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`Final Report
`
`Identification and Quantification of
`
`Phospholipids in Beaudoin Oil
`
`No. Lab: O9-1651
`
`Sponsored by
`
`Neptune Technologies and
`
`Bioressources
`
`December 22, 2009
`
`By:
`
`Earl L. White, Ph.D.
`
`President & CEO
`
`Kenyon Hood, B.S.
`Chemist
`
`Contains Confidential Business Information
`
`NEP877ITC-00267599
`
`Subject to the Protective Order
`
`1
`
`1
`
`AKBM 1098
`
`AKBM 1098
`
`

`
`Introduction
`
`A sample labeled Beaudoin Oil No. Lab: 09-1651 was submitted in a brown opaque 250 mL
`
`bottle to MDx BioAna|ytica| Laboratory for LC/MS and LC—MS/MS analysis to determine the
`
`identify of three classes of phospholipids (PL) present and to obtain relative concentrations of
`
`each lipid with the hope of obtaining quantitative information about phospholipids containing
`
`eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the sn—1 and sn—2 positions
`
`of
`
`the glycerol backbone.
`
`The PLs
`
`of
`
`interest
`
`included phosphatidylcholine
`
`(PC),
`
`phosphophatidylethanolamine (PE) and phosphatidylinositol
`
`(Pl). These PL classes were
`
`believed to be present at 85%, 7% and 8% for PC, PE and PL, respectively.
`
`Method
`
`The Beaudoin Oil sample No. Lab: 09-1651 was analyzed by LC/MS.
`
`Initially the sample was
`
`dissolved in ethyl acetate (~1mg/mL). A white precipitate was observed in the bottom of the
`
`flask. The supernatant was removed and the precipitate dried under a stream of nitrogen and
`
`found to be water soluble.
`
`The ethyl acetate solution was evaporated to dryness and
`
`reconstituted to a concentration of 100 ng/uL in 20% ACN with 0.1% formic acid (FA) and
`
`subjected to LC/MS. Subsequently, chloroform was used to dissolve the oil sample. A 5 uL
`
`aliquot of this sample was injected onto a C18 column and separated on a Waters MicroMass
`
`CapLC and detected with a Waters MicroMass Q—TOF Micro quadrupole time—of—f|ight mass
`
`spectrometer with nano electrospray ionization (ESI).
`
`The instrument was operated in the full
`
`scan positive ion mode for these samples. Continuum mass spectra were acquired over the
`
`mass range of 400-2000 amu with a scan time of 1.80 s and an inter—scan delay of 0.1 s. The
`
`capillary, sample cone and extraction cone voltage were 3654, 30 and 2.5 V, respectively. The
`
`source temperature was held at 120°C, the cone N2 gas flow was 50 L/h and the collision
`
`energy was held constant at 10 V. The HPLC column was a CVC Micro-Tech C18WS—150EU
`
`nano column. HPLC/MS procedure; mobile phase A (0.1% FA H2O:ACN, 95:5%) was held at
`
`20% B (ACN: 0.1% FA in H20, 95:5%) for 4 min and then programmed to 70% B at 5 min, 80%
`
`at 15 min then to 100% B at 30 min and held for 36 min. The flow rate was 7 uL/min with a split
`
`ratio of about 6:1 yielding a flow through the column of approximately 600 nL/min. For MS/MS
`
`analysis, the Q—TOF was operated in the survey scan mode where data—dependent MS/MS
`
`analysis was perform on all ions detected above a pre—determined intensity threshold. Optimum
`
`collision energies for compound dissociation were determined.
`
`Results:
`
`There were approximately 45 PLs detected in the oil sample and approximately 16 with a
`
`relative percentage 21%. A list of phospholipids detected in the Beaudoin oil sample labeled
`
`No. Lab: 09-1651 is given in the table below with relative percentages based on area under the
`
`curve with the caveat that all PLs do not necessarily respond the same. Therefore, these
`
`values should be considered an approximate representation of the various PLs and classes of
`
`PLs only. Absolute quantification could be obtained by comparing these areas with those of an
`
`l
`
`2
`
`2
`
`Contains Confidential Business Information
`
`NEP877ITC-00267600
`
`Subject to the Protective Order
`
`

`
`authentic standard for each PL. Also, the sample contained major polyethylene glycol (PEG)
`
`contaminants which inhibited confirmation by MS/MS for several of the PLs. Other PLs are
`
`contained within these chromatograms at
`
`lower concentrations. This list
`
`is by no means
`
`exhaustive, but does contain most of the major PLs.
`
`Figure 1 shows a representative total ion chromatogram for a 5 uL injection of a 0.5 ug/uL oil
`
`sample dissolved in CHCI3. Peaks appearing in the first 12 minutes of the chromatogram were
`
`primarily low molecular weight PLs which contained essentially OH in the sn—1 or -2 position. A
`
`full scan summed mass spectrum for the time 9.5—12.5 min of the chromatogram is shown in
`
`Figure 2. Figure 3 shows the portion of the spectrum that contains the major low molecular
`
`weight PLs ranging from about 416 to 570 Da. The zoomed portion of the spectrum shown in
`
`Figure 4 depicts PEGs in the range of 600-900 Da. The zoomed region shown in Figure 5
`
`represents dimers of the low MW PLs. Dimers and trimers (Figure 6) are characteristic for PLs
`
`and are often used as diagnostic ions for confirmation. Representative full scan ESI specta for
`
`major peaks in the chromatogram are shown in Figures 7 through 18. Figures 8,10 and 13 are
`
`zooms of portions of spectra that contain PLs. Figures 7, 9, 11 and 12 all contain PEGs in the
`
`MW range of 600-1000 Da. Figure 11 has two different ranges of PEGs. Those in the 800 Da
`
`region caused some inteference with sample and data analysis. Two major PLs were detected
`
`in each of the peaks at 19.4 and 20.1 min as shown in Figures 14 and 15.
`
`Peak areas were calculated from the molecular ions for each of the major peaks shown in the
`
`spectra.
`
`Ion chromatograms for these m/z’s are shown in Figures 19 through 28. The bottom
`
`chromatogram for each figure represents the tota|—ion chromatogram for that region of the
`
`chromatogram. Areas from these chromatograms were entered into the table and used to
`
`calculate relative % for each peak representing the identified PL.
`
`Note that some
`
`chromatograms contain more than one integrated peak (Figures 22A, B & C, 25A, 26A & B,
`
`27C, and 28B). These peaks represent different PLs or even classes of PL with the same
`
`nominal molecular weight. Figure 29 is a representative MS/MS spectrum for a PL with a
`
`molecular ion at m/z 826.5. Fragment ions detected at m/z 524.3 and 542.3 are indicative of
`
`302 and 284 Da losses from the molecular ion which indicate loss of C20:5 and C18:0 fatty
`
`acids from the protonate molecular ions.
`
`There were
`
`no
`
`phosphatidylinositols detected in
`
`the
`
`oil
`
`sample,
`
`however
`
`several
`
`phosphatidylserines were detected.
`
`Contains Confidential Business Information
`
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`
`3
`
`3
`
`

`
`Table 1
`
`Phospholipids Detected in Beaudoin Oil Sample
`
`PL Class
`PC
`
`fl mg
`10.28
`454
`
`Phosgholipid
`C1320/OH
`
`C1822/OH
`
`C1620/OH
`
`C1420/OH
`
`C16:0/OH
`
`C1621/OH+No*
`
`C1821/OH
`
`C2025/OH
`
`C22:6/OH
`
`C1820/OH
`
`C1821/OH
`
`C20:2/OH
`
`C20:1/OH
`
`PE
`
`PC
`
`PC
`
`PC
`
`PC
`
`PC
`
`PC W/EPA
`
`PC w/DHA
`
`PC
`
`PE
`
`PS
`
`PC
`
`C2226/OH+N<:1*
`
`C22:6/OH
`Unideniified
`
`C1620/C22:2
`
`PC w/DHA
`
`PC w/DHA
`
`PC
`
`11.27
`
`10.73
`
`10.1
`
`11.3
`
`9.9
`
`11.7
`
`10.4
`
`1 1.0
`
`10.4
`
`12.7
`
`1 1.0
`
`13.1
`
`11.0
`
`14.7
`16.6
`
`18.3
`
`478
`
`480
`
`468
`
`496
`
`516
`
`522
`
`542
`
`568
`
`524
`
`524
`
`550
`
`550
`
`590
`
`578
`606
`
`800
`
`Area
`149
`
`127
`
`142
`
`642
`
`2535
`
`1318
`
`1013
`
`4406
`
`1650
`
`169
`
`122
`
`57
`
`77
`
`15
`
`208
`28
`
`204
`
`Relative%
`0.60
`
`0.51
`
`0.57
`
`2.58
`
`10.20
`
`5.31
`
`4.08
`
`17.74
`
`6.64
`
`0.68
`
`0.49
`
`0.23
`
`0.31
`
`0.06
`
`0.84
`0.11
`
`0.82
`
`0.07
`
`C2025/C12:0
`
`C2025/C18:3
`Unideniified
`
`PC W/EPA
`
`PC W/EPA
`
`17.9
`
`18.3
`22.7
`
`724
`
`802
`844
`
`18
`
`119
`10
`
`0.48
`0.04
`
`C20:4 C12:0
`PC
`18.9
`726
`216
`0.87
`
`C2025/C20:5
`PC w/EPA & EPA
`18.9
`826
`C14:0 C20:5
`PC
`19.4
`752
`904
`3.64
`
`C2226/C20:5**
`
`PC w/DHA & EPA
`
`C21 :0/C20:5**
`
`PC W/EPA
`
`C 12:0/22:4*
`
`PC
`
`C22:6/C13:0**
`
`PC w/DHA
`
`C1620/C20:5**
`
`C2226/C16:0**
`
`C2015/C18:1**
`
`C2025/C16:0
`
`C2226/C16:1**
`
`PC W/EPA
`
`PE w/DHA
`
`PE w/EPA
`
`PC W/EPA
`
`PS w/DHA
`
`C2226/C16:0**
`
`PC w/DHA
`
`C1810/C18:0**
`_ e_;**
`
`PC
`._--A
`
`-
`
`19.5
`
`19.5
`
`20.5
`
`20.5
`
`20.9
`
`21.3
`
`21.9
`
`21.0
`
`21.5
`
`22.0
`
`20.9
`
`852
`
`852
`
`754
`
`764
`
`764
`
`764
`
`764
`
`780
`
`806
`
`806
`
`790
`'9
`
`452
`
`849
`
`61
`
`69
`
`i9
`
`18
`
`3861
`
`656
`
`1610
`
`63
`A
`
`1.82
`
`3.42
`
`0.25
`
`0.28
`
`0.08
`
`0.07
`
`15.54
`
`2.64
`
`6.48
`
`0.25
`I
`
`
`
`878
`
`C2226/C22:6
`
`PC w/DHA & DHA
`
`C2226/C14:0**
`
`C2025/C16:1**
`
`PC w/DHA
`
`PC w/EPA
`
`19.8
`
`20.2
`
`778
`
`778
`
`158
`
`329
`
`0.64
`
`1.32
`
`Contains Confidential Business Information
`
`NEP877ITC-00267602
`
`Subject to the Protective Order
`
`4
`
`4
`
`

`
`C1620/C2220
`
`C2220/C1621
`
`C22:6/C18:1
`
`C20:5/C18:O**
`
`C2226/C16:O**
`
`C2015/C21:0**
`
`C20:4/C18:1
`
`C2023/C1822
`C20:5/C20:6**
`
`C2226/C18:O**
`
`PC
`
`PS
`
`PC W/DHA
`
`PC w/EPA
`
`PS w/DHA
`
`PE W/EPA
`PC
`
`PC
`
`PC W/EPA
`
`PC W/DHA
`
`21.7
`
`24.5
`
`22.5
`
`21.9
`
`23.4
`
`24.0
`
`24.4
`
`25.4
`24.8
`
`24.8
`
`81 8
`
`81 8
`
`832
`
`808
`
`808
`
`808
`
`808
`
`808
`834
`
`834
`
`29
`
`49
`
`601
`
`245
`
`1 50
`
`1 98
`
`1 34
`
`87
`328
`
`0.12
`
`0.20
`
`2.42
`
`0.99
`
`0.60
`
`0.80
`
`0.54
`
`0.35
`1 .32
`
`Total
`
`24841
`
`100.0
`
`* — Unconfirmed
`
`** — Could not differentiate
`
`PC — Phosphatidylcholine
`
`PE — Phosphatidylethanolamine
`
`PS — Phosphatidylserine
`
`EPA — Eicosapentaenoic acid
`DHA — Docosahexanoic acid
`
`Contains Confidential Business Information
`
`NEP877ITC-00267603
`
`Subject to the Protective Order
`
`5
`
`5
`
`

`
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`
`Figure 1. Represenioiive ’ro’rc1|—ion chromoiogrom for 500 ng/pL of Beoudoin Oil # O9-
`1651.
`
`Contains Confidential Business Information
`
`NEP877ITC-00267604
`
`Subject to the Protective Order
`
`6
`
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`
`period of ihe chromdiogrdrn in Figure 1
`dissolved in CHCI3.
`
`for The 500 fig/LIL Bedudoin Oil # 09-1651
`
`Contains Confidential Business Information
`
`NEP877ITC-00267605
`
`Subject to the Protective Order
`
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`Contains Confidential Business Information
`
`NEP877ITC-00267606
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`Subject to the Protective Order
`
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`NEP877ITC-00267609
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`
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`chromciiogrcim in Figure 1 for ihe 500 fig/LJL Beoudoin Oil # 09-1651 dissolved in CHCI3.
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`Contains Confidential Business Information
`
`NEP877ITC-00267610
`
`Subject to the Protective Order
`
`12
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`Contains Confidential Business Information
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`NEP877ITC-00267611
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`Subject to the Protective Order
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`13
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`Full scan ESI moss specirum for peak of i4.8 min in The chromoiogrom in
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`Figure 1 for The 500 ng/uLBeoudoir1 Oil # 09-1651 dissolved in CHCI3.
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`Contains Confidential Business Information
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`NEP877ITC-00267612
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`Subject to the Protective Order
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`14
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`ohromoiogrom in Figure 1 forihe 500 rig/uL Beoudoin Oil # 09-1651 dissolved in CHCI3.
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`NEP877ITC-00267613
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`Figure 1 for The 500 ng/uL Beciudoin Oil # 09-1651 dissolved in CHCI3.
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`Contains Confidential Business Information
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`NEP877ITC-00267614
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`Figure 1 for The 500 ng/uL Beciudoin Oil # 09-1651 dissolved in CHCI3.
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`NEP877ITC-00267615
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`ohromoiogrom in Figure 1 forihe 500 rig/uL Beoudoin Oil # 09-1651 dissolved in CHCI3.
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`Contains Confidential Business Information
`
`NEP877ITC-00267616
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`Subject to the Protective Order
`
`18
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`Figure 1 for The 500 fig/LJL Beciudoin Oil # 09-1651 dissolved in CHCI3.
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`NEP877ITC-00267617
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`Subject to the Protective Order
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`19
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`Figure 1 for The 500 fig/LJL Beciudoin Oil # 09-1651 dissolved in CHCI3.
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`Contains Confidential Business Information
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`NEP877ITC-00267618
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`20
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`Full sccm ESI moss specirum for peak of 20.5 min in The chromoiogrom in
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`Figure 1 for The 500 ng/uL Beciudoin Oil # 09-1651 dissolved in CHCI3.
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`20
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`NEP877ITC-00267619
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`inleptune Eeaucioin Ciiil {'1u::_|iuL in 2C1‘?-isiii-i£ii~=lj§»
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`Full sccm ESI moss specirum for peak of 20.9 min in The chromoiogrom in
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`Figure 1 for The 500 ng/uL Beciudoin Oil # 09-1651 dissolved in CHCI3.
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`NEP877ITC-00267620
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`Figure 1 for The 500 fig/LJL Beciudoin Oil # 09-1651 dissolved in CHCI3.
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`NEP877ITC-00267621
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`Subject to the Protective Order
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`

`
`iwleptune Eeaudoin Cril {1ug1TuL in 2ifJ‘?:zi;.ii'-‘aC;i-ij;a
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`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/pL Bedudoin Oil # 09-1651 dissolved in
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`NEP877ITC-00267622
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`iwleptune Eeaudoin Cril {1ug11uL in 2-IfJ‘?:r:;.1i'-‘1C;I"-lj;a
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`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/11L Bedudoin Oil # 09-1651 dissolved in
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`NEP877ITC-00267623
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`iwleptune Eeaudoin Cril {1ug1iuL in 2ifJ‘?:zi;.ii'-‘aC;i-lj;a
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`dhd (D) ToTdl ion <:hromdTogrdm for The 500 rig/pL Bedudoih Oil # 09-1651 dissolved in
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`NEP877ITC-00267624
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`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 fig/TJL Bedudoin Oil # 09-1651 dissolved in
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`NEP877ITC-00267625
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`iwleptune Eeaudoin Cril {'1ug1JuL in 2ifJ‘?:zi;.ii'-‘aC;i-lj;a
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`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/pL Bedudoin Oil # 09-1651 dissolved in
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`27
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`NEP877ITC-00267626
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`iwleptune Eeaudoin 1."Z+i|{'1u::_11’uL in 2T:}€:‘X;3r€:‘1{:l~"l::3
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`
`13.13
`?52'33
`
`.
`
`Figure 24.
`
`Ion chromdiogrdrns for PLs wiTh I\/\Ws dT (A) m/z 751, (B) m/z 826, (C) m/z 726
`
`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/11L Bedudoin Oil # 09-1651 dissolved in
`
`CHCI3. Top number is reTenTion Tirne, middle is m/z dnd boTTorn is The pedk dred (for Top
`
`Three c:hrorndTogrdms).
`
`28
`
`29
`
`29
`
`Contains Confidential Business Information
`
`NEP877ITC-00267627
`
`Subject to the Protective Order
`
`

`
`iwleptune Eeaudoin Cril {'1ug1JuL in 2ifJ‘?:zi;.ii'-‘aC;i-lj;a
`
`23
`09
`
`3
`
`—
`
`23.5?
`254.3?
`5i
`
`21.2?’
`222 22 21.33
`12
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`
`A
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`20.!-iE| _
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`1””
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`23.3:-'_ 21.11
`233.33
`233.33
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`0
`
`$315395 21.33
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`23533 22.33 22 22
`23.13
`212323;
`0'37’
`15-35
`-
`-
`233.32
`3232.332/2222
`223.33
`x.
`EI?E.3E= 2
`Time
`10.00
`17.00
`10.00
`19.00
`20.00
`21.00
`22.00
`23.00
`
`13.43
`?52'33
`
`.
`
`Figure 25.
`
`Ion <:hromdTogrdrns for PLs wiTh I\/\Ws dT (A) m/z 764, (B) m/z 754, (C) m/z 852
`
`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/pL Bedudoin Oil # 09-1651 dissolved in
`
`CHCI3. Top number is reTenTion Tirne, middle is m/z dnd boTTorn is The pedk dred (for Top
`
`Three c:hrorndTogrdms).
`
`29
`
`30
`
`30
`
`Contains Confidential Business Information
`
`NEP877ITC-00267628
`
`Subject to the Protective Order
`
`

`
`iwleptune Eeaudoin Cril {'1ug1JuL in 2ifJ‘?:zi;.ii'-‘aC;i-lj;a
`
`1””
`
`2:19? _
`7030.34
`53
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`22.0:-'
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`21 .0E|;i30E.3E;1E10
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`20.00
`21.00
`22.00
`23.00
`24.00
`
`21.29
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`£30.35
`
`'
`
`Figure 26.
`
`Ion <:hromdTogrdrns for PLs wiTh i\/\Ws dT (A) m/z 790, (B) m/z 806, (C) m/z 780
`
`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/pL Bedudoin Oil # 09-1651 dissolved in
`
`CHCI3. Top number is reTenTion Tirne, middle is m/z dnd boTTorn is The pedk dred (for Top
`
`Three c:hrorndTogrdms).
`
`30
`
`31
`
`31
`
`Contains Confidential Business Information
`
`NEP877ITC-00267629
`
`Subject to the Protective Order
`
`

`
`Neptune Eeaudoin 1."Z+i|{'1u::_11’uL in 21fJ‘?:r:;.1i'-"e11ii;I"-lj;a
`
`1 '3'”
`
`“£2.
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`:1
`
`2'2§é1D3g4_
`1132
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`212.15 _
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`A
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`
`22.92 _ 21.11
`232.34 232.313
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`1943
`18.89 25233
`1325.31
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`15 35
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`22.25
`3213.39 23.22
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`232.12 21139
`/
`331.3»:
`Time
`10.00
`20.00
`22.00
`24.00
`26.00
`20.00
`
`Figure 27.
`
`Ion <:hromdTogrdrns for PLs wiTh I\/\Ws dT (A) m/z 878, (B) m/z 778, (C) m/z 818
`
`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/11L Bedudoin Oil # 09-1651 dissolved in
`
`CHCI3. Top number is reTenTion Tirne, middle is m/z dnd boTTorn is The pedk dred (for Top
`
`Three c:hrorndTogrdms).
`
`31
`
`32
`
`32
`
`Contains Confidential Business Information
`
`NEP877ITC-00267630
`
`Subject to the Protective Order
`
`

`
`iwleptune Eeaudoin Cril {'1ug1JuL in 2ifJ‘?:zi;.ii'-‘4C;i-lj;a
`
`33
`320
`
`A
`
`-34
`
`0
`
`100
`
`21.99 _
`
`24.41
`
`23.33 _
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`1””
`
`2IZI.9F"_21.1T
`233.34
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`
`D
`
`.443
`« 54;-
`”
`
`23.22
`33242
`24.23
`4’
`334.34
`Time
`20.00
`22.00
`24.00
`20.00
`20.00
`
`Figure 28.
`
`Ion <:hromdTogrdrns for PLs wiTh I\/\Ws dT (A) m/z 834, (B) m/z 808, (C) m/z 832
`
`dnd (D) ToTd|—ion <:hromdTogrdrn for The 500 ng/pL Bedudoin Oil # 09-1651 dissolved in
`
`CHCI3. Top number is reTenTion Tirne, middle is m/z dnd boTTorn is The pedk dred (for Top
`
`Three c:hrorndTogrdms).
`
`32
`
`33
`
`33
`
`Contains Confidential Business Information
`
`NEP877ITC-00267631
`
`Subject to the Protective Order
`
`

`
`Ifileptune Beaudoin Oil 08-1851 {O.5ug:_;fuL}
`
`ml]
`
`8284828
`
`C 1 8:0
`
`C20:5 (EPA)
`
`826.5—284=C 1 8:0
`
`82841555.,
`
`82838485,.
`
`828338141
`
`325-3D?3~.
`
`822.4838
`/
`
`,..82?".8288
`
`-5
`
`826.5—302=C20:5
`
`
`
`
`
`/
`
`524.2828 ..542.31 48
`
`525.5555
`
`828.2F"84H/
`
`588
`
`558
`
`888
`
`858
`
`T88
`
`T58
`
`888
`
`858
`
`l'l"I:'Z
`
`Figure 29. RepresenToTive MS/MS speoTrurn for phospholipid wiTh C1 rnoleoulor weighT of
`
`825.48 from peak 0T 38 min in The 500 ng/uL Beoudoin Oil # 09-1651 dissolved in CHCI3.
`
`This spe<:Trurn represenTs c1 C2025/Cl8:O PL C15 indi<:c1Ted by CID ions represenTing loss of
`These rnosses from The rnoleculor ion.
`
`33
`
`34
`
`34
`
`Contains Confidential Business Information
`
`NEP877ITC-00267632
`
`Subject to the Protective Order
`
`(EPA)