EPA/EPA eluted first at a retention time of approximately 2.5 min, and PC-
`
`
`
`DHA/DHA eluted at approximately 2.8 min.
`
`
`
`Figure 38. 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.
`
`
`- 101 -
`
`
`
`00000101
`
`

`
`
`
`Figure 39. 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
`
`correspoding 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
`
`
`- 102 -
`
`SRM.
`
`
`
`
`
`00000102
`
`

`
`
`
`Figure 40. 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.
`
`
`
`96. 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-
`
`
`- 103 -
`
`00000103
`
`

`
`
`
`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 41 through 58.
`
`
`- 104 -
`
`
`
`00000104
`
`

`
`
`
`Figure 41. 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.
`
`
`
`
`- 105 -
`
`00000105
`
`

`
`
`
`
`
`Figure 42. 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.
`
`
`
`
`- 106 -
`
`00000106
`
`

`
`
`
`Figure 43. 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.
`
`
`
`
`- 107 -
`
`00000107
`
`

`
`
`
`Figure 44. 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.
`
`
`
`
`- 108 -
`
`00000108
`
`

`
`
`
`Figure 45. 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.
`
`
`
`
`- 109 -
`
`00000109
`
`

`
`
`
`Figure 46. 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.
`
`
`- 110 -
`
`
`
`
`
`00000110
`
`

`
`
`
`Figure 47. 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.
`
`
`- 111 -
`
`
`
`00000111
`
`

`
`
`
`
`
`Figure 48. 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.
`
`
`- 112 -
`
`00000112
`
`

`
`
`
`Figure 49. 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.
`
`
`
`
`- 113 -
`
`00000113
`
`

`
`
`
`
`
`Figure 50. 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.
`
`
`
`
`
`
`- 114 -
`
`00000114
`
`

`
`
`
`
`
`Figure 51. 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.
`
`
`- 115 -
`
`00000115
`
`

`
`
`
`Figure 52. 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.
`
`
`
`
`- 116 -
`
`00000116
`
`

`
`
`
`Figure 53. 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.
`
`
`
`
`- 117 -
`
`00000117
`
`

`
`
`
`
`
`Figure 54. 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.
`
`
`
`
`- 118 -
`
`00000118
`
`

`
`
`
`Figure 55. 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.
`
`
`- 119 -
`
`
`
`00000119
`
`

`
`
`
`Figure 56. 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.
`
`
`
`
`- 120 -
`
`00000120
`
`

`
`
`
`Figure 57. 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.
`
`
`
`
`- 121 -
`
`00000121
`
`

`
`
`
`
`
`Figure 58. 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.
`
`
`
`
`- 122 -
`
`00000122
`
`

`
`
`
`
`
`97. 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 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 is shown in Figure 59, 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 60.
`
`
`- 123 -
`
`
`
`00000123
`
`

`
`
`
`Figure 59. 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 µl of a 5 µM 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.
`
`
`
`
`- 124 -
`
`00000124
`
`

`
`
`
`Figure 60. 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 µl of 5 µM 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.
`
`
`
`
`- 125 -
`
`00000125
`
`

`
`
`
`98.
`
`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.
`
`99. Based on my analyses above, I conclude that the prior art Fujita
`
`Reference inherently discloses to one of ordinary skill in the art at least at the time
`
`of the alleged invention a krill extract comprising the Claimed Phospholipid as
`
`required by each of the claims of the ‘351 patent. Each of the Fujita-hexane, Fujita-
`
`hexane-ethanol, and Fujita-once-through extraction methods disclosed in Fujita
`
`necessarily results in extracts containing multiple Claimed Phospholipids.
`
`100. Based on my analyses above, I conclude that the prior art Rogozhin
`
`reference also inherently discloses to one of ordinary skill in the art at least at the
`
`time of the alleged invention a krill extract comprising the Claimed Phospholipid
`
`as required by each of the claims of the ‘351 patent. The lipid extraction method in
`
`Example 1 of that patent necessarily results in extracts containing multiple
`
`Claimed Phospholipids.
`
`101. I am personally aware of how the Fujita-hexane, Fujita-hexane-
`
`ethanol, Fujita-once-through, and Rogozhin patent extracts were made. At least
`
`
`- 126 -
`
`00000126
`
`

`
`
`
`each of these prior art extracts necessarily is suitable for human consumption. The
`
`Rogozhin extract was made with nothing other than krill and water as the solvent
`
`and therefore is unquestionably fit for human use and eating. Moreover, as
`
`described in Fujita itself, krill is a food product and hexane is a solvent used for
`
`food preparation including extraction of vegetable oils. Ethanol too is fit for
`
`eating; it is the alcohol commonly found in liquors and beers. Moreover, as
`
`discussed above, the solvents used in the Fujita Reference were evaporated. There
`
`is nothing in these extracts that makes them unfit for human use or eating by
`
`humans.
`
`102. Based on my analyses above, I conclude that the prior art Fujita
`
`Reference anticipates and therefore invalidates at least claim 1 of the ‘351 Patent.
`
`103. Based on my analyses above, I conclude that the prior art Rogozhin
`
`patent anticipates and therefore invalidates at least claim 1 of the ‘351 Patent.
`
`104. I note that the named inventor of ‘351 patent represented to the
`
`USPTO during the reexamination of the ‘348 Patent that her allegedly inventive
`
`extract was distinguishable from the prior art because it “uniquely contained
`
`substantial amounts of phosphoplipids bearing omega-3 fatty acids (such as EPA
`
`and DHA) and was suitable for human consumption.” I see that Neptune made
`
`
`- 127 -
`
`00000127
`
`

`
`
`
`similar statements throughout prosecution and reexamination of the ‘351 patent
`
`and the ‘348 Patent.2 My analysis shows that this line of arguments is not correct.
`
`105. At the Respondents’ request, the extracts I created were distributed
`
`into test tubes and labeled. At Respondents’ request, I sent the following samples
`
`to Avanti Polar Lipids and Chemir Analytical Services:
`
`Sample Name Extraction Method
`
`to
`
`Shipped
`
`VB2 8/8/13 FH
`
`Fujita Hexane
`
`VB8 8/9/13 FHE Fujita Hexane
`
`Ethanol
`
`VB 1 8/8/13 FH Fujita Hexane
`
`VB 7 8/9/13
`
`Fujita Hexane
`
`FHE
`
`Ethanol
`
`Chemir
`
`Chemir
`
`Avanti
`
`Avanti
`
`
`
`106. I understand that the ’351 patent purports to claim priority to
`
`Provisional Application No. 60/307,842 (the Provisional Application; Ex. 1062)
`
`The Provisional Application (filed on July 27, 2001) is the earliest application to
`
`2
`(April 2, 2012 Response to Office Action, ‘351 patent (Ex. 1061, p. 34) (“Thus, as the
`extraction process of Beaudoin does not produce extracts comprising PLs of Formula I, as
`instantly claimed in the present application in any relevant amount, Beaudoin cannot
`anticipate the claimed extract.”)).
`
`
`- 128 -
`
`00000128
`
`

`
`
`
`which the Asserted Patents claim priority. I further understand that a patent may
`
`claim priority to a provisional application only if, among other requirements, the
`
`provisional application provides an adequate written description of the invention. I
`
`conclude that the Provisional Application does not provide an adequate written
`
`description for the Claimed Phospholipid for any of the Asserted Claims, and thus
`
`the Asserted Claims are not entitled to the benefit of the Provisional Application
`
`filing date.
`
`107. The Provisional Application contains no drawing of any phospholipid
`
`molecule. The Provisional Application also does not include any discussion of any
`
`two fatty acids being attached to any particular phospholipid molecule.
`
`108. The Provisional Application does not contain Example 1 of the ‘351
`
`patent or any discussion of Sample #804.3 Tables 2, 8, 9, and 10 of the ‘351
`
`
`3 The ‘351 patent specification contains three Examples. The patents state that Example 1
`“illustrates the isolation and molecular characterization of the phospholipids from the extract.”
`(‘351 patent, col.22 ll.36-37.) In this Example, a Sample referred to as “#804” was analyzed.
`Sample #804 is described as containing “large amounts of phospholipids, mainly: PC (438.48
`mg/g lipid) and PE (183.15 mg/g lipid).” (Id., col.22 ll. 39-42.) The ‘351 patent does not
`identify what method was used to quantify the recited phospholipids. Example 1 next describes a
`series of steps taken to analyze phospholipids in Sample #804. These steps can be generally
`summarized as follows. First, Example 1 reports that “[t]o obtain large quantities of PC and PE,
`separation was done by Thin Layer Chromatography.” (Id., col.22 ll. 46-49.) Example 1 reports
`that PC and PE were separated into separate bands on a TLC plate. Merely separating
`compounds into bands on a TLC plate, however, does not identify the compounds. The
`Example reports no use of any control or standard during this TLC experiment.
`
`
`
`- 129 -
`
`00000129
`
`

`
`
`
`patent are also absent from the Provisional Application. Figures 1-3 of the ‘351
`
`patent also are not in the Provisional Application.
`
`109. The Provisional Application includes a Table 5 that does not appear in
`
`the ‘351 patent:
`
`
`
`110. Table 5 of the Provisional Application identifies fatty acids in the
`
`“phospholipid mixture” of the claimed extract. This table does not show what
`
`particular fatty acids are attached to any particular phospholipid molecule and thus
`
`does not show the presence of the Claimed Phospholipid. This table noticeably
`
`identifies no DHA in the phosphatidyl ethanolamine fraction of the extract, and no
`
`DHA or EPA in the phosphatidyl inositol fraction of the extract. One of skill
`
`reading this table would understand it to affirmatively indicate there are no
`
`detected amounts of the following Claimed Phospholipids in the extract:
`
`
`- 130 -
`
`00000130
`
`

`
`
`
`Sn1: DHA
`
`Sn2: EPA
`
`Sn1: EPA
`
`Sn2: DHA
`
`Sn1: EPA
`
`Sn2: EPA
`
`Sn1: DHA
`
`Sn2: DHA
`
`Sn1: DHA
`
`Sn2: EPA
`
`Sn1: EPA
`
`Sn2: DHA
`
`Sn1: DHA
`
`Sn2: DHA
`
`
`
`(Phosphatidylinositol or “PI”)
`
`
`
`—CH2CH2NH3
`
`(Phosphatidylethanolamine or “PE”)
`
`
`
`111. The Provisional Application also includes Figures 2 and 3A through
`
`3K that are not included in the ‘351 patent. Figure 2 is nearly illegible. It is
`
`described as follows: “an HPLC profile of the flavonoid fraction of the
`
`- 131 -
`
`00000131
`
`

`
`
`
`composition of the present invention eluted with methanol. The HPLC shows
`
`aglycone [sic] peak and two or more glucosides.” (Provisional Application, Figure
`
`2) Figure 2 does not show the presence of a phospholipid molecule having any
`
`particular structure, much less any of the Claimed Phospholipids.
`
`112. Figures 3A to 3K are also nearly illegible. They are identified in the
`
`‘351 patent as “mass spectra (molecular analysis) of fatty acids attached to
`
`phospholipids in the composition of the present invention.” (Id., Figure 3A-3K)
`
`Figures 3A to 3K do appear to be mass spectra. However, they do not show any
`
`particular fatty acids attached to any particular phospholipid molecules. They also
`
`do not show any combination of fatty acids attached to any molecule. Instead,
`
`these spectra appear to be mass spectra of free fatty acid derivatives that are
`
`unbound to any molecule.
`
`113. The Provisional Application also does not discuss any “novel
`
`phospholipid.”
`
`114. At least the following portions of the ’351 Patent are also not included
`
`in the Provisional Application: col.1 ll.43-col.3 l.65, col.4 ll.6-14, 16-35; col.5 l.1-
`
`33; col.6 l.33-col.13 l.2; col.16 l.1-45; col. 17 l.31-39; col. 18 l. 25-30; col.19
`
`l.l.10-67; col.20 ll.34-41; col.20 l.65-col.22 l.21; col.22 l.34-col.24 l.45; col.25
`
`l.50-col.40 l.38.
`
`
`- 132 -
`
`00000132
`
`

`
`
`
`115. The ’351 patent purports to claim priority to the application for the
`
`’348 patent and the PCT Application under 35 U.S.C. §§ 120 and 365(c).
`
`116. In my opinion the ’351 patent is not entitled to claim priority to the
`
`Provisional Application.
`
`117. 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.
`
`
`- 133 -
`
`00000133
`
`

`
`
`
`Date: September 2?, 2013
`
`
`
`Dr. Richard B. van Breemen
`
`Attachments:
`
`Attachment A —
`
`Curriculum Vitae, Other Cases
`
`
`
`00000134
`
`
`- 134 -
`-134-
`
`00000134
`
`

`
`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)
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`1979-1980 Honors research, Department of Chemistry, Oberlin College
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`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)
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`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
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`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.
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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 dyes. Textile Chemist and Colorist, 22, 23-28 (1990).
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`19. Martin LB, Schreiner AF, van Breemen RB. Characterization of cisplatin adducts of
`oligonucleotides by fast atom bombardment mass spectrometry. Anal. Biochem. 193, 6-15
`(1991).
`van Breemen RB, Martin LB, Le JC. "Continuous-flow fast atom bombardment mass
`spectrometry of oligonucleotides." J. Am. Soc. Mass Spectrom. 2, 157-163 (1991).
`van Breemen RB, Canjura FL, Schwartz SJ. High-performance liquid chromatography-
`continuous-flow mass spectrometry of chlorophyll derivatives. J. Chromatogr. 542, 373-383
`(1991).
`van Breemen RB, Bartlett MG, Tsou Y, Culver C, Swaisgood H, Unger SE. Degradation of
`peptide drugs by immobilized digestive proteases. Drug Metab. Dispos. 19, 683-690 (1991).
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`van Breemen RB, Canjura FL, Schwartz SJ. Identification of chlorophyll derivatives by mass
`spectrometry. J. Agric. Food Chem. 39, 1452-1456 (1991).
`Freeman HS, Hao Z, Sokolowska-Gajda J, van Breemen RB. Matrix selection in the FAB
`mass spectrometric analysis of synthetic dyes. Dyes and Pigments, 16, 317-327 (1991).
`van Breemen RB, Huang C-H, Bumgardner CL. Continuous-flow fast atom bombardment
`and field desorption mass spectrometry of oligomers of 3,3,3-trifluorophenylpropyne. Anal.
`Chem. 63, 2577-2580 (1991).
`26. Bumgardner CL, Huang C-H, van Breemen RB. Characterization of oligomers of 3,3,3-
`trifluoro-1-phenylpropyne and 1-phenylpropyne by mass spectrometry. J. Fluor. Chem. 56,
`175-187 (1992).
`Schmitz HH, van Breemen RB, Schwartz SJ. Applications of fast atom bombardment mass
`spectrometry (FAB-MS) and continuous-flow FAB-MS to carotenoid analysis. Methods
`Enzymol. 213, 322-336 (1992).
`van Breemen RB, Davis RG. Rates of peptide proteolysis measured using liquid
`chromatography and continuous-flow fast atom bombardment mass spectrometry. Anal.
`Chem. 64, 2233-2237 (1992).
`van Breemen RB, Schmitz HH, Schwartz SJ. Continuous-flow fast atom bombardment
`liquid chromatography/mass spectrometry of carotenoids. Anal. Chem. 65, 965-969 (1993).
`Saljoughian M, Williams PG, Morimoto H, Goodlett DR, van Breemen RB. Tritiated
`diimide: A regio- and stereo-selective tritium labeling reagent. J. Chem. Soc., Chem.
`Commun. 414-416 (1993).
`31. Oommen TV, Petrie EM, van Breemen RB, Haney CA. Analysis of furanic compounds from
`cellulose aging by GC-MS, and attempts to correlate with degree of polymerization. CIGRE
`Paper 110-02, CIGRE Symposium on Diagnostic and Maintenance Techniques. Berlin,
`Germany, April 19-21. 1993.
`32. Blackburn RK, van Breemen RB. Degradation of the cyclic peptide antibiotic lysobactin by
`immobilized digestive proteases. Drug Metab. Dispos. 21, 573-579 (1993).
`van Breemen RB, Tsou Y, Connolly G. Oxidation of dicyclopentadiene in surface water.
`Biol. Mass Spectrom. 22, 579-584 (1993).
`Spanos GA, Schwartz SJ, van Breemen RB, Huang C-H. High-performance liquid
`chromatography with light-scattering detection and desorption chemical-ionization tandem
`mass spectrometry of milk fat triacylglycerols. Lipids, 30, 85-90 (1995).
`van Breemen RB, Schmitz HH, Schwartz SJ. Fast atom bombardment tandem mass
`spectrometry of carotenoids. J. Agric. Food Chem. 43, 384-389 (1995).
`van Breemen RB, Jiang O, Tidwell RR, Hall JE, Brewer TG. Fast atom bombardment
`tandem mass spectrometry of the anti-parasitic agent pentamidine and its oxygenated
`metabolites. J. Mass Spectrom. 30, 549-556 (1995).
`van Breemen RB. Electrospray liquid chromatography-mass spectrometry of carotenoids.
`Anal. Chem. 67, 2004-2009 (1995).
`38. Thomas VR, Schreiner AF, van Breemen R, Xie TY, Chen CL, Gratzl JS. Photolytic
`dechlorination of 4-chlorophenol using an ArF* excimer laser. Holzforschung, 49, 139-145
`(1995).
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`van Breemen RB. Advances in carotenoid analysis: Electrospray liquid chromatography-
`mass spectrometry using C30 reversed phase HPLC. Carotenoid News, 5, 8-9 (1995).
`van Breemen RB, Huang CH, Lu ZZ, Rimando A, Fong HHS, Fitzloff JF. Electrospray
`liquid chromatography/mass spectrometry of ginsenosides. Anal. Chem. 67, 3985-3989
`(1995).
`van Breemen RB. Innovations in carotenoid analysis using liquid chromatography/mass
`spectrometry. Anal. Chem. 68, 299A-304A (1996). *This paper was featured on the cover
`page of this issue of Analytical Chemistry.
`van Breemen RB, Huang C-H. High performance liquid chromatography-electrospray mass
`spectrometry of retinoids. FASEB J. 10, 1098-1101 (1996).
`van Breemen RB, Huang C-H, Tan Y, Sander LC, Schilling AB. Liquid
`chromatography/mass spectrometry of carotenoids using atmospheric pressure chemical
`ionization. J. Mass Spectrom. 31, 975-981 (1996).
`Shen L, Pisha E, Huang Z, Pezzuto JM, Krol E, Alam Z, van Breemen RB, Bolton JL.
`Bioreductive activation of catechol estrogen-ortho-quinones: Aromatization of the B ring in
`4-hydroxyequilenin markedly alters quinoid formation and reactivity. Carcinogenesis, 18,
`1093-1101 (1997).
`van Breemen RB, Huang C-H, Nikolic D, Woodbury CP, Zhao YZ, Venton DL. Pulsed
`ultrafiltration electrospray mass spectrometry: A new method for screening combinatorial
`libraries. Anal. Chem. 69, 2159-2164 (1997).
`46. Zhao YZ, van Breemen RB, Nikolic D, Huang C-R, Woodbury CP, Schilling A, Venton DL.
`Screening solution-phase combinatorial libraries using pulsed ultrafiltration/ electrospray
`mass spectrometry. J. Med. Chem. 40, 4006-4012 (1997).
`Shen L, Qiu S, van Breemen RB, Zhang F, Chen Y, Bolton JL. Reaction of the Premarin
`metabolite 4-hydroxyequilenin semiquinone radical with 2’-deoxyguanosine: Formation of
`unusual cyclic adducts. J. Am. Chem. Soc. 119, 11126-11127 (1997).
`van Breemen RB. Liquid chromatography/mass spectrometry of carotenoids. Pure Appl.
`Chem. 69, 2061-2066 (1997).
`49. Zhang HQ, Dixon RP, Marletta MA, Nikolic D, van Breemen R, Silverman RB. Mechanism
`of inactivation of neuronal nitric oxide synthase by Nω-allyl-L-arginine. J. Am. Chem. Soc.
`119, 10888-10902 (1997).
`50. Bolton J