(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1013
`
`

`

`Article
`
`animal source. Molecular species profiles were determined using
`liquid chromatography—electrospray ionization— tandem mass
`spectrometry (LC—ESl—Msz) by fragmenting lithiatcd adducts
`ofthc molecular species of PC.
`This analytical method can be advantageously used by the food
`industry to screen both new potential sources of bioactive
`ingredients and the quality of their raw materials.
`
`MATERIALS AND METHODS
`
`Materials. All solvents used for high-performance liquid chromatog—
`raphy (HPLC) analyses were HPLC—grade. Methanol was purchased from
`VWR (Strasbourg, France), and acetonitrile was purchased from Sigma—
`Aldrich (Steinheim, Germany). Chloroform (Sigma—Aldrich, Steinheim,
`Germany), methanol, and ri-heptane (Carlo Erba, Val de Reuil, France)
`used for lipid extraction and PC purification were of analytical grade.
`Wash solution was prepared from sodium chloride of analytical grade and
`Ultrapure water (Millipore, Molsheim, France). Soy PC standard was
`purchased from Avanti Polar Lipids (Alabaster, AL). Hens" eggs (6.5%
`PC), soybeans (Glycine max) (0.7% PC), and 0): liver (1.7% PC) were
`purchased from a local retailer. Krill oil (29.0% PC) has been kindly
`provided by Ncsiec SA (Lausanne, Switzerland).
`Sample Preparation and Lipid Extraction, Preparation was differ-
`ent for each sample depending upon its physical stale. Both soy beans and
`mi liver were grinded. but only ox liver was lyophilized after grinding. Egg
`yolk was lyophilized but without grinding. Krill oil was fractionated and
`analyzed as such without further preparation. Grinding was achieved
`under cryogenic conditions (liquid nitrogen, 3 steps of 5 min) using a 6870
`Freezer/Mill (Spex CertiPrep, Stanmore, U.K.). Total lipids were ex—
`tracted according to Folch et a1. (12), with minor modifications. A total of
`l g of the treated food matrix was suspended in 30 mL of a CHCl3/CH30H
`(2: 1, v/v) mixture and shaken mechanically for 20 min. The suspension was
`centrifuged at 8500g for 10 min, and the supernatant was removed and
`washed with 5 mL of sodium chloride aqueous solution (1%, w/v). The
`organic phase containing the lipid fraction was removed. evaporated
`under vacuum, and dried under a gentle stream of NZ. The total lipid
`extract was weighed and dissolved in 1 mL of CHCl3.
`Liquid Chromatography.
`Flash Chromatography. A 150 mg
`aliquot of the total lipid extract (from krill oil, soy, or egg) was subjected
`to preparative flash Chromatography (CombiFlash retrieve, Serlabo,
`Entraigues stir la Sorgue, France) onto a 15 g silica cartridge precondi—
`tioned with n—heptane. Flution was performed at a flow rate of 10ml./min
`using 60 mL of CHClg, then 120 mL of CHClg/CH30H/H20 (351654
`v/vi‘v) mixture, and finally 120 mL ofcthanol for a column wash. Fractions
`(_ 10 mL) were collected and tested by thin—layer chromatography (TLC)
`using silica plates, CHCly'CHgOH/HZO (3526524, v/v/v) as an eluent, and
`phosphomolybdic acid in ethanol (20:80, vj’v) to visualize compounds. PC
`was identified in comparison of its retention factor (R,) to the one of soy
`PC (Avanti Polar Lipids, Alabaster, AL) as a reference. Fractions
`containing pure PC were pooled and evaporated under vacuum.
`HPLC. A chromatographic system made of a 616 controller, a 2424
`ELS deteclor, and a 7l7Plus autosampler (Vl’aters, Saint Quentin Falla—
`vier, France) was used to perform the chromatographic analyses. High—
`purity nitrogen from a nitrogen generator (Domnik Hunter, Villefranche—
`sur—Saone. France) was used as a nebulizing gas at a pressure 0145 psi. The
`drift tube temperature was set at 45 °C. Separations were performed at
`room temperature using a 20 min linear gradient ranging from CHCl3,
`CHgOH (88:12, vjv) to CHClg/CH3OH/l M formic acid adjusted to pH 3
`with triethylamine (28:60:12, vrv/v) (13), This chromatographic system
`and this gradient, were both used for PC purification and to check purity of
`the isolated PC.
`To isolate PC from ox liver, a 100 mg aliquot of the ox liver lipidic
`extract was injected on a 250 >< 10 mm, 10 run normal-phase Liehrospher
`column (Interchim, Montlucon, France). A total on/3 ofthc mobile-phase
`flow, which was set at a rate of 2 mL/min, was diverted, ahead of the ELSD
`inlet, to a fraction collector. The PC peak was identified in comparison of
`its retention time to the standard one, and time windows were set to collect
`the PC peak.
`The purity of PC isolated from each matrix was checked by injecting a
`fraction of the purified samples on a 150 x 3 mm. 3 pm Luna normal phase
`(Phenomenex, Le Pecq, France) using a flow rate 0105 mL/min. Purity (P)
`
`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`of the isolated PC was calculated as follows:
`
`6015
`
`
`1
`P:"’Cxioo
`.4‘
`
`where AFC is the PC peak area and A, is the total of peak areas on the
`chromatogram.
`Separation and [den tification ofMo/ecular Species by LC7
`ESI—AMS. Molecular species of PC were determined using a Prostar
`HPLC system made of two 210 solvent delivery modules, a 4l0 auto—
`sampler, and a 1200 L triple quadrupole mass spectrometer fitted with an
`ESI source (Varian, Les Ulis, France). High—purity nitrogen (Domnik
`Hunter) was used as a nebulizing gas, set at 46 psi, and as a drying gas, set
`at 300 °C. Separation was performed on a C 18 reverse-phase 250 X 3 mm,
`3 film lsis Nucleodur column (Macherey—Nagel) using an isoeratic flow of
`CH3CNJ°CH3OH (40:60, vfiv) containing 0.1% NH4OH at a rate of 1 mL/
`min. A split system however allowed the HPl.C effluent to enter the mass
`spectrometer at a flow rate of0.2 mL/min. To help the identification ofthc
`molecular species, 3 mL of 10 mM lithium formate was added to l L of
`mobile phase. Major detected ions were iM+ Li]’ referring to the ino—
`lecular ion with a lithium adduct. Acquisition was performed in positive
`mode in a mass range between m/z 450 and 900. mfz ([M + Li]+) were
`fragmented in positive mode (M52), which was used to identify FA linked
`to the PC glycerol backbone. The mass range was set between 171/: 200 and
`600 for fragmentation products. The dwell time was set to 0.2 s for each
`m/z. Argon was used as a collision gas, and collision energy was set to 30 V.
`The percentage of each molecular species was calculated as follows:
`
`0/ :—
`A (peak(m/z))
`ZA(peaks) X100
`1”
`where Atipeak(m,‘z)) refers to the peak area of the selected mfz and
`2.4(peaks) refers to the sum of all peak areas. Analyses were performed
`in triplicate, with results expressed as mean in standard deviation (SD).
`Gas Chromatographic Analysis of FA. A 3400 Varian gas
`chromatograph equipped with a flame ionization detector was used for
`the analysis of FA methyl esters (FAMEs) after transesterification of
`
`Table 1. FA Profiles 01 Purified PC from Soybeans, Egg Yolk, Krill Oil, and Ox
`Livera
`
`egg yolk PC
`krill oil PC
`ox liver PC
`
`FAs
`soy PC (%)b
`(9/5)”
`he)"
`not”
`nd‘
`nd
`2.2 :: 0.09
`nd
`nd
`0.3 W 0.01
`0.4 W 0.02
`ntl
`
`0.03
`34.1
`0.2
`26.8 :: 0.1
`20.3 :: 0.6
`ml
`1.6 :: 0.03
`1.8 :: 0.08
`ml
`0.2 :: 0.01
`nd
`0.2 :: 0.06
`0.5 :: 0.4
`
`0.2
`0.02
`13.6
`4.8
`1.6 :: 0.05
`85.4 :: 0.02
`0.4
`9.4
`0.08
`29.3
`5.7 :1: 0.03
`11.7 :1: 0.5
`57.6 :: 0.10
`16.0 :: 0.2
`3.0 :: 0.02
`21.2 :: 0.7
`5.5 :: 0.01
`nd
`1.7 :i: 0.01
`[10
`nd
`nd
`0.1 a: 0.05
`nd
`I'ICl
`nd
`0.6 :I: 0.01
`1.5 :I: 0.08
`nd
`nd
`2.4 fl: 0.01
`nd
`nd
`nd
`nd
`2.5 :1: 0.07
`
`4.5 :: 0.02
`nd
`0.6 :i: 0.01
`5.8 :i: 0.2
`nd
`0.8 :: 0.01
`nd
`nd
`nd
`mi
`1.3 :1: 0.02
`nd
`nd
`nd
`0.8 3: 0.01
`nd
`0.5 :: 0.01
`nd
`nd
`nd
`nd
`nd
`0.5 :i: 0.01
`nd
`
`nd
`nd
`34.1 ,2 0.07
`nd
`
`0.7
`0.01
`0.8
`16.4 :: 0.1
`nd
`0.3
`48.0
`26.1
`31.2
`57.2
`9.4
`30.9
`9.9
`13.3
`
`19.9
`
`
`
`
`
`
`14:0
`15:0
`16:0
`16:1 (ii-7)
`17:0
`18:0
`18:1 (rt—9)
`18:2 (11—6)
`1323111'3)
`20:0
`20:1 (Fl-11)
`20:2 (ii-5)
`20:3 (ii-3)
`2014 (11-5)
`22:0
`22:1 (ii-13)
`22:2 (rt—6)
`24:0
`24:1 ln—9)
`20:5 (11-3)
`22:5 (11-3)
`2 saturated FAs
`2 monounsaturated
`FAs
`
`z PUFAs 29.5 64.5 21.1 59.0
`
`
`
`
`
`
`
`
`
`
`
`a Results (n = 3) are expressed as mean + SD. t'Percentage of the total peak
`area of FAs. Cnrl = not detecled.
`
`000002
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:21)
`
`AKER877IT000740545
`
`

`

`Le Grandois et al.
`
`step, fractions containing PC were pooled and purity was checked
`by HPLC—evaporative light—scattering detector (ELSD). Purity
`levels for PC isolated from each food sample were as follows:
`98.7% for egg yolk, 98.0% for krill oil, 95.0% for soy, and 90.1%
`for on liver. These levels were sufficient for the subsequent
`determinations of FA profiles and molecular species in PC
`samples.
`FA Profiles of Purified PC. Table 1 shows FA profiles of PC
`purified from the studied food matrices as determined from the
`normalized data of GC—FID analysis. Soy PC was naturally
`shown to contain higher proportions of unsaturated PA, as
`compared to PC from animal sources. For instance, 18:2
`(576%) and 18:3 (6.6%) were particularly abundant in soy PC.
`Some saturated FA, such as 22:0 (0.8%) and 24:0 (0.5%), were
`also present in soy PC, while they were not found in PC from egg
`yolk, 0x liver, and krill oil. Conversely, 20:4 was identified in egg
`yolk PC (4.5%) and ox liver PC (5.8%), while it was not detected
`in soy PC.
`As far as egg yolk PC is concerned, 16:0 (341%) and 18:1
`(29.3%) were the two main FA identified. followed by 18:2
`(16.0%) and 18:0 (13.6%) (Table l), which is in agreement with
`a previous report (14). The same FAs were predominant in ox
`liver but at different levels: 18:0 (36.4%). 18:2 (21.2%), 16:0
`(20.3%), and 18:1 (11.7%). Minor FA were 20:1 (1.5%) and 20:3
`(2.591;) in ox liver and 16:1 (1.6%) and DHA (0. %) in egg yolk.
`
`Krill oil PC contained the largest variety of FAs in comparison
`
`
`to other matrices but with *PA (34.1%), 16:0 (26.8911), and DHA
`(16.4%) being the three major FAs. This result is in accordance
`
`25
`
`MCoglnts
`,l
`l
`20 ~
`1
`15 i
`
`‘10
`
`MCounts
`
`(C)
`
`:8
`
`Retention time (min)
`
`11
`
`
`
`
`10
`
`(B)
`
` 5
`
`1'0
`
`2b
`15
`Retention time (min)
`
`55
`
`3
`
`iii
`
`5
`
`20
`1b
`10
`Retention time (min)
`
`25
`
`50
`
`so
`
`40
`
`30
`
`20
`
`Figure 1. Chromatograms of purified PC: (A) soy, (B) ox liver, (C) egg yolk, and (D) krill oil using LCeESHVIS. Separation was periormed using isocratic
`conditions: CchN/CHSOH (40:60) containing 0.1% NH4OH at a flow rate of 1 mL/min onto a reverse-phase column (250 x 3 mm, 3pm). Letters are assigned
`to majoridentified peaks: at, 18:3—18:3-PG; b, 18:3—18:2-PG; c, 18:2—18:2-PC; d, 16:0—18:3-PC; 9, 18:1 —18:2-PC; 1, 16:0—1S:2-PC; g, 16:0—18:1-PC; h,
`18:0—18:2-PC +13:1—18:1-PC;i,18:0—18:1-PC;j,1B:O—20:4-PC; k, 18:0—20:3—PC;I,18:1—20:4-PC;m,18:2—20:3-PC;n, 16:0—20:5-PC;0,16:0—18:0-
`PC; p, 16:0—22:6-PC; q, 18:1—22:6-F’C; r, 18:0—22:6-F’C; s, 14:0—20:5-PC; 1, 20:5—20:5-PC; u, 20:5—22:6-PC; v, 22:6—22:6-F’C; w, 18:1—20:5-PC; x,
`18:0—20:5—PC; and y, 18:1—20:4—PC.
`
`000003
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:22)
`
`AKER877IT000740546
`
`6016
`
`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`
`purified PC. Separation was made by a CP Sil 88 column [(88%
`cyanopropyl)—arylpolysiloxane, 100 m X 0.25 mm, 0.2 pm, Varian].
`Samples (1 ,uL) were injected. Carrier gas was helium of high purity
`(99.99.9504)). The injector and detector were set to 230 °C, and the column
`was set at 60 °C, held for 5 min. and raised to 165 cC (rate of 15 “C/min).
`The temperature of 165 0C was held for l min and then raised to 225 °C
`(rate of2 °C/min). The final temperature was maintained for 30 min. Peaks
`were identified by a comparison to standards (FAME mix C4—C24.
`Sigma—Aldrich, Saint-Quentin—Fallavier, France). Purified PC were trans—
`esterified using KOH (0.5 M) in CH3OH by vortexing for 2 min at room
`temperature. After decantation, FAM E were extracted using n—heptane.
`Gas chromatography (GC) data were normalized, and the percentage of
`each FA was calculated as follows:
`
`n/ :
`[0
`
`.
`A(peak(m/z))
`ZA(peaks) X 100
`
`where A(peak) refers to the area of each identified peak and 2:4(pcaks)
`refers to the sum of areas of each identified peak. Each analysis was
`
`performed in triplicate. All results are expressed as mean 3: SD.
`
`RESULTS AND DISCUSSION
`
`Purification of PC. PC was separated from non—polar lipids and
`other PLs using either flash chromatography or preparative
`HPLC. While flash chromatography was fast and easy. it was
`ineffective with food samples containing phosphatidylserine or
`sphingomyeline. Preparative HPLC on the other hand allowed
`for the isolation of PC from such samples as ox liver for instance,
`which contained both of these PLs. After each chromatographic
`MCounts
`
`150
`
`125
`
`f
`
`(A)
`
`100
`
`75
`
`50
`
`i
`
`5
`10
`1 5
`20
`25
`Retention time (min)
`h
`
`MCOU (S
`
`

`

`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`449
`
`6017
`
` 100%
`
`uC
`a) 75%
`CO
`1:C
`
`(a) 07/2 764
`
`400
`{77/12
`
`600
`
`B(
`
`U 50%
`a;
`.2
`
`25%
`
`E §
`
`0%
`
`200
`
`100%
`
`(17) 171/2872
`
`
`
`200
`
`400
`m/z
`
`500
`
`595
`
`1<
`
`5‘75
`
`(c)m/z 784
`
`yj
`1
`
`447
`
`Article
`
`with a previous study showing that these three FAs were very
`abundant in krill oil (15). including krill PC (14). Saito et al. showed
`
`that, even if krill PA composition varied according to the period of
`
`
`capture, its PC was always rich in ‘PA, DHA, and 16:0 ([6).
`The relative rates of saturated, monounsaturated, and PUFAs
`were very different among food matrices (Table 1:). PUFAs were
`most abundant in soy PC (64. 0/0) and in krill oil PC (59.0%) but
`with the latter containing far higher amounts of LC—PUFAs,
`namely, EPA and DHA, which is in line with the usual composi—
`tion of food products from marine sources (1). Saturated FAs on
`the other hand were predominant in animal source foods: 31.2%
`in krill oil PC, 48.0% in egg yolk PC. and 57.2% in ex liver PC.
`These data on FA distribution profiles in purified PC were later
`used in a comparative analysis of the molecular species determi—
`nations to check if the identified structures in each PC matched its
`determined FA composition,
`PC Molecular Species. Clirmnatngraphir Separation. Separa—
`tion was achieved using a 3 pm column, and MS chromatograms are
`given in Figure 1. With less than 10 chromatographic peaks, soy PC
`(Figure 1A) and egg yolk PC (Figure 1C) had a simple composition
`compared to ox liver PC (Figure 1B) and krill PC (Figure 1D). which
`showed at least 15 peaks. Judging by the retention times, the same
`molecular species could be present in different foods. It is particu—
`larly true for the animal soru‘ce ones: egg yolk and ox liver, where
`several peaks have the same retention times.
`Identification ofPC A/[olecular Species. Molecular species of
`PC were detected as m/: of lithium adducts [M -i- Li] +. Lithium
`was added to the chromatographic mobile phase and was shown
`not to interfere with the separation, which allowed for the
`combination of an effective chromatographic separation and an
`improved structural identification. Lithium has already been used
`in previous studies to identify molecular species of PC but with no
`chromatographic separation beforehand (1 7—19). To identify PC
`molecular species.
`lithium adducts were fragmented in N152,
`which was previously shown to yield three main fragments:
`[MLi — FA]+ corresponding to the loss of a FA group, [MLi —
`FALi]+ corresponding to the loss of a lithium salt of an FA
`group, and [MLi — TMA — FA]+ corresponding to the loss of a
`trimethylamirie group (TMA, 59 Da) and a FA group (I 7). The
`obtained fragmentation patterns of lithium adducts proved in fact
`to be effective in identifying various PC species.
`including
`symmetrical, asymmetrical, saturated, and unsaturated ones (I 7).
`The identification performed in this study did not
`take into
`account the position of the FA in the glycerol moiety and was
`in most cases tentative.
`
`
`
`Representative MS2 fragmentation spectra of the molecular
`species of PC are presented in Figure 2.
`Figure 2a shows the MS2 spectrum of the species m/z 764
`(RT : 13.2 min). One of its two FAs was identified as 16:0 based
`on m/z 449 and 502, which correspond respectively to fragments
`[M -i — (16:0) — TMAit and [MLi — (16:0)Liit. The second FA
`was identified as 18:2 based on m/: 484, which corresponds to
`fragment [MLi— (18:Z)]+. An additional fragment. m/z 575,
`ma ehed the mass ofthe PC minus its phosphocholine head with
`a lithium adduct (1 D. Consequently, the molecule detected as a
`lithium adduct with m/z 764 (RT : 13.2 min) was identified as
`[(16:0—18:2)PC + Li]+.
`Figure 2b shows the MS2 fragmentation spectrum of the
`molecular species 111/: 812 (RT = 8.6 min), which was identified
`as [(16:0—22:6)PC + Li] ‘
`. The two ions obtained, m/z 478 and
`556, corresponded in fact respectively to fragments [M Li 7 (22:6)—
`Li]+ and [MLi — (160)]: showing DHA and 16:0 as the two
`FAs of the molecule.
`
`A third example is given in Figure 2c with the MS2 spectrum of
`m/z 784 (RT : 6.0 min) containing three fragments that allowed
`
`03
`as
`8 75%
`'UC
`
`50%
`
`25%
`
`0%
`
`100%
`
`.
`75A)
`
`500/
`..
`
`25%
`
`0%
`
`3 g
`
`3id
`5.5a)
`0:
`
`§
`g
`
`C3 g
`
`HE E
`
`3
`
`
`
`200
`
`400
`m/z
`
`600
`
`tons obtained in positive mode in Est—MS2 between 200 and
`Figure 2.
`6001mm fragmentation of Iithiated adduets: (a) fragmentation of m/z 764,
`(b) fragmentation of m/z 812, and (c) fragmentation of m/z 784 and their
`corresponding structures.
`
`for its identification as [(18:3—18:3)PC+ Li]+, Two of these
`fragments, m/z 506 and 447, were identified as [MLi— (1823)]+
`and [MLi — TMA — (18:3)]+, which pointed to 18:3 as the sole
`FA. The third fragment, m/z 595, corresponded. as above for m/z
`575, to the loss of the phosphocholine head with a lithium adduct.
`This approach was applied to each m/z detected with purified PC
`samples and allowed for the identification of the constitutive
`molecular species for each food investigated.
`PC Molecular Species Profiles. Table 2 gives the relative
`distributions of the various PC molecular species in the foods
`investigated. As far as soy PC is concerned, the major species was
`
`000004
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:23)
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`AKER877ITC00740547
`
`

`

`Le Grandois et al.
`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`6018
`
`Table 2. Molecular Species Profiles of Each Purified PCa
`
`m/z [M + Li]
`structure”
`retention time (min)
`soy PC (%)c
`egg yolk PC ("/u)C
`krill oil PC (“Alf
`ox liver PC (”81‘
`
`nd
`rid
`nd
`nd
`ml
`20.2 :: 0.7
`nd
`
`
`
`ndd
`0.9 3: 0.2
`nd
`m
`2.8 :: 0.4
`20.8 :: 10
`nd
`
`
`
`
`
`7386
`740.6
`758.6
`
`762.6
`764.6
`
`(180—458)Pe
`(16:0—16:0)PC
`(14:0—20:5)PC
`n4n—2ane
`(16:0—18:3)PC
`(16:0—18:2)PC
`(16:1—18:1)PC
`
`1L5
`17.4
`5.5
`60
`10.4
`13.2
`13.6
`
`768.6
`778.6
`780.6
`784.6
`
`786.6
`
`788.6
`
`790.6
`
`782.6
`
`794.6
`812.6
`
`814.6
`
`816.6
`
`818.6
`
`832.6
`836.6
`8386
`840.6
`
`
`
`
`
`nd
`4 5:01
`nd
`17.5
`(16:0—18:0)PC
`rid
`rid
`rid
`15.5
`(17:0—18:2)PC
`rid
`nd
`nd
`20.9
`(17:0—18:1)PC
`rid
`rid
`0.7 Ii: 0.2
`6.0
`(18:3—18:3)PC
`1.4 i 0.0
`nd
`nd
`5.8
`(14:0—22:6)PC
`1.3 :1: 0.3
`nd
`rid
`6.5
`(16:1 —20:5)PC
`l'iC
`rid
`7.7 3: 1.2
`8.6
`(18:2:18:3)PC
`32.4 :: 0.3
`rid
`rid
`8.1
`(16:0—20:5)PC
`1.0:: 0.1
`3.3:1: 0.2
`34.0 :1: 2.0
`11.0
`18:2—18:2)PC
`4.1 :: 0.1
`nd
`rid
`8.7
`16:0—20:4)PC
`1.03:0.1
`3.1 :l:0.3
`16.3 Ii: 0.2
`13.3
`18:1—18:2)PC
`4.1 Ii: 0.1
`nd
`nd
`12.6
`16:0—20:3)PC
`6.6+0.9
`83-1-07
`6.3+0.3
`18.7
`18:0—18:2)PC
`rid
`3.3:1:0.7
`3.2 :: 0.4
`17.8
`18:1—18:1)PC
`rid
`8.9 :: 0.5
`1.6 :: 0.2
`22.4
`18:0*18:1)PC
`
`11.9 :00
`2.8::01
`rid
`8.6
`16:0—22:6)PC
`rid
`nd
`nd
`8.2
`18:2—20:4)PC
`7.4 :: 0.4
`not
`nd
`7.5
`18:1 —20:5)PC
`1.3 :: 0.4
`nd
`nd
`9.1
`18:0—20:5)PC
`1.9 :01
`lid
`lid
`9.7
`1821—2024)PC
`l’iC
`rid
`nd
`10.8
`(18:2—20:3)PC
`
`l'id
`4 5:: 0.3
`nd
`15.5
`18:0—20:4)PC
`rid
`nd
`nd
`15.7
`(18:1:20:3)PC
`ric
`rid
`rid
`16.0
`(18:0—20:3)PC
`rid
`rid
`rid
`16.4
`(18:2—20:1)PC
`rid
`1.7 :: 0.0
`rid
`rid
`4.4
`(20:5—20:5)PC
`0.1 :: 0.1
`rid
`rid
`rid
`9.4
`(20:4—20:4)PC
`n
`n
`033:06
`nd
`as
`(181—228Pc
`lie
`1.1 :1: 0.1
`1.1 :1: 0.3
`rid
`12.0
`(18:0—22:6)PC
`0.8 :: 0.0
`rid
`rid
`rid
`13.2
`(20:3:20:3)PC
`rid
`rid
`nd
`rid
`8.2
`(20:1 —20:5)PC
`2.21:: 0.2
`rid
`nd
`nd
`15.2
`(20:4—20:1)PC
`842.6
`nd
`asgzon
`nd
`nd
`46
`(2es—22eiPc
`8586
`
`884.6 rid (22:6—22:6)PC 5.0 nd nd 0.8 a: 0.0
`
`
`
`
`
`
`”'1 Results {n = 3) are expressed as mean :: SD. bThe position of fatty acids in the glycerol moiety has not been determined. “Percentages of all identified molecularspecies.
`”rid : not detected.
`
`273:01
`rid
`3.2 :: 0.0
`nd
`tic
`2.6 :: 0.0
`1.1::0.1
`
`
`
`nd
`0.7 :: 0.0
`rid
`02:00
`rid
`10.9 :: 0.2
`rid
`
`nd
`0.7 :: 0 3
`0.9 :: 0.0
`0 2 :: 0.0
`n
`ne
`rid
`0.3 :: 0.0
`3.3 :: 0.3
`rid
`3.5:: 0.4
`2.8 :: 0.4
`34.8+0.5
`rid
`15.9 :: 0.5
`nd
`0.4 :: 0.0
`rid
`mi
`1.6:: 0.6
`1.2 :: 0.7
`5.7:: 0.3
`
`5.5 :: 0.2
`
`
`
`(18:2—18:2)PC (34.0%), followed by (16:0—18:2)PC (20.8%) and
`(18:1—18:2)PC (16.3%), while minor ones included (18:0—18z2)—
`PC (3.2%), (16:0—18:3)PC (2.89/8),(18:0—18:1)PC (1.6%), and
`(l8:3—l8:3)PC (0.7%). Similar patterns have already been re—
`ported for soy PC (20 :22). Species determination data for soy PC
`is in agreement with its FA profile (Table l), with high amounts of
`16:0, 18:0, 18:1, 18:2, and 18:3 and 18:2 being by far the
`predominant one.
`The major molecular species in egg yolk PC was identified as
`(16:0—18:1)PC (39.7%), followed by (16:0—18:2)PC (20.2%),
`(l8:0*l8:2)1‘C (8.9%), and(l8:0:l8:l)PC (8.3%). Minor species
`included (18:2—18:2)PC (3.3%), (18:1—18:2)PC(3.1%), (16:0—
`22:6)PC (2.8%), and (18:0—22:6)PC (1.1%). The data reported
`here for egg yolk are to a certain extent in agreement with a
`previous report by Paeetti et a1. (14), essentially showing a similar
`pattern and (16:0—18:1)PC as the predominant species. Some
`quantitative differences that were observed, especially with minor
`molecular species, may be attributed to differences in hens“ diet,
`which affects FA composition, On the basis of the PC molecular
`species, 16:0 is the most abundant FA, being part of two species
`
`that accounted for ca. 60% of all species. This is in accordance
`with the FA profile of egg yolk PC, which showed that 16:0
`represented 34.1% of the total FA content (Table I). To a lesser
`extent, a similar observation was made for 18:1.
`The four main molecular species in ex liver were the same as in
`the other animal source food, egg yolk, albeit
`in different
`proportions: (18:0—18:2)PC (34.3%), (1820—18:1)PC (15.9%),
`(16:0—18:2)PC (10.9%),
`and (16:0—18:1)PC (9.1%). This
`matched the FA pattern, which showed 18:0 (364%), 18:2
`(21.2%), 16:0 (203%), and 18:1 (11.7%) as the dominant FAs
`in ex liver (Table 1). Bovine liver PC molecular species have been
`determined by Rang et al. (20) and Dobson et al. (23). With a
`content 0121,5904), (18:0—21:3)PC was found by Bang et al. to be
`the major molecular species. What is striking about this identi—
`fication is that 21:3, which was not present in our determinations,
`is an extremely rare FA. Dobson et al. on the other hand showed a
`pattern much closer to our findings, with (18:0—1822)PC, (18:0—
`l8:l)PC, and (16:0—18:1)PC being the main species and no
`(18:0—21:3)PC being detected (23). These remarks let us think
`that the identification performed by Bang et a1.
`is probably
`
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`AKER877ITCOO740548
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`

`Article
`
`wrong. Minor species found in ox liver included (18:0—20:3)—
`PC + (18:2-20:1)PC (5.5%). (18:0—20:4)PC + (18:1—20:3)PC
`(5.7%),
`(18:1—18:2)PC (3.5%),
`(16:0-20:4)PC (3.3%), and
`(1620—20:3)PC (2.8%) and were essentially the same as those
`identified in other studies. Some few differences observed with
`
`minor molecular species could be ascribed to the animal origin
`and diet, which affect the FA content (14). For molecular species
`that could not be adequately separated by chromatography, e.g.,
`(18:0—20:3)PC and (18:2—20:1)PC, the calculated content took
`the two structures into account (1‘able 2).
`As far as krill oil is concerned, to our knowledge, this is the first
`time the identification of PL molecular species is investigated,
`although FA profiles have already been determined for phospha—
`tidylethanolamine, PC, and TAG in krill (Euphazrsiapaczfica) (16).
`The major PC molecular species was determined as (16:0—20:5)—
`PC and represented 32.4% of all PC. Other species, such as
`(16:0—22:6)PC (11.9%), (18:1—20:5)PC (7.4%), (18:0—18:2)PC
`(6.6%), (16:0—18:1)PC (5.9%), and (16:0—20:4)PC (4.1%), were
`less abundant. Once again, the patterns of molecular species and
`FA coincided, because both determinations indicated that EPA,
`DHA, and 16:0 were the predominant FAs. Interestingly, minor
`molecular species of krill oil PC included two [(14:0—20:5)PC
`(3.2%) and (l4:0—22:6)PC (1.4%)] with 14:0, the presence of
`which is a common feature of marine oils.
`
`In this study, an effective method of separation and identifica—
`tion of PC molecular species was reported. This method used
`lithium formate to form lithiated adducts. MS2 fragmentation of
`these adducts allowed for the identification of FA linked to the
`glycerol backbone of PC molecular species. As an example of
`application, the relative contents of individual molecular species
`were determined, which allowed for a sound comparison of PC
`structures from foods of plant, animal, and marine origins,
`represented by soy, ox liver, egg yolk, and krill oil. Soy lecithin,
`mainly made of PC and already in use as a food additive and a
`nutritional complement, was therefore shown to be highly rich in
`essential FAs but not in their LC—PUFA metabolites. Krill oil PC
`
`showed an opposite pattern in comparison to soy PC, with very
`
`
`low amounts of 18:2 or 18:3 detected (_ < 5%) but with *PA and
`DHA being the most abundant among the foods tested ( > 50%).
`This study developed is an interesting contribution to the in—
`vestigation of new sources rich in LC—PUl—‘A—PLs. As an
`
`example, krill oil was shown as a potential source for food
`
`
`supplementation with *PA and DHA.
`
`ABBREVIATIONS USED
`
`
`
`
`
`
`
`
`long—chain polyunsaturated fatty acid; PA or
`LC-PUFA,
`20:5 (ii—3), eicosapentaenoic acid: DHA or 22:6 (n—3), doeosahex—
`aenoic acid; PL, phospholipid; PUFA—PL, PUFA—rich PL; PC,
`
`ohosphatidylcholine; TAG, triacylglycerol; LC, liquid chroma—
`
`
`tography; *SI, electrospray ionization; MS, mass spectrometry;
`
`<LSD, evaporative light—scattering detector; GC, gas chroma—
`tography;
`:ID, flame ionization detector; 14:0, myristie acid;
`16:0, palmitic acid; 16:1 (n—7), palmitolcic acid; 17:0, hcptadcea—
`noic acid; 18:0, stearic acid; 18:1 (it—9), oleic acid; 18:2 (it—6),
`linoleic acid; 18:3 (n—3), u—linolenic acid; 20:1 (n—ll), gadoleic
`acid; 20:3 (n—3), dihomo—y—linolenic acid; 20:4 (n—6), arachidonic
`acid; 22:0, behenic acid; 24:0, lignoceric acid.
`
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`
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`
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`AKER877IT000740549
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`6020
`
`J. Agric. Food Chem, Vol. 57, No. 14, 2009
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`Le Grandois et al.
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`l\'. Analysis of phospholipid molecular
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`
`Received March 17, 2009. Revised manuscript received Vlay 11, 2009.
`Accepted May 17, 2009.
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`000007
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:26)
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`AKER877ITC00740550
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