IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`
`In re Ex Parte Reexamination of U.S. Patent No. 8,278,351
`
`
`Entitled:
`
`NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING
`POLYUNSATURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`2 October 2012 to Sampalis
`
`
`Issued:
`
`Control No.: Not yet assigned
`
`Filed:
`
`Examiner:
`
`
`
`October 2, 2012
`
`Not yet assigned
`
`DECLARATION OF RICHARD B. VAN BREEMEN, PH.D., IN
`SUPPORT OF EX PARTE REEXAMINATION
`OF U.S. PATENT NO. 8,278,351
`
`
`EFS WEB Filed
`
`Mail Stop Ex Partes Reexam
`Commissioner for Patents
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`
`I, Richard B. van Breemen, Ph.D., hereby declare and say:
`
`1.
`
`I am Professor of Medicinal Chemistry and Pharmacognosy at the College of Pharmacy
`
`of the University of Illinois at Chicago. I was promoted to this position in 2000. I hold the
`
`administrative position of Assistant to the Director of the Research Resources Center at the
`
`University of Illinois at Chicago. In this position, I provide advice regarding campus needs in
`
`the area of mass spectrometry, and my laboratory serves as a central campus resource in mass
`
`spectrometry. I am the Director of the UIC/NIH Center for Botanical Dietary Supplements
`
`Research. I am also on the editorial boards of the scientific journals Biomedical Chromatography
`
`and Assay and Drug Development Technologies. A copy of my Curriculum Vitae is attached as
`
`Exhibit 1 and sets forth my education, teaching positions, honors, various memberships in
`
`000001
`
`

`

`professional societies, selected speaking engagements, listing of my previous and present
`
`graduate students and postdoctoral research associates, and listings of my patents and
`
`publications.
`
`2.
`
`I was asked by Aker Biomarine ASA to review Declarations and references submitted by
`
`Neptune Bioresources and Technologies (Neptune) in the co-pending re-examination
`
`proceedings for U.S. Pat. No. 8,030,348 (in which I submitted a Declaration very similar to this
`
`Declaration) and the very similar Declarations and references submitted during the prosecution
`
`of this patent. I was also asked to use ultrahigh performance liquid chromatography-tandem
`
`mass spectrometry (UHPLC-MS-MS) to analyze lipid fractions prepared by Mr. Haugsgerd from
`
`two species of krill, Euphausia superba and Euphausia pacifica. I am being compensated for
`
`this analysis.
`
`The 2011 White Declaration Demonstrates the Presence of the Claimed Phospholipid
`
`Species in Beaudoin Krill Extracts
`
`3.
`
`Tables 1 and 2 of the 31 May 2011 White Declaration demonstrate the presence of
`
`phospholipids species detected as protonated molecules of m/z 826 and m/z 852 in fractions from
`
`each of the sample sets tested. These masses are consistent with phosphatidylcholines containing
`
`two eicosapentainoic acid groups (PC-EPA/EPA) (m/z 826) and one EPA group plus one
`
`docosahexaenoic acid group (PC-EPA/DHA) (m/z 852), respectively. Neptune’s experts, Dr.
`
`Yeboah and Dr. Shahidi both recognize and acknowledge this fact. As stated by Dr. Yeboah in
`
`¶36 of his 16 March 2012 Declaration:
`
`The species detected at m/z values of 826 and 852 represent amounts in a range on the
`order of only 0.1 to 2.8% of the phospholipids of the oil. I understand that phospholipids
`represent about 40% of the total lipids in krill oil and therefore, the raw data of Tables 1
`and 2 of the White Declaration shows that the amount of phospholipids carrying two and
`EPA and DHA in the total Beaudoin oil is only about 0.05 to 1.1%.
`
`Likewise, Dr. Shahidi stated in ¶22 of his 29 March 2012 Declaration:
`
`As Beaudoin reports an oil potentially with a small amount of the phospholipid
`containing two of EPA and DHA (i.e. about 0.1 to 1%), it is my opinion that this is not a
`biologically effective amount. As the claims of the '348 patent [sic] are directed to
`biologically effective amounts of this composition, they are distinct from Beaudoin.
`
`000002
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`

`

`Both Dr. Yeboah and Dr. Shahidi agree that the Beaudoin samples generated by Neptune and
`
`analyzed by Dr. White contain the claimed phospholipid species with two of EPA and DHA (PC-
`
`EPA/EPA and PC-DHA/DHA).
`
`4.
`
`Dr. Yeboah and Dr. Shahidi both argue that amounts of the claimed phospholipid species
`
`detected by Dr. White are de minimis or not a “biologically effective amount.” However, the
`
`2011 White Declaration provides no data comparing the amount of the claimed phospholipid
`
`species in extracts produced according to the ‘351 Patent to those present in the Beaudoin
`
`extracts. Likewise, the ‘351 patent itself contains no disclosure or data that can be used to
`
`determine the amount of the claimed phospholipid species in the ‘351 patent extracts.
`
`5.
`
`The ‘351 patent contains no data that identifies the presence or amount of any particular
`
`phospholipid species such a phospholipid containing EPA and/or DHA at both sn-1 and sn-2
`
`positions. Tables 2-5 of the ‘348 patent (as well as the dependent claims) clearly show that
`
`phospholipids of the krill extracts contain a complex mixture of fatty acids. The only reasonable
`
`assumption from these data is that only a small percentage of the phospholipids in the extracts
`
`actually produced and described in the ‘351 Patent contain two of EPA and DHA as claimed.
`
`Krill phospholipid extracts are complex mixtures of many different phospholipid species. The
`
`identities of these species is described in a recent paper, Winther et al., Elucidation of
`
`Phosphatidylcholine Composition in Krill Oil Extracted from Euphausia superba, Lipids
`
`46(1):25-36 (2011). I note that Neptune’s expert, Dr. Yeboah (Yeboah Declaration ¶34), relied
`
`on this paper in his analysis of the fatty acid content of the phospholipids in the 2011 White
`
`Declaration. As can be seen in Table 2 of Winther et al., there are many different phospholipids
`
`in a krill extract, including phospholipids with one of either DHA or EPA at one of the sn-1 or
`
`sn-2 positions and another non-omega 3 fatty acid at the other position, as well as phospholipids
`
`that do not have an omega 3 fatty acid attached. Phospholipids with two of EPA or DHA at the
`
`sn-1 and sn-2 positions (i.e., phospholipid species corresponding the claims) make up only a very
`
`small portion of the total phospholipid species in the extract. Winther et al., which was relied on
`
`by Dr. Yeboah for calculation of fatty acid percentages in the 2011 White declaration,
`
`corroborates the fact the claimed phospholipid species are only a few of many present in krill
`
`phospholipid extracts and that the relative percentage of the claimed phospholipid species is low
`
`in a krill extract. In summary, the available data indicate that any krill oil extract will only have
`
`small amounts of phospholipids with EPA and DHA at both sn-1 and sn-2 positions. There is no
`
`000003
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`

`

`information to the contrary. The de minimis arguments presented by Dr. Yeboah and Dr. Shahidi
`
`are without merit because they have not addressed the issue of how much of the claimed
`
`phospholipid species is actually in the ‘351 extracts.
`
`6.
`
`Dr. Yeboah admits that there are several errors in the 2011 White Declaration as
`
`originally submitted in the parent ‘348 patent in 2011 (Yeboah Declaration ¶¶27-34). I agree
`
`with Dr. Yeboah that Figure 10 does show a positive ion electrospray tandem mass spectrum
`
`consistent with phosphatidylcholine containing EPA at both sn-1 and sn-2 positions. According
`
`to the 2011 White Declaration, Figure 10 indicates a phospholipid containing EPA (C20:5) and
`
`stearic acid (C18:0). However, when calculating the molecular mass of a phosphatidylcholine
`
`with EPA (20:5) in one position and stearic acid (18:0) in the other, the molecular mass would be
`
`807.6, which would be detected as a protonated molecule of m/z 808.6. Instead, the protonated
`
`molecule of the phospholipid in Figure 10 is m/z 826.5, which is consistent with a
`
`phosphatidylcholine containing EPA at both sn-1 and sn-2 positions (PC-EPA/EPA). This
`
`assignment of the phospholipid PC-EPA/EPA is also supported by fragment ions of m/z 524.3
`
`and m/z 542.3 in Figure 10 of the White Declaration, which correspond to loss of EPA (-302),
`
`[MH-302]+, and loss of dehydrated EPA, [MH-284]+, respectively.
`
`7.
`
`Dr. Yeboah argues that the data in Figure 10 of the White Declaration should not be
`
`considered because the sample identified as “previous Beaudoin Oil” in the legend for Figure 10
`
`is not actually a Beaudoin oil. Dr. Yeboah goes to great length to distinguish the protocol used
`
`to generate the oil analyzed in Figure 10 from the Beaudoin protocol used for the other samples
`
`analyzed by Dr. White. I note that all the data in the White Declaration indicate the fact that
`
`different protocols give the same result – all extracts contained the claimed phospholipid species.
`
`8.
`
`In addition to the errors identified by Dr. Yeboah, it is my opinion that Dr. White also
`
`erred in not analyzing positive controls such as PC-DHA/DHA and PC-EPA/EPA. In ¶11, Dr.
`
`White reports that the phospholipids with protonated molecules of m/z 826 and m/z 852 were
`
`detected, which were consistent with PC-EPA/EPA and PC-EPA/DHC. However, Dr. White
`
`suggests that the tandem mass spectra of these ions were inconsistent with these structures. How
`
`does he know that the tandem mass spectra he obtained, such as that shown in Figure 11, are not
`
`PC-EPA/DHC unless he compared them with standards as positive controls? Dr. White
`
`addresses this shortcoming in his Supplemental Declaration in which he states that he did use a
`
`000004
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`

`

`commercial phospholipid standard set as a positive control (White Supplemental Decl. ¶¶10-12).
`
`However, this standard set did not include the target phospholipids with two of EPA or DHA and
`
`thus has no value in establishing utility of the experimental set up to actually detect the target
`
`phospholipids. If a positive control of PC-EPA/EPA had been analyzed, it would have been
`
`clear that Figure 10 represents a phospholipid with EPA at both of the sn-1 and sn-2 positions
`
`(PC-EPA/EPA) and not stearic acid and EPA as concluded by Dr. White.
`
`My Own Analysis of Beaudoin Extracts Produced by the Revised Protocol Confirms the
`
`Presence of the Claimed Phospholipid Species
`
`9.
`
`A total of 18 krill oil fractions in glass tubes were received in my laboratory from Aker
`
`Biomarine and stored at -80 °C until analysis using UHPLC-MS-MS. The samples and their
`
`descriptions are summarized in Table 1 and represent acetone fractions, ethanol fractions and
`
`ethyl acetate fractions of oil from two species of krill, E. superba and E. pacifica. Some samples
`
`were not heated, some were heated to 60 °C or 70 °C, and other samples were heated to 125 °C
`
`during processing. In preparation for UHPLC-MS-MS analysis, 1 ml methanol was added to
`
`each glass tube, which was then vortex mixed for 3 min, sonicated for 3 min, and then diluted
`
`100-fold with 50% aqueous methanol. Aliquots of 5
`
`l each were injected onto the UHPLC-MS-
`
`MS system. Standards of PC-EPA/EPA and PC-DHA/DHA (purity > 99.0%) were purchased
`
`from Sigma-Aldrich (St. Louis, MO), and a standard solution containing 1 M of each
`
`compound was prepared in methanol. Aliquots of 1
`
`l of the standard solution were injected on
`
`the UHPLC-MS-MS system.
`
`10.
`
`I used UHPLC-MS-MS for the identification of PC-EPA/EPA, PC-DHA/DHA, and PC-
`
`EPA/DHA in krill oil samples, because UHPLC-MS-MS is among the most selective of all
`
`analytical techniques. Ultrahigh pressure liquid chromatography (UHPLC) provides higher
`
`chromatographic resolution than does HPLC. Selectivity means the ability to measure a
`
`particular analyte without interference from other compounds. I used a Shimadzu (Kyoto, Japan)
`
`LCMS-8030 triple quadrupole mass spectrometer equipped with a Shimadzu Nexera UHPLC
`
`system and Shimadzu XR-ODS III reversed phase column (1.6 µm, 2.0 x 50 mm). The mobile
`
`phase consisted of 80% methanol containing 5 mM aqueous ammonium formate for 0.5 min
`
`followed by a 1 min linear gradient to 100% methanol containing 5 mM ammonium formate.
`
`000005
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`

`

`After 2 min, the mobile phase was restored to the original conditions before the next analysis.
`
`PC-EPA/EPA, PC-DHA/DHA, and PC-EPA/DHA were detected using positive ion electrospray
`
`and SRM. Mass spectrometer parameters included the use of nitrogen drying gas at 20 L/min,
`
`nebulizing gas at 3 L/min and a heat block temperature of 400 °C. Nitrogen was used as the
`
`collision gas for collision-induced dissociation during product ion tandem mass spectrometry.
`
`11.
`
`The positive ion electrospray mass spectra of PC-EPA/EPA and PC-DHA/DHA
`
`standards are shown in Figure 1, and the positive ion electrospray product ion tandem mass
`
`spectra of these standards are shown in Figure 2. PC-EPA/EPA and PC-DHA/DHA formed
`
`abundant protonated molecules of m/z 826 and m/z 878, respectively (Figure 1), which were used
`
`as precursors during tandem mass spectrometry. During product ion tandem mass spectrometry,
`
`each phospholipid fragmented to form an ion of m/z 184, which was the only abundant ion each
`
`tandem mass spectrum (Figure 2). Therefore, the SRM transitions of m/z 826 to m/z 184 and m/z
`
`878 to m/z 184 were used for the selective detection of PC-EPA/EPA and PC-DHA/DHA as they
`
`eluted from the UHPLC column. Although no standard was available, the SRM transition of m/z
`
`852 to m/z 184 was used to monitor PC-DHA/EPA based on the theoretical mass of its
`
`protonated molecule and its predicted major fragment ion. The UHPLC-MS-MS SRM analysis
`
`of a mixture of PC-EPA/EPA and PC-DHA/DHA standards is shown in Figure 3. PC-EPA/EPA
`
`eluted first at a retention time of approximately 2.5 min, and PC-DHA/DHA eluted at
`
`approximately 2.8 min.
`
`12. UHPLC-MS-MS was used to measure the 18 krill oil samples (Table 1), which included
`
`9 samples from E. pacifica and 9 samples from E. superba that had been heated to 60 C, 70 C
`
`or 125 C, or not heated at all. Solvent blanks were injected and analyzed between UHPLC-MS-
`
`MS analyses of each krill oil sample. PC-EPA/EPA and PC-DHA/DHA were detected in all 18
`
`krill oil samples. PC-DHA/EPA was also detected in all 18 krill oil samples and eluted between
`
`PC-DHA/DHA and PC-EPA/EPA during UHPLC-MS-MS. No phospholipid peaks were
`
`detected in any of the solvent blank analyses, which proved that there was no sample carry over
`
`between analyses. The UHPLC-MS-MS chromatograms showing the detection of all three
`
`phospholipids in the 18 krill oil samples are shown in Figures 4 through 21.
`
`13.
`
`PC-EPA/EPA and PC-DHA/DHA standards were spiked into some of the krill oil
`
`samples, which were then reanalyzed. The PC-EPA/EPA and PC-DHA/DHA standards coeluted
`
`000006
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`

`

`with the krill oil phospholipid peaks at ~2.5 and ~2.8 min, respectively, instead of eluting as
`
`separate peaks. Based on coelution with standards during UHPLC, the formation of protonated
`
`molecules of identical masses, and the formation of identical fragment ions of m/z 184 during
`
`tandem mass spectrometry, the phospholipids eluting at ~2.5 and ~2.8 min during UHPLC-MS-
`
`MS analysis of the krill oil samples were identified as PC-EPA/EPA and PC-DHA/DHA,
`
`respectively. For example, UHPLC-MS-MS chromatograms for the analysis of spiked krill oil
`
`from an ethyl acetate fraction of E. pacifica that had been heated at 70 °C are shown in Figure
`
`22, and UHPLC-MS—MS chromatograms for the analysis of spiked krill oil from an ethanol
`
`fraction of E. superba that had been heated at 125 °C are shown in Figure 23.
`
`14.
`
`In conclusion, the phospholipids PC-DHA/DHA, PC-EPA/EPA and PC—DHA/EPA were
`
`detected and identified using UHPLC-MS-MS in all 18 krill oil samples tested regardless of
`
`species, extraction solvent or heat treatment. Identification of these phospholipids in the krill oil
`
`samples was based on molecular mass, tandem mass spectrometric fragmentation and
`
`chromatographic coelution with standards.
`
`15.
`
`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 false statements may jeopardize the validity of the application or any
`
`patent issued thereon.
`
`Respectfully submitted,
`
`WW
`
`0 ‘__OOH12L9~QL2
`
`Richard B. van Breemen
`
`Date
`
`000007
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`000007
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`

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`Table 1. Krill oil samples analyzed using UHPLC-MS-MS detection for the detection and
`identification of phosphatidyl cholines (PC) containing two EPA residues, two DHA residues or
`one EPA and one DHA residue. Note that all three types of phosphatidyl cholines were detected
`in every sample (see Figures 4 - 21 for the corresponding UHPLC-MS-MS chromatograms).
`
`Sample
`
`Solvent
`
`Heat treatment
`
`Oil amount
`(mg)
`
`Phosphatidyl cholines
`detected
`
`E. pacifica I
`
`Acetone
`
`Not heated
`
`E. pacifica I
`
`Acetone
`
`60 C
`
`E. pacifica I
`
`Acetone
`
`125 C
`
`E. pacifica IIa
`
`Ethanol
`
`Not heated
`
`E. pacifica IIa
`
`Ethanol
`
`70 C
`
`E. pacifica IIa
`
`Ethanol
`
`125 C
`
`E. pacifica IIb
`
`Ethyl acetate
`
`Not heated
`
`E. pacifica IIb
`
`Ethyl acetate
`
`70 C
`
`E. pacifica IIb
`
`Ethyl acetate
`
`125 C
`
`E. superba I
`
`Acetone
`
`Not heated
`
`E. superba I
`
`Acetone
`
`60 C
`
`E. superba I
`
`Acetone
`
`125 C
`
`E. superba IIa
`
`Ethanol
`
`Not heated
`
`E. superba IIa
`
`Ethanol
`
`70 C
`
`E. superba IIa
`
`Ethanol
`
`125 C
`
`E. superba IIb
`
`Ethyl acetate
`
`Not heated
`
`E. superba IIb
`
`Ethyl acetate
`
`70 C
`
`E. superba IIb
`
`Ethyl acetate
`
`125 C
`
`
`
`
`
`
`
`53.0
`
`53.6
`
`56.4
`
`54.2
`
`76.5
`
`46.8
`
`53.5
`
`41.1
`
`65.9
`
`59.6
`
`60.1
`
`60.5
`
`46.2
`
`81.0
`
`69.2
`
`56.9
`
`58.4
`
`61.5
`
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`PC-EPA/EPA, PC-DHA/DHA,
`PC-DHA/EPA
`
`000008
`
`

`

`Figure 1. Positive ion electrospray mass spectra of the phosphatidyl choline standards PC-
`DHA/DHA and PC-EPA/EPA. Protonated molecules of each phospholipid were detected as the
`base peaks of each spectrum at m/z 878 and m/z 826, respectively.
`
`
`
`
`
`
`
`
`
`
`
`000009
`
`

`

`Figure 2. Positive ion electrospray product ion tandem mass spectra of PC-DHA/DHA and PC-
`EPA/EPA. The protonated molecules of m/z 878 and m/z 826 corresponding to PC-DHA/DHA
`and PC-EPA/EPA, respectively, were used as precursor ions. Based on these tandem mass
`spectra, the abundant product ion of m/z 184 was used for subsequent measurements of these
`phospholipids during SRM.
`
`
`
`
`
`0000010
`
`

`

`Figure 3. UHPLC-MS-MS chromatogram of a mixture of PC-EPA/EPA and PC-DHA/DHA
`standards obtained using positive ion electrospray and SRM of the transition m/z 826 to m/z 184
`for PC-EPA/EPA and SRM of the transition m/z 878 to m/z 184 for PC-DHA/DHA. The
`
`retention times of PC-EPA/EPA and PC-DHA/DHA were 2.5 min and 2.8 min, respectively.
`
`
`
`
`
`0000011
`
`

`

`Figure 4. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an acetone fraction of
`krill oil from E. pacifica that had been not been heat treated. Note that PC-EPA/EPA was
`
`detected at a retention time of 2.5 min, PC-DHA/DHA was detected eluting at 2.8 min, and
`
`PC-DHA/EPA eluted in between the other phospholipids at a retention time of 2.7 min.
`
`
`
`
`
`
`
`
`
`0000012
`
`

`

`Figure 5. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an acetone fraction of
`
`krill oil from E. pacifica that had been heated at 60 C. Note that PC-EPA/EPA was detected at a
`
`retention time of 2.5 min, PC-DHA/DHA was detected eluting at 2.8 min, and PC-DHA/EPA
`
`eluted in between the other phospholipids at a retention time of 2.7 min.
`
`
`
`0000013
`
`

`

`Figure 6. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an acetone fraction of
`
`krill oil from E. pacifica that had been heated at 125 C. Note the detection of peaks
`
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`0000014
`
`

`

`Figure 7. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethanol fraction of
`krill oil from E. pacifica that was not heat treated. Note the detection of peaks corresponding to
`PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`0000015
`
`

`

`Figure 8. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethanol fraction of
`
`krill oil from E. pacifica that had been heated at 70 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`0000016
`
`

`

`Figure 9. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethanol fraction of
`
`krill oil from E. pacifica that had been heated at 125 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`
`
`
`
`
`
`0000017
`
`

`

`Figure 10. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethyl acetate
`fraction of krill oil from E. pacifica that had not been heat treated. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`
`
`
`
`0000018
`
`

`

`Figure 11. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethyl acetate
`
`fraction of krill oil from E. pacifica that had been heated at 70 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`0000019
`
`

`

`Figure 12. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethyl acetate
`
`fraction of krill oil from E. pacifica that had been heated at 125 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`0000020
`
`

`

`Figure 13. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an acetone fraction
`of krill oil from E. superba that had not been heated. Note the detection of peaks corresponding
`to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`
`
`0000021
`
`

`

`Figure 14. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an acetone fraction
`
`of krill oil from E. superba that had been heated at 60 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`0000022
`
`

`

`Figure 15. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an acetone fraction
`
`of krill oil from E. superba that had been heated at 125 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`0000023
`
`

`

`Figure 16. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethanol fraction
`of krill oil from E. superba that had not been heated. Note the detection of peaks corresponding
`to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`0000024
`
`

`

`Figure 17. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethanol fraction
`
`of krill oil from E. superba that had been heated at 70 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`0000025
`
`

`

`Figure 18. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethanol fraction
`
`of krill oil from E. superba that had been heated at 125 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`0000026
`
`

`

`Figure 19. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethyl acetate
`fraction of krill oil from E. superba that had not been heated. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`0000027
`
`

`

`Figure 20. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethyl acetate
`
`fraction of krill oil from E. superba that had been heated at 70 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`0000028
`
`

`

`Figure 21. Positive ion electrospray UHPLC-MS-MS with SRM analysis of an ethyl acetate
`
`fraction of krill oil from E. superba that had been heated at 125 C. Note the detection of peaks
`corresponding to PC-EPA/EPA, PC-DHA/DHA and PC-DHA/EPA.
`
`
`
`
`
`
`
`
`
`
`
`
`
`0000029
`
`

`

`Figure 22. UHPLC-MS-MS chromatograms for the analysis of spiked krill oil from an ethyl
`
`acetate fraction of E. pacifica that had been heated at 70 C. The krill oil sample was spiked with
`
`0.5
`
`l of a 5 M solution of PC-DHA/DHA and PC-EPA/EPA. Note that the peaks eluting at
`
`2.5 min and 2.8 min increased in height but not the peak at 2.7 min corresponding to PC-
`
`DHA/EPA.
`
`
`
`
`
`0000030
`
`

`

`Figure 23. UHPLC-MS-MS chromatograms of krill oil from an ethanol fraction of E. superba
`
`that had been heated at 125 C and spiked with 1.0
`
`l of 5 M PC-DHA/DHA and PC-
`
`EPA/EPA. Note that the peaks eluting at 2.5 min and 2.8 min increased in height but not the
`
`peak at 2.7 min, which corresponded to PC-DHA/EPA.
`
`
`
`
`
`0000031
`
`

`

`EXHIBIT 1
`EXHIBIT 1
`
`0000000
`
`0000032
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`

`

`April 2012
`
`RICHARD BRUCE VAN BREEMEN
`Professor of Medicinal Chemistry
`Department of Medicinal Chemistry and Pharmacognosy
`College of Pharmacy
`University of Illinois at Chicago
`833 South Wood Street
`Chicago, Illinois 60612-7231
`EDUCATION
`1980-1985
`Ph.D. 1985, in Pharmacology
`
`Johns Hopkins University School of Medicine
`
`Baltimore, Maryland.
`1976-1980
`B.A. 1980, in Chemistry, with honors, German minor
`
`Oberlin College, Oberlin, Ohio.
`
`
`Telephone: (312) 996-9353
`FAX:
`(312) 996-7107
`email: breemen@uic.edu
`
`PROFESSIONAL EXPERIENCE
`
`
`2000-present Professor of Medicinal Chemistry and Pharmacognosy
`College of Pharmacy, University of Illinois at Chicago, Chicago, IL
`2010-present Assistant Head for Curricular Affairs, Department of Medicinal Chemistry and
`Pharmacognosy, University of Illinois at Chicago, Chicago, IL
`2002-present Academic Director, Mass Spectrometry and Proteomics, Research Resources
`Center, University of Illinois at Chicago, Chicago, IL
`2001-present Assistant to the Director of the Research Resources Center, University of Illinois
`at Chicago, Chicago, IL
`2006-present Editorial Board, Biomedical Chromatography
`2010-present Editorial Board, Assay and Drug Development Technologies
`2010-present Editor-in-Chief emeritus, Combinatorial Chemistry & High Throughput
`Screening
`1997-2010
`Editor-in-Chief, Combinatorial Chemistry & High Throughput Screening
`1994-2000 Associate Professor of Medicinal Chemistry
`
`
`
`College of Pharmacy, University of Illinois at Chicago, Chicago, IL
`1986-1993 Assistant Professor of Chemistry, Member of the Biotechnology Faculty
`
`Director of the Mass Spectrometry Laboratory for Biotechnology Research
`
`North Carolina State University, Raleigh, NC.
`1985-1986
`Postdoctoral Fellow. Middle Atlantic Mass Spectrometry Laboratory, NSF
`Regional Instrumentation Facility, Johns Hopkins University School of Medicine,
`Baltimore, MD. (Laboratory of Catherine Fenselau and Robert Cotter)
`Visiting Assistant Professor of Chemistry
`North Carolina State University, Raleigh, NC.
`Predoctoral research, Dept. of Pharmacology Johns Hopkins University School
`of Medicine. Baltimore, MD. Dissertation: "Electrophilic Reactions of 1-O-Acyl
`Glucuronides." (Advisor, Catherine Fenselau)
`1
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`1980-1985
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`0000033
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`April 2012
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`2.
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`3.
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`1979-1980 Honors research, Department of Chemistry, Oberlin College
`
`Oberlin, OH. Synthesized spirocyclic lactones as analogs of insect pheromones
`1979
`Research assistant, Department of Pharmacology, Toxicology Center, Univ. of
`Iowa, Iowa City, IA. Quantitation of metabolites of diphenylhydantoin in human
`serum using GC/MS. (Laboratory of L.J. Fischer)
`
`
`
`
`HONORS AND AWARDS
`NIH Predoctoral Fellowship, 1980-1984
`Phi Lambda Upsilon (Chemistry Honor Society)
`Award for Outstanding Paper at the Third North American Meeting of the International Society
`for the Study of Xenobiotics. San Diego, CA, October 21-25,1990
`Named by the Editors of Spectroscopy as one of 19 “Bright Young Stars” in analytical
`spectroscopy. (“Perspectives on 10 Years in Spectroscopy,” Spectroscopy, 10, 52-61, 1995)
`Invited speaker, 11th International Carotenoid Conference, Leiden, The Netherlands, 1996
`Invited speaker, International Convention on Pharmaceutical Sciences Commemorating the 50th
`Anniversary of the Pharmaceutical Society of Korea. Seoul, Korea, 2001
`Outstanding Paper Award, 88th American Oil Chemists Society Annual Meeting & Expo.
`Seattle, WA, May 11-14, 1997
`Linus Pauling Institute Seminar Series Speaker. Oregon State University, Corvallis, OR,
`February 16, 2006
`University Scholar Award. University of Illinois, 2003-2006
`AOAC Presidential Task Force on Dietary Supplements, 2009-2011
`AOAC International Harvey W. Wiley Award, 2008
`AOAC International Outstanding Dietary Supplement Methods Panel Chair, 2010
`
`PUBLICATIONS
`
`1. Papers
`Cotter RJ, van Breemen R, Yergey J, Heller D. Thermal, laser, and fast atom desorption. Int.
`1.
`J. Mass Spectrom. Ion Proc. 46, 395-8 (1983).
`van Breemen RB, Snow M, Cotter RJ. Time resolved laser desorption mass spectrometry, I.
`Desorption of preformed ions. Int. J. Mass Spectrom. Ion Phys. 49, 35-50 (1983).
`Gibson W, van Breemen R, Fields A, LaFemina R, Irmiere A. D,L-α-
`Difluoromethylorinithine inhibits human cytomegalovirus replication. J. Virology. 50, 145-
`54 (1984).
`van Breemen RB, Tabet J-C, Cotter RJ. Characterization of oxygen-linked glucuronides by
`laser desorption mass spectrometry. Biomed. Mass Spectrom. 11, 278-83 (1984).
`van Breemen RB, Fenselau C. Acylation of albumin by 1-O-acyl glucuronides. Drug Metab.
`Dispos. 13, 318-20 (1985).
`van Breemen RB, Fenselau CC, Dulik DM. Activated Phase II metabolites: Comparison of
`alkylation by 1-O-acyl glucuronides and acyl sulfates. in Biological Reactive Intermediates
`III, ed. by JJ. Kocsis, DJ. Jollow, CM. Whitmer, JO Nelson, and R. Snyder. pp. 423-9,
`Plenum Publishing Corp., New York, 1986.
`
`4.
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`5.
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`6.
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`2
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`0000034
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`April 2012
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`7.
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`8.
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`9.
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`10.
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`11.
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`12.
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`13.
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`14.
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`15.
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`17.
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`18.
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`van Breemen RB, Fenselau C. Reaction of 1-O-acyl glucuronides with 4-p-
`(nitrobenzyl)pyridine. Drug Metab. Dispos. 14, 197-201 (1986).
`van Breemen RB, Fenselau C, Mogilevsky W, Odell GB. Reaction of bilirubin glucuronides
`with serum albumin. J. Chromatogr. 383, 387-92 (1986).
`van Breemen RB, Fenselau C, Cotter RJ, Curtis A.J., Connolly G. Derivatives of
`dicyclopentadiene in ground water. Biomed. Environ. Mass Spectrom. 14, 97-102 (1987).
`Fenselau C, van Breemen R, Bradow G, Stogniew M. Acyl-linked glucuronides as reactive
`metabolites. Fed. Proc. 46, 2436-9 (1987).
`van Breemen RB, Martin LB, Schreiner AF. Comparison of electron impact, desorption
`chemical ionization, field desorption, and fast atom bombardment mass spectra of nine
`monosubstituted Group VI metal carbonyls. Anal. Chem. 60, 1314-8 (1988).
`van Breemen RB, Stogniew M, Fenselau C. Characterization of acyl-linked glucuronides by
`electron impact and fast atom bombardment mass spectrometry. Biomed. Environ. Mass
`Spectrom. 17, 97-103 (1988).
`van Breemen RB. Fast atom bombardment mass spectrometry with B/E linked scanning of
`ether- and thiophenol-linked glucuronides. In Cellular and Molecular Aspects of
`Glucuronidation, ed. by G. Siest, J. Magdalou, B. Burchell, vol. 173, pp. 211-9, Colloque
`INSERM/John Libbey Eurotext Ltd., 1988.
`van Breemen RB, Le JC. Enhanced sensitivity of peptide analysis by fast atom bombardment
`mass spectrometry using nitrocellulose as a substrate. Rapid Commun. Mass Spectrom. 3, 20-
`4 (1989).
`van Breemen RB, Martin LB, Schreiner AF. Formation of Negative ions of monosubstituted
`Group VIB pentacarbonyls during fast atom bombardment mass spectrometry. Org. Mass
`Spectrom. 25, 3-8 (1990).
`16. Goodlett DR, Armstrong FB, Creech RJ, van Breemen RB. Formylated peptides from
`cyanogen bromide digests identified by fast atom bombardment mass spectrometry. Anal.
`Biochem. 186, 116-20 (1990).
`van Breemen RB, Wheeler JJ, Boss WF. Identification of carrot inositol phospholipids by
`fast atom bombardment mass spectrometry. Lipids, 25, 328-334 (1990).
`Freeman HS, van Breemen RB, Esancy J F, Hao Z, Ukponmwan DO, Hsu WN. Fast atom
`bombardment and desorption chemical ionization mass spectrometry in the analysis of
`involatile textile