2,
`and S6-S11. The response of the standard, injected at an interval of every three samples, were
`then compared to system suitability results.
`3. System suitability was determined by six replicate injections of the 0.127 ppm standard during
`the second round of analyses.
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000016
`
`

`

`10 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`4. The standard at 0.127 ppm was used as a control during the HPLC analyses. The response of the
`standard, injected at an interval of every three samples, were then compared to system
`suitability results.
`5. Accuracy test was performed on 0.85 ppm astaxanthin solution which was fitted to both
`calibration curves. The results are shown in Table 3.
`
`
`
`Table 3 Accuracy test of 0.85 ppm astaxanthin solution.
`% recovery with 347
`% recovery with 352
`ppm astaxanthin
`ppm astaxanthin
`calibration curve
`calibration curve
`98.64%
`99.22%
`
`Sample
`concentration
`
`0.85 ppm
`
`
`Spiked samples:
`1. One out of three samples were randomly selected for spike/recovery experiments.
`2. The spike level was targeted at ca. 100% with respect to the concentration of astaxanthin in the
`sample.
`3. Ca. 2 mL of the sample stock solution was treated with ca.1 mL of the appropriate diluted stock
`solution (i.e. 2 mL (1.45 g) S1 solution (ca. 0.068 ppm astaxanthin) was treated with 1 mL
`(0.7767 g) 0.138 ppm diluted stock solution to give a spike level of 109%). Results are shown in
`Table 4.
`
`
`
`Table 4 Spike Recovery Results
`Sample
`Spike Level
`S1
`109%
`S6
`138%
`S18
`87%
`
`Recovery
`90.3%
`87.5%
`80.4%
`
`
`HPLC Injection Sequence:
`
`The injection sequence on the instrument began with a solvent blank followed by six replicate system
`suitability injections, blank, standards for the calibration curve, accuracy check (0.85 ppm sample) and then the
`samples (which included sample spikes). Blanks and check standards were injected during the course of the
`sample analyses (ca. every 6 sample injections) to monitor whether or not the instrument was running properly.
`
`Representative Chromatographs:
`The following chromatographs shown below use the gradient in Table 2:
`1. astaxanthin ester purchased from USP in Figure 1;
`2. astaxanthin ester USP digested with cholesterol esterase in Figure 2
`3. astaxanthin USP 1 ppm solution in Figure 3.
`Digestion of astaxanthin ester resulted in three major peaks at ca. 9, 10 and 13 mins. The retention times and
`relative ratios resembled results reported in the literature12 where the:
`1. 9 min peak was 13-cis-astaxanthin;
`2. 10 min peak was all-trans-astaxanthin;
`3. 13 min peak was 9-cis-astaxanthin13.
`In an effort to confirm the nature of the three peaks, the LC method was transferred to an LC/MS system and
`modified by substituting phosphoric acid with formic acid.14 The chromatograph is shown in Figure 4.
`
`
`12 Fuji Chemical Industry Co., Ltd. “Spectrophotometric and HPLC Analysis Method for Determining Astaxanthin Content in
`AstaREAL®-P2AF.”
`13 This peak was not observed in the Astaxanthin USP standard.
`14 Phosphoric acid is not compatible with LC/MS, signals from phosphate clusters overwhelm signals from the analyte.
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`0000017
`
`

`

`11 | P a g e - S e p t e m b e r 1 6 , 2 0 1 3
`
`
`
`Formic acid in the gradient resulted in shorter retention times but the chromatograph showed an elution
`profile similar to Figure 2. The ion profiles for all three peaks in Figure 4 were similar with m/z: [M+H] = 597.4,
`[M+Na] = 619.4 and fragments at 402.2, 386.2, 128.2 and 88.1 (Figure 5, Figure 6 and Figure 7). This supported
`that the compounds have the same molecular weight, are closely related to one another and likely to be
`geometric isomers as reported in the literature.
`
`Figure 1 Astaxanthin ester “as received” from USP.
`
`
`Figure 2 Astaxanthin ester USP digested with cholesterol esterase.
`
`
`Figure 3 Astaxanthin USP, 1 ppm
`
`
`Figure 4 Chromatograph of digested astaxanthin ester
`
`
`
`
`
`
`
`2672 METRO BOULEVARD │ MARYLAND HEIGHTS, MISSOURI 63043│℗ 314.291.6620│WWW.CHEMIR.COM
`
`V1DJU114
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`14
`
`16
`
`18
`
`20
`
`22
`
`24
`
`26
`
`28
`
`Retention Time (mi