standard and reanalyzed using UHPLC-MS/MS (Figure 8). The standard coeluted
`
`with the phospholipid in the extract thereby identifying this phospholipid in the
`
`Fujita hexane/ethanol extract as PC-DHA/DHA.
`
`(x10,000,000)
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`IT-TOF MS
`Fujita Hexane/Ethanol
`Extraction
`
`14487372
`
`1.0
`1.5
`(x10,000,000)
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Spiked with PC-DHA/DHA
`standard
`
`16080782
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`5.0
`5.5
`Retention time (min)
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 8. Positive ion electrospray high resolution IT-TOF UHPLC-MS
`
`computer-reconstructed mass chromatograms of the Fujita hexane ethanol
`
`extract showing the detection of peaks corresponding to PC-EPA/EPA (m/z
`
`826.53), PC-DHA/EPA (m/z 852.55) and PC-DHA/DHA (m/z 878.56)
`
`eluting at approximately 5.2, 5.4 and 5.6 minutes, respectively (top). The
`
`
`- 51 -
`
`0000051
`
`
`
`with the phospholipid in the extract thereby identifying this phospholipid in the
`
`Fujita hexane/ethanol extract as PC-DHA/DHA.
`
`(x10,000,000)
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`IT-TOF MS
`Fujita Hexane/Ethanol
`Extraction
`
`14487372
`
`1.0
`1.5
`(x10,000,000)
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Spiked with PC-DHA/DHA
`standard
`
`16080782
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`5.0
`5.5
`Retention time (min)
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 8. Positive ion electrospray high resolution IT-TOF UHPLC-MS
`
`computer-reconstructed mass chromatograms of the Fujita hexane ethanol
`
`extract showing the detection of peaks corresponding to PC-EPA/EPA (m/z
`
`826.53), PC-DHA/EPA (m/z 852.55) and PC-DHA/DHA (m/z 878.56)
`
`eluting at approximately 5.2, 5.4 and 5.6 minutes, respectively (top). The
`
`
`- 51 -
`
`0000051
`
`
`
`
`
`extract was spiked with a PC-DHA/DHA standard and then reanalyzed
`
`(bottom). Note that the area of the peak corresponding to PC-DHA/DHA
`
`increased confirming the identity of PC-DHA/DHA in the extract.
`
`Inten. (x10,000,000)
`
`826.5336(1)
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`IT-TOF MS
`Fujita Hexane/Ethanol
`Extraction
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`852.5491(1)
`
`828.5552(1)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`878.5665(1)
`
`0.0
`800
`810
`Inten. (x10,000,000)
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`810
`800
`Inten. (x1,000,000)
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`804.5517(1)
`
`0.0
`800
`
`838.5699(1)
`
`854.5682(1)
`
`810
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`894.5939(1)
`890
`
`m/z
`
`
`
`Figure 9. High resolution accurate mass measurements of the peaks
`
`corresponding to PC-EPA/EPA (top) eluting at a retention time of
`
`approximately 5.2 min (Figure 8), PC-EPA/DHA (middle) eluting at a
`
`retention time of approximately 5.4 min (Figure 8), and PC-DHA/DHA
`
`(bottom) eluting at a retention time of approximately 5.6 min (Figure 8).
`
`
`- 52 -
`
`0000052
`
`
`
`
`
`—— : 878.50>184.10(+) PC-DHA/DHA
`······· : 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ : 852.60>184.10(+) PC-DHA/EPA
`
`UHPLC-MS-MS triple quadrupole
`Fujita Hexane/Ethanol Extraction
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`
`
`600000
`
`500000
`
`400000
`
`300000
`
`200000
`
`100000
`
`0
`1.0
`
`Figure 10. Positive ion electrospray UHPLC-MS/MS analysis of the
`
`Claimed Phospholipids in the Fujita Hexane/Ethanol Extract. A triple
`
`quadrupole mass spectrometer was used with collision-induced dissocation
`
`and selected reaction monitoring (SRM) of the transitions indicated.
`
`
`
`68. Further analysis of the Claimed Phospholipids in the Fujita hexane
`
`ethanol extract was carried out using positive ion electrospray UHPLC-MS/MS
`
`with collision-induced dissociation and selected reaction monitoring (SRM) on a
`
`triple quadrupole tandem mass spectrometer. The SRM transitions that were used
`
`for analysis were m/z 826 to m/z 184 for PC-EPA/EPA, m/z 852 to m/z 184 for PC-
`
`EPA/DHA, and m/z 878 to m/z 184 for PC-DHA/DHA. A chromatogram for this
`
`analysis is shown in Figure 10.
`
`
`- 53 -
`
`0000053
`
`
`
`
`
`69. Figures 11-13 below reflect the result of my analysis of the Fujita
`
`once-through extract, Sample No. vB O. As shown in the Figures, each of the
`
`following three Claimed Phospholipid species was detected in the extract: PC-
`
`DHA/DHA, PC-EPA/EPA, and PC-EPA/DHA. Using UHPLC-MS on the high
`
`resolution IT-TOF mass spectrometer (Figure 11), PC-EPA/EPA eluted first at
`
`approximately 5.2 minutes and was measured at m/z 826.5338 (Figure 12). Since
`
`the theoretical mass of PC-EPA/EPA is 826.5386 (Δm = -6 ppm), the elemental
`
`composition of this phospholipid in the Fujita once-through extract was determined
`
`to be identical to that of PC-EPA/EPA. PC-EPA/DHA eluted next from the
`
`UHPLC-MS system at a retention time of approximately 5.4 minutes (Figure 11)
`
`and was measured at m/z 852.5493 (Figure 12). Since the theoretical mass of PC-
`
`EPA/DHA is 852.5543 (Δm = -6 ppm), the elemental composition of this
`
`phospholipid in the Fujita hexane extract was determined to be identical to that of
`
`PC-EPA/DHA. PC-DHA/DHA eluted at a retention time of approximately 5.6 min
`
`(Figure 11) and was measured at m/z 878.5668 (Figure 12). Since the theoretical
`
`mass of PC-DHA/DHA is 878.5699 (Δm = -3 ppm), the elemental composition
`
`was determined to be identical to that of PC-DHA/DHA. Furthermore, the Fujita
`
`once-through extract was spiked with a PC-DHA/DHA standard and reanalyzed
`
`using UHPLC-MS/MS (Figure 11). The standard coeluted with the phospholipid in
`
`
`- 54 -
`
`0000054
`
`
`
`the extract thereby identifying this phospholipid in the Fujita once-through extract
`
`
`
`as PC-DHA/DHA.
`
`(x10,000,000)
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`IT-TOF MS
`Fujita Once-through
`Extraction
`
`13785683
`
`1.0
`1.5
`(x10,000,000)
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Spiked with PC-DHA/DHA
`standard
`
`14698433
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`5.5
`5.0
`4.5
`Retention time (min)
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 11. Positive ion electrospray high resolution IT TOF UHPLC-
`
`MS computer-reconstructed mass chromatograms of the Fujita once-
`
`through extract showing the detection of peaks corresponding to PC-
`
`EPA/EPA (m/z 826.53), PC-DHA/EPA (m/z 852.55) and PC-
`
`DHA/DHA (m/z 878.56) eluting at approximately 5.2, 5.4 and 5.6
`
`minutes, respectively (top). The extract was spiked with a PC-
`
`DHA/DHA standard and then reanalyzed (bottom). Note that the area
`
`- 55 -
`
`0000055
`
`
`
`
`
`of the peak corresponding to PC-DHA/DHA increased confirming the
`
`identity of PC-DHA/DHA in the extract.
`
`Inten. (x10,000,000)
`
`826.5338(1)
`
`5.0
`
`2.5
`
`IT-TOF MS
`Fujita Once-Through
`Extraction
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`852.5493(1)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`878.5668(1)
`
`0.0
`800
`810
`Inten. (x10,000,000)
`
`1.5
`
`1.0
`
`0.5
`
`802.5392(1)
`
`0.0
`800
`810
`Inten. (x1,000,000)
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`804.5508(1)
`
`0.0
`800
`
`810
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`
`
`854.5670(1)
`
`Figure 12. High resolution accurate mass measurements of the peaks
`
`corresponding to PC-EPA/EPA (top) eluting at a retention time of
`
`approximately 5.2 min (Figure 11), PC-EPA/DHA (middle) eluting at
`
`a retention time of approximately 5.4 min (Figure 11), and PC-
`
`DHA/DHA (bottom) eluting at a retention time of approximately 5.6
`
`min (Figure 11).
`
`
`- 56 -
`
`0000056
`
`
`
`
`
`—— : 878.50>184.10(+) PC-DHA/DHA
`······· : 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ : 852.60>184.10(+) PC-DHA/EPA
`
`UHPLC-MS-MS triple quadrupole
`Fujita Once-Through Extraction
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`
`
`700000
`
`600000
`
`500000
`
`400000
`
`300000
`
`200000
`
`100000
`
`0
`1.0
`
`Figure 13. Positive ion electrospray UHPLC-MS/MS analysis of the
`
`Claimed Phospholipids in the Fujita once-through extract. A triple
`
`quadrupole mass spectrometer was used with collision-induced dissociation
`
`and selected reaction monitoring (SRM) of the transitions indicated.
`
`
`
`70. Further analysis of the Claimed Phospholipids in the Fujita once-
`
`through extract was carried out using positive ion electrospray UHPLC-MS/MS
`
`with collision-induced dissociation and selected reaction monitoring (SRM) on a
`
`triple quadrupole tandem mass spectrometer. The SRM transitions that were used
`
`for analysis were m/z 826 to m/z 184 for PC-EPA/EPA, m/z 852 to m/z 184 for PC-
`
`EPA/DHA, and m/z 878 to m/z 184 for PC-DHA/DHA. A chromatogram for this
`
`analysis is shown in Figure 13.
`
`
`- 57 -
`
`0000057
`
`
`
`
`
`71. Figures 14-16 below reflect the result of my analysis of the Rogozhin
`
`extract, Sample No. vB Canada. As shown in the Figures, each of the following
`
`three Claimed Phospholipid species were detected in the extract: PC-DHA/DHA,
`
`PC-EPA/EPA, and PC-EPA/DHA. Using UHPLC-MS on the high resolution IT-
`
`TOF mass spectrometer (Figure 14), PC-EPA/EPA eluted first at approximately
`
`5.2 minutes and was measured at m/z 826.5342 (Figure 15). Since the theoretical
`
`mass of PC-EPA/EPA is 826.5386 (Δm = -5 ppm), the elemental composition of
`
`this phospholipid in the Rogozhin extract was determined to be identical to that of
`
`PC-EPA/EPA. PC-EPA/DHA eluted next from the UHPLC-MS system at a
`
`retention time of approximately 5.4 minutes (Figure 14) and was measured at m/z
`
`852.5495 (Figure 15). Since the theoretical mass of PC-EPA/DHA is 852.5543
`
`(Δm = -6 ppm), the elemental composition of this phospholipid in the Rogozhin
`
`extract was determined to be identical to that of PC-EPA/DHA. PC-DHA/DHA
`
`eluted at a retention time of approximately 5.6 min (Figure 14) and was measured
`
`at m/z 878.5666 (Figure 15). Since the theoretical mass of PC-DHA/DHA is
`
`878.5699 (Δm = -4 ppm), the elemental composition was determined to be
`
`identical to that of PC-DHA/DHA. Furthermore, the Rogozhin extract was spiked
`
`with a PC-DHA/DHA standard and reanalyzed using UHPLC-MS/MS (Figure 14).
`
`The standard coeluted with the phospholipid in the extract thereby identifying this
`
`phospholipid in the Rogozhin extract as PC-DHA/DHA.
`
`
`- 58 -
`
`0000058
`
`
`
`
`
`
`
`(x10,000,000)
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`IT-TOF MS
`Rogozhin (Canada)
`Extraction
`
`13099287
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`1.0
`1.5
`(x10,000,000)
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`Spiked with PC-DHA/DHA
`standard
`
`15159837
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.5
`4.0
`5.5
`5.0
`Retention time (min)
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 14. Positive ion electrospray high resolution IT-TOF UHPLC-
`
`MS computer-reconstructed mass chromatograms of the Rogozhin
`
`extract showing the detection of peaks corresponding to PC-EPA/EPA
`
`(m/z 826.53), PC-DHA/EPA (m/z 852.55) and PC-DHA/DHA (m/z
`
`878.56) eluting at approximately 5.2, 5.4 and 5.6 minutes,
`
`respectively (top). The extract was spiked with a PC-DHA/DHA
`
`standard and then reanalyzed (bottom). Note that the area of the peak
`
`
`- 59 -
`
`0000059
`
`
`
`
`
`corresponding to PC-DHA/DHA increased confirming the identity of
`
`PC-DHA/DHA in the extract.
`
`Inten. (x10,000,000)
`
`826.5342(1)
`
`IT-TOF MS
`Rogozhin Extract
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`852.5495(1)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`878.5666(1)
`
`5.0
`
`2.5
`
`0.0
`810
`800
`Inten. (x10,000,000)
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`810
`800
`Inten. (x1,000,000)
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`804.5502(1)
`
`0.0
`800
`
`810
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`
`
`854.5670(1)
`
`Figure 15. High resolution accurate mass measurements of the peaks
`
`corresponding to PC-EPA/EPA (top) eluting at a retention time of
`
`approximately 5.2 min (Figure 14), PC-EPA/DHA (middle) eluting at
`
`a retention time of approximately 5.4 min (Figure 14), and PC-
`
`DHA/DHA (bottom) eluting at a retention time of approximately 5.6
`
`min (Figure 14).
`
`
`- 60 -
`
`0000060
`
`
`
`
`
`—— 878.50>184.10(+) PC-DHA/DHA
`······· 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ 852.60>184.10(+) PC-DHA/EPA
`
`UHPLC-MS-MS triple quadrupole
`Rogozhin
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`
`
`700000
`
`600000
`
`500000
`
`400000
`
`300000
`
`200000
`
`100000
`
`0
`1.0
`
`Figure 16. Positive ion electrospray UHPLC-MS/MS analysis of the
`
`Claimed Phospholipids in the Rogozhin extract. A triple quadrupole
`
`mass spectrometer was used with collision-induced dissociation and
`
`selected reaction monitoring (SRM) of the transitions indicated.
`
`72. Further analysis of the Claimed Phospholipids in the Rogozhin extract
`
`was carried out using positive ion electrospray UHPLC-MS/MS with collision-
`
`induced dissociation and selected reaction monitoring (SRM) on a triple
`
`quadrupole tandem mass spectrometer. The SRM transitions that were used for
`
`analysis were m/z 826 to m/z 184 for PC-EPA/EPA, m/z 852 to m/z 184 for PC-
`
`EPA/DHA, and m/z 878 to m/z 184 for PC-DHA/DHA. A chromatogram for this
`
`analysis is shown in Figure 16.
`
`
`- 61 -
`
`0000061
`
`
`
`
`
`73. Figures 17-19 below reflect the result of my analysis of “Beaudoin
`
`Extract P0” received from Dr. Budge’s laboratory. As shown in the Figures, each
`
`of the following three Claimed Phospholipid species was detected in the extract:
`
`PC-DHA/DHA, PC-EPA/EPA, and PC-EPA/DHA. Using UHPLC-MS on the high
`
`resolution IT-TOF mass spectrometer (Figure 17), PC-EPA/EPA eluted first at
`
`approximately 5.2 minutes and was measured at m/z 826.5351 (Figure 18). Since
`
`the theoretical mass of PC-EPA/EPA is 826.5386 (Δm = -4 ppm), the elemental
`
`composition of this phospholipid in the Beaudoin extract P0 was determined to be
`
`identical to that of PC-EPA/EPA. PC-EPA/DHA eluted next from the UHPLC-MS
`
`system at a retention time of approximately 5.4 minutes (Figure 17) and was
`
`measured at m/z 852.5518 (Figure 18). Since the theoretical mass of PC-EPA/DHA
`
`is 852.5543 (Δm = -3 ppm), the elemental composition of this phospholipid in the
`
`Beaudoin extract P0 was determined to be identical to that of PC-EPA/DHA. PC-
`
`DHA/DHA eluted at a retention time of approximately 5.6 min (Figure 17) and
`
`was measured at m/z 878.5672 (Figure 18). Since the theoretical mass of PC-
`
`DHA/DHA is 878.5699 (Δm = -3 ppm), the elemental composition was determined
`
`to be identical to that of PC-DHA/DHA. Furthermore, the Beaudoin extract P0 was
`
`spiked with a PC-DHA/DHA standard and reanalyzed using UHPLC-MS/MS
`
`(Figure 17). The standard coeluted with the phospholipid in the extract thereby
`
`identifying this phospholipid in the Beaudoin P0 extract as PC-DHA/DHA.
`
`
`- 62 -
`
`0000062
`
`
`
`
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`3.5
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`
`
`(x10,000,000)
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`IT-TOF MS
`BEA P0
`
`12166126
`
`1.5
`1.0
`(x10,000,000)
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Spiked with PC-DHA/DHA
`standard
`
`18234615
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`5.5
`5.0
`4.5
`Retention time (min)
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 17. Positive ion electrospray high resolution IT-TOF UHPLC-
`
`MS computer-reconstructed mass chromatograms of the Beaudoin
`
`extract P0 showing the detection of peaks corresponding to PC-
`
`EPA/EPA (m/z 826.53), PC-DHA/EPA (m/z 852.55) and PC-
`
`DHA/DHA (m/z 878.56) eluting at approximately 5.2, 5.4 and 5.6
`
`minutes, respectively (top). The extract was spiked with a PC-
`
`DHA/DHA standard and then reanalyzed (bottom). Note that the area
`
`
`- 63 -
`
`0000063
`
`
`
`
`
`of the peak corresponding to PC-DHA/DHA increased confirming the
`
`identity of PC-DHA/DHA in the extract.
`
`Inten. (x10,000,000)
`
`826.5351(1)
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`IT-TOF MS
`Beaudoin Extract P0
`
`0.0
`810
`800
`Inten. (x10,000,000)
`
`2.0
`
`1.0
`
`0.0
`810
`800
`Inten. (x1,000,000)
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`804.5538(1)
`
`0.0
`800
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`852.5518(1)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`896.7609(1)
`
`878.5672(1)
`
`828.5514(1)
`
`854.5682(1)
`
`872.5994(1)
`866.5672(1)
`
`894.5969(1)
`
`896.7653(1)
`
`810
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`
`
`Figure 18. High resolution accurate mass measurements of the peaks
`
`corresponding to PC-EPA/EPA (top) eluting at a retention time of
`
`approximately 5.2 min (Figure 17), PC-EPA/DHA (middle) eluting at
`
`a retention time of approximately 5.4 min (Figure 17), and PC-
`
`DHA/DHA (bottom) eluting at a retention time of approximately 5.6
`
`min (Figure 17).
`
`
`- 64 -
`
`0000064
`
`
`
`
`
`—— : 878.50>184.10(+) PC-DHA/DHA
`······· : 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ : 852.60>184.10(+) PC-DHA/EPA
`
`UHPLC-MS-MS triple quadrupole
`Beaudoin Extract P0
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`
`
`200000
`
`175000
`
`150000
`
`125000
`
`100000
`
`75000
`
`50000
`
`25000
`
`0
`1.0
`
`Figure 19. Positive ion electrospray UHPLC-MS/MS analysis of the
`
`Claimed Phospholipids in the Beaudoin extract P0. A triple quadrupole mass
`
`spectrometer was used with collision-induced dissociation and selected
`
`reaction monitoring (SRM) of the transitions indicated.
`
`
`
`74. Further analysis of the Claimed Phospholipids in the Beaudoin extract
`
`P0 was carried out using positive ion electrospray UHPLC-MS/MS with collision-
`
`induced dissocation and selected reaction monitoring (SRM) on a triple quadrupole
`
`tandem mass spectrometer. The SRM transitions that were used for analysis were
`
`m/z 826 to m/z 184 for PC-EPA/EPA, m/z 852 to m/z 184 for PC-EPA/DHA, and
`
`m/z 878 to m/z 184 for PC-DHA/DHA. A chromatogram for this analysis is shown
`
`in Figure 19.
`
`
`- 65 -
`
`0000065
`
`
`
`
`
`75. Figures 20-22 below reflect the result of my analysis of “Beaudoin
`
`Extract P1” received from Dr. Budge’s laboratory. As shown in the Figures, each
`
`of the following three Claimed Phospholipid species was detected in the extract:
`
`PC-DHA/DHA, PC-EPA/EPA, and PC-EPA/DHA. Using UHPLC-MS on the high
`
`resolution IT-TOF mass spectrometer (Figure 20), PC-EPA/EPA eluted first at
`
`approximately 5.2 minutes and was measured at m/z 826.5335 (Figure 21). Since
`
`the theoretical mass of PC-EPA/EPA is 826.5386 (Δm = -6 ppm), the elemental
`
`composition of this phospholipid in the Beaudoin extract P1 was determined to be
`
`identical to that of PC-EPA/EPA. PC-EPA/DHA eluted next from the UHPLC-MS
`
`system at a retention time of approximately 5.4 minutes (Figure 20) and was
`
`measured at m/z 852.5491 (Figure 21). Since the theoretical mass of PC-EPA/DHA
`
`is 852.5543 (Δm = -6 ppm), the elemental composition of this phospholipid in the
`
`Beaudoin extract P1 was determined to be identical to that of PC-EPA/DHA. PC-
`
`DHA/DHA eluted at a retention time of approximately 5.6 min (Figure 20) and
`
`was measured at m/z 878.5665 (Figure 21). Since the theoretical mass of PC-
`
`DHA/DHA is 878.5699 (Δm = -4 ppm), the elemental composition was determined
`
`to be identical to that of PC-DHA/DHA. Furthermore, the Beaudoin extract P1 was
`
`spiked with a PC-DHA/DHA standard and reanalyzed using UHPLC-MS/MS
`
`(Figure 20). The standard coeluted with the phospholipid in the extract thereby
`
`identifying this phospholipid in the Beaudoin extract P1 as PC-DHA/DHA.
`
`
`- 66 -
`
`0000066
`
`
`
`(x10,000,000)
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`
`
`IT-TOF MS
`BEA P1
`
`18480256
`
`1.0
`1.5
`(x10,000,000)
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Spiked with PC-DHA/DHA
`standard
`
`20435308
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`5.5
`5.0
`4.5
`Retention time (min)
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 20. Positive ion electrospray high resolution IT-TOF UHPLC-
`
`MS computer-reconstructed mass chromatograms of the Beaudoin
`
`extract P1 showing the detection of peaks corresponding to PC-
`
`EPA/EPA (m/z 826.53), PC-DHA/EPA (m/z 852.55) and PC-
`
`DHA/DHA (m/z 878.56) eluting at approximately 5.2, 5.4 and 5.6
`
`minutes, respectively (top). The extract was spiked with a PC-
`
`DHA/DHA standard and then reanalyzed (bottom). Note that the area
`
`- 67 -
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`
`
`0000067
`
`
`
`
`
`of the peak corresponding to PC-DHA/DHA increased confirming the
`
`identity of PC-DHA/DHA in the extract.
`
`Inten. (x10,000,000)
`
`826.5335(1)
`
`7.5
`
`5.0
`
`2.5
`
`IT-TOF MS
`Beaudoin Extract P1
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`852.5512(1)
`
`840.5524(1)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`878.5661(1)
`
`0.0
`810
`800
`Inten. (x10,000,000)
`
`3.0
`
`2.0
`
`1.0
`
`810.5127(1)
`
`0.0
`810
`800
`Inten. (x1,000,000)
`
`5.0
`
`2.5
`
`804.5529(1)
`
`854.5684(1)
`
`872.6000(1)
`866.5718(1)
`
`894.5951(1)
`
`810
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`
`
`0.0
`800
`
`Figure 21. High resolution accurate mass measurements of the peaks
`
`corresponding to PC-EPA/EPA (top) eluting at a retention time of
`
`approximately 5.2 min (Figure 20), PC-EPA/DHA (middle) eluting at
`
`a retention time of approximately 5.4 min (Figure 20), and PC-
`
`DHA/DHA (bottom) eluting at a retention time of approximately 5.6
`
`min (Figure 20).
`
`
`- 68 -
`
`0000068
`
`
`
`
`
`—— :878.50>184.10(+) PC-DHA/DHA
`······· :826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ : 852.60>184.10(+) PC-DHA/EPA
`
`UHPLC-MS-MS triple quadrupole
`Beaudoin Extract P1
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`
`
`750000
`
`500000
`
`250000
`
`0
`1.0
`
`Figure 22. Positive ion electrospray UHPLC-MS/MS analysis of the
`
`Claimed Phospholipids in the Beaudoin extract P1. A triple quadrupole mass
`
`spectrometer was used with collision-induced dissociation and selected
`
`reaction monitoring (SRM) of the transitions indicated.
`
`
`
`76. Further analysis of the Claimed Phospholipids in the Beaudoin extract
`
`P1 was carried out using positive ion electrospray UHPLC-MS/MS with collision-
`
`induced dissocation and selected reaction monitoring (SRM) on a triple quadrupole
`
`tandem mass spectrometer. The SRM transitions that were used for analysis were
`
`m/z 826 to m/z 184 for PC-EPA/EPA, m/z 852 to m/z 184 for PC-EPA/DHA, and
`
`m/z 878 to m/z 184 for PC-DHA/DHA. A chromatogram for this analysis is shown
`
`in Figure 22.
`
`
`- 69 -
`
`0000069
`
`
`
`
`
`77. Figures 23-25 below reflect the result of my analysis of “Beaudoin
`
`Extract P2” received from Dr. Budge’s laboratory. As shown in the Figures, each
`
`of the following three Claimed Phospholipid species was detected in the extract:
`
`PC-DHA/DHA, PC-EPA/EPA, and PC-EPA/DHA. Using UHPLC-MS on the high
`
`resolution IT-TOF mass spectrometer (Figure 23), PC-EPA/EPA eluted first at
`
`approximately 5.2 minutes and was measured at m/z 826.5343 (Figure 24). Since
`
`the theoretical mass of PC-EPA/EPA is 826.5335 (Δm = 1 ppm), the elemental
`
`composition of this phospholipid in the Beaudoin extract P2 was determined to be
`
`identical to that of PC-EPA/EPA. PC-EPA/DHA eluted next from the UHPLC-MS
`
`system at a retention time of approximately 5.4 minutes (Figure 23) and was
`
`measured at m/z 852.5509 (Figure 24). Since the theoretical mass of PC-EPA/DHA
`
`is 852.5543 (Δm = -4 ppm), the elemental composition of this phospholipid in the
`
`Beaudoin extract P2 was determined to be identical to that of PC-EPA/DHA. PC-
`
`DHA/DHA eluted at a retention time of approximately 5.6 min (Figure 23) and
`
`was measured at m/z 878.5661 (Figure 24). Since the theoretical mass of PC-
`
`DHA/DHA is 878.5699 (Δm = -4 ppm), the elemental composition was determined
`
`to be identical to that of PC-DHA/DHA. Furthermore, the Beaudoin extract P2 was
`
`spiked with a PC-DHA/DHA standard and reanalyzed using UHPLC-MS/MS
`
`(Figure 23). The standard coeluted with the phospholipid in the extract thereby
`
`identifying this phospholipid in the Beaudoin extract P2 as PC-DHA/DHA.
`
`
`- 70 -
`
`0000070
`
`
`
`(x10,000,000)
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`
`
`IT-TOF MS
`BEA P2
`
`18173226
`
`1.0
`1.5
`(x10,000,000)
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`······ 826.53 (PC-EPA/EPA)
`- - - - 852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Spiked with PC-DHA/DHA
`standard
`
`19006919
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`5.5
`5.0
`4.5
`Retention time (min)
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 23. Positive ion electrospray high resolution IT-TOF UHPLC-
`
`MS computer-reconstructed mass chromatograms of the Beaudoin
`
`extract P2 showing the detection of peaks corresponding to PC-
`
`EPA/EPA (m/z 826.53), PC-DHA/EPA (m/z 852.55) and PC-
`
`DHA/DHA (m/z 878.56) eluting at approximately 5.2, 5.4 and 5.6
`
`minutes, respectively (top). The extract was spiked with a PC-
`
`DHA/DHA standard and then reanalyzed (bottom). Note that the area
`
`
`- 71 -
`
`0000071
`
`
`
`
`
`of the peak corresponding to PC-DHA/DHA increased confirming
`
`the identity of PC-DHA/DHA in the extract.
`
`Inten. (x10,000,000)
`
`826.5335(1)
`
`IT-TOF MS
`Beaudoin Extract P2
`
`5.0
`
`2.5
`
`0.0
`810
`800
`Inten. (x10,000,000)
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`810.5097(1)
`
`0.0
`810
`800
`Inten. (x1,000,000)
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`804.5518(1)
`
`0.0
`800
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`852.5509(1)
`
`840.5523(1)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`878.5661(1)
`
`836.5279(1)
`
`866.5720(1)
`
`854.5672(1)
`
`894.5955(1)
`
`810
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`
`
`Figure 24. High resolution accurate mass measurements of the peaks
`
`corresponding to PC-EPA/EPA (top) eluting at a retention time of
`
`approximately 5.2 min (Figure 23), PC-EPA/DHA (middle) eluting at
`
`a retention time of approximately 5.4 min (Figure 23), and PC-
`
`DHA/DHA (bottom) eluting at a retention time of approximately 5.6
`
`min (Figure 23).
`
`
`- 72 -
`
`0000072
`
`
`
`
`
`—— : 878.50>184.10(+) PC-DHA/DHA
`······· : 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ : 852.60>184.10(+) PC-DHA/EPA
`
`UHPLC-MS-MS triple quadrupole
`Beaudoin Extract P2
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`
`
`800000
`
`700000
`
`600000
`
`500000
`
`400000
`
`300000
`
`200000
`
`100000
`
`0
`1.0
`
`Figure 25. Positive ion electrospray UHPLC-MS/MS analysis of the
`
`Claimed Phospholipids in the Beaudoin extract P2. A triple quadrupole mass
`
`spectrometer was used with collision-induced dissociation and selected
`
`reaction monitoring (SRM) of the transitions indicated.
`
`
`
`78. Further analysis of the Claimed Phospholipids in the Beaudoin extract
`
`P2 was carried out using positive ion electrospray UHPLC-MS/MS with collision-
`
`induced dissociation and selected reaction monitoring (SRM) on a triple
`
`quadrupole tandem mass spectrometer. The SRM transitions that were used for
`
`analysis were m/z 826 to m/z 184 for PC-EPA/EPA, m/z 852 to m/z 184 for PC-
`
`EPA/DHA, and m/z 878 to m/z 184 for PC-DHA/DHA. A chromatogram for this
`
`analysis is shown in Figure 25.
`
`
`- 73 -
`
`0000073
`
`
`
`
`
`79. Figures 26-28 below reflect the result of my analysis of “Beaudoin
`
`Extract SU0” received from Dr. Budge’s laboratory. As shown in the Figures,
`
`each of the following three Claimed Phospholipid species was detected in the
`
`extract: PC-DHA/DHA, PC-EPA/EPA, and PC-EPA/DHA. Using UHPLC-MS on
`
`the high resolution IT-TOF mass spectrometer (Figure 26), PC-EPA/EPA eluted
`
`first at approximately 3.9 minutes and was measured at m/z 826.5415 (Figure 27).
`
`Since the theoretical mass of PC-EPA/EPA is 826.5386 (Δm = 3 ppm), the
`
`elemental composition of this phospholipid in the Beaudoin extract SU0 was
`
`determined to be identical to that of PC-EPA/EPA. PC-EPA/DHA eluted next from
`
`the UHPLC-MS system at a retention time of approximately 4.1 minutes (Figure
`
`26) and was measured at m/z 852.5540 (Figure 27). Since the theoretical mass of
`
`PC-EPA/DHA is 852.5543 (Δm = 0 ppm), the elemental composition of this
`
`phospholipid in the Fujita hexane extract was determined to be identical to that of
`
`PC-EPA/DHA. PC-DHA/DHA eluted at a retention time of approximately 4.5 min
`
`(Figure 26) and was measured at m/z 878.5701 (Figure 27). Since the theoretical
`
`mass of PC-DHA/DHA is 878.5699 (Δm = 0 ppm), the elemental composition was
`
`determined to be identical to that of PC-DHA/DHA. Furthermore, the Beaudoin
`
`extract SU0 was spiked with a PC-DHA/DHA standard and reanalyzed using
`
`UHPLC-MS/MS (Figure 28). The standard coeluted with the phospholipid in the
`
`
`- 74 -
`
`0000074
`
`
`
`
`
`extract thereby identifying this phospholipid in the Beaudoin extract SU0 as PC-
`
`DHA/DHA.
`
`(x10,000,000)
`
`······ 826.53 (PC-EPA/EPA)
`- - - -852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Bea SU0
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 26. Positive ion electrospray high resolution IT TOF UHPLC-
`
`MS computer-reconstructed mass chromatograms of the Beaudoin
`
`extract SU0 showing the detection of peaks corresponding to PC-
`
`EPA/EPA (m/z 826.53), PC-DHA/EPA (m/z 852.55) and PC-
`
`DHA/DHA (m/z 878.56) eluting at approximately 3.9, 4.1 and 4.5
`
`minutes, respectively.
`
`
`
`1.00
`
`0.75
`
`0.50
`
`0.25
`
`0.00
`
`
`- 75 -
`
`0000075
`
`
`
`
`
`
`
`1.0
`
`0.5
`
`Inten. (x10,000,000)
`
`826.5415(1)
`
`848.5199(1)
`
`Bea SU0
`
`800.5249(1)
`806.5052(1)
`
`822.5057(1)
`
`0.0
`810
`800
`Inten. (x1,000,000)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`802.5430(1)
`
`810.5426(1)
`815.4728(1)
`
`852.5540(1)
`
`874.5348(1)
`
`Bea SU0
`
`826.5432(1)
`
`832.5230(1)
`
`848.5213(1)
`
`0.0
`810
`800
`Inten. (x1,000,000)
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`800
`
`800.5213(1)
`
`Bea SU0
`
`878.5701(1)
`
`810.5352(1)
`
`826.5369(1)
`
`838.5703(1)
`
`852.5521(1)
`
`874.5360(1)
`
`894.5967(1)
`
`810
`
`820
`
`830
`
`840
`
`850
`
`860
`
`870
`
`880
`
`890
`
`m/z
`
`
`
`Figure 27. High resolution accurate mass measurements of the peaks
`
`corresponding to PC-EPA/EPA (top) eluting at a retention time of
`
`approximately 3.9 min (Figure 26), PC-EPA/DHA (middle) eluting at
`
`a retention time of approximately 4.1 min (Figure 26), and PC-
`
`DHA/DHA (bottom) eluting at a retention time of approximately 4.5
`
`min (Figure 26).
`
`
`- 76 -
`
`0000076
`
`
`
`
`
`—— 878.50>184.10(+) PC-DHA/DHA
`······· 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ 852.60>184.10(+) PC-DHA/EPA
`
`UHPLC-MS-MS triple quadrupole
`BEA SU0
`
`4606931
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`—— 878.50>184.10(+) PC-DHA/DHA
`······· 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ 852.60>184.10(+) PC-DHA/EPA
`
`Spiked with PC-DHA/DHA
`standard
`
`5284835
`
`1000000
`
`750000
`
`500000
`
`250000
`
`0
`1.0
`
`1000000
`
`750000
`
`500000
`
`250000
`
`0
`1.0
`2.0
`3.0
`4.0
`5.0
`6.0
`7.0
`FIGURE 28. Positive ion electrospray UHPLC-MS/MS of Beaudoin extract
`
`min
`
`SU0 before and after spiking with a PC-DHA/DHA standard. Note that the
`
`area of the peak corresponding to PC-DHA/DHA increased confirming the
`
`identity of PC-DHA/DHA in the extract.
`
`80. Further analysis of the Claimed Phospholipids in the Beaudoin extract
`
`SU0 was carried out using positive ion electrospray UHPLC-MS/MS with
`
`collision-induced dissociation and selected reaction monitoring (SRM) on a triple
`
`quadrupole tandem mass spectrometer. The SRM transitions that were used for
`
`analysis were m/z 826 to m/z 184 for PC-EPA/EPA, m/z 852 to m/z 184 for PC-
`
`EPA/DHA, and m/z 878 to m/z 184 for PC-DHA/DHA. A chromatogram for this
`
`analysis is shown in Figure 29.
`
`
`
`
`- 77 -
`
`0000077
`
`
`
`
`
`Bea SU0
`
`—— 878.50>184.10(+) PC-DHA/DHA
`······· 826.40>184.05(+) PC-EPA/EPA
`
`̶̶̶̶ ̶̶̶̶ ̶̶̶̶ 852.60>184.10(+) PC-DHA/EPA
`
`2.0
`
`3.0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`min
`
`
`
`2500000
`
`2000000
`
`1500000
`
`1000000
`
`500000
`
`0
`1.0
`
`Figure 29. Positive ion electrospray UHPLC-MS/MS analysis of the
`
`Claimed Phospholipids in the Beaudoin extract SU0. A triple quadrupole
`
`mass spectrometer was used with collision-induced dissociation and selected
`
`reaction monitoring (SRM) of the transitions indicated.
`
`
`
`81. Figures 30-33 below reflect the result of my analysis of “Beaudoin
`
`Extract SU1” received from Dr. Budge’s laboratory. As shown in the Figures,
`
`each of the following three Claimed Phospholipid species was detected in the
`
`extract: PC-DHA/DHA, PC-EPA/EPA, and PC-EPA/DHA. Using UHPLC-MS on
`
`the high resolution IT-TOF mass spectrometer (Figure 30), PC-EPA/EPA eluted
`
`first at approximately 3.9 minutes and was measured at m/z 826.5430 (Figure 31).
`
`Since the theoretical mass of PC-EPA/EPA is 826.5386 (Δm = 5 ppm), the
`
`elemental composition of this phospholipid in the Beaudoin extract SU1was
`
`
`- 78 -
`
`0000078
`
`
`
`
`
`determined to be identical to that of PC-EPA/EPA. PC-EPA/DHA eluted next from
`
`the UHPLC-MS system at a retention time of approximately 4.1 minutes (Figure
`
`30) and was measured at m/z 852.5540 (Figure 31). Since the theoretical mass of
`
`PC-EPA/DHA is 852.5543 (Δm = 0 ppm), the elemental composition of this
`
`phospholipid in the Beaudoin extract SU1was determined to be identical to that of
`
`PC-EPA/DHA. PC-DHA/DHA eluted at a retention time of approximately 4.4 min
`
`(Figure 30) and was measured at m/z 878.5685 (Figure 31). Since the theoretical
`
`mass of PC-DHA/DHA is 878.5699 (Δm = -1 ppm), the elemental composition
`
`was determined to be identical to that of PC-DHA/DHA. Furthermore, the
`
`Beaudoin extract SU1was spiked with a PC-DHA/DHA standard and reanalyzed
`
`using UHPLC-MS/MS (Figure 32). The standard coeluted with the phospholipid in
`
`the extract thereby identifying this phospholipid in the Beaudoin SU1 extract as
`
`PC-DHA/DHA.
`
`(x1,000,000)
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`1.0
`
`······ 826.53 (PC-EPA/EPA)
`- - - -852.55 (PC-DHA/EPA)
`—— 878.56 (PC-DHA/DHA)
`
`Bea SU1
`
`1.5
`
`2.0
`
`2.5
`
`3.0
`
`3.5
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`6.0
`
`6.5
`
`7.0
`
`7.5
`
`8.0
`
`8.5
`
`
`
`Figure 30. Positive ion electrospray hig
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