6014
`
`J. Agric. Food Chem. 2009, 57, 6014—6020
`DO|:10.1 021/]1‘9009035
`
`JOURNAL
`
`AG RICULTURAL_AND
`
`FOOD CHEMISTRY
`
`Investigation of Natural Phosphatidylcholine Sources:
`
`Separation and Identification by Liquid
`Chromatography—Electrospray Ionization—Tandem Mass
`Spectrometry (LC—ESl—MSZ) of Molecular Species
`
`JULIE LE GRANDOISJ' ERIC MARCi-iIONI,*’T MINJIE ZHAOj FRANCESCA GIUFFRIDAF
`SAlD ENNAHAR,T AND FRANCOISE BINDLERT
`
`+Laboratoire de Chimie Analytique et Sciences de 1"Aliment, lPHC—DSA, Universite de Strasbourg,
`CN RS, 74, route du Rhin, 67400 lllkirch, France, and iiNestle’ Research Center, Nestec Limited, Post Office
`Box 44, CH—1000 Lausanne 26, Switzerland
`
`
` his study is a contribution to the exploration of natural phospholipid (PL) sources rich in long-chain
`polyunsaturated fatty acids (LC-PUFAs) with nutritional interest. Phosphatidylcholines (PCs) were
`purified from total
`lipid extracts of different food matrices, and their molecular species were
`
`separated and identified by liquid chromatography—electrospray ionization—tandem mass spectro-
`
`
`metry (LC- SI M82). Fragmentation of lithiated adducts allowed for the identification of fatty acids
`linked to the glycerol backbone. Soy PC was particularly rich in species containing essential fatty
`acids, such as (18:2—18:2)F’C (34.0%), (16:0—18:2)
`3C (20.8%), and (18:1—18:2)PC (16.3%). PC
`from anima sources (ox liver and egg yolk) contained
`major molecular species, such as (16:0—18z2)
`PC, (16:0—18:1)PC, (18:0—18:2)PC, or (18:0—18:1)PC. Finally, marine source (krill oil), which was
`
`
`
`source for food supplementation with LC-PUFA—PLs, particularly eicosapentaenoic acid ( PA) and
`
`docosahexaenoic acid (DHA).
`
`
`particularly rich in (16:0—20:5)PC and (16:0—22:6) 3C, appeared to be an interesting potential
`
`KEYWORDS: Phosphatidylcholine; PUFA; supplementation; molecular species; lithium; LC—ESl—MS
`
`INTRODUCTION
`
`Polyunsaturated fatty acids (PUFAs) play very important roles
`in many aspects of human health, in particular in reducing risks of
`cardiovascular diseases,
`inflammation, hypertension, allergies,
`and immune and renal disorders (1, 2), Among these,
`linoleic
`acid (18:2) and d—linolenic acid (18:3) are considered as essential
`fatty acids (FAs), because they have to be necessarily supplied by
`the diet and cannot be synthesized by the human organism.
`
`Moreover, they are the precursors of long—chain PUFAs (LC—
`
`
`PUFAs), in particular eicosapentaenoic acid ( PA) and docosa—
`hexaenoic acid (DHA), which are essential for brain and retina
`development (3—5). It is still unclear though whether the con—
`sumption of the precursors (18:3 and 18:2)
`is sufficient
`to
`synthesize the necessary amounts of LC—PUFAs or if diet
`supplementation with EPA and DHA may be required. In fact,
`while some reports showed infant formulas supplemented with
`18:3 giving retina development comparable to human breast milk
`(containing DHA) (6, 7), others showed reduced visual maturity
`
`with infant formulas supplemented with 18:3 compared to for—
`
`
`mulas that were supplemented with )HA (4,8). However, there is
`agreement among investigators on the crucial importance of
`PUFAs in the diet and their essential role in human nutrition.
`
`
`*To whom correspondence should be addressed. Telephone: + 33—
`390—244—326.
`Fax:
`+ 33—390—244—325.
`E—mail:
`eric.marchi0ni@
`pharma.u—strasbg.fr.
`
`
`
`The natural molecular forms of PUFAs are typically triacyl—
`glycerols (TAGS) and phospholipids (PLs). While TAGs are quite
`a homogeneous group of lipids, PLs can be divided into three
`classes: glycerophospholipids, ether glycerolipids, and sphingo—
`phospholipids. Glycerophospholipids represent the most wide—
`spread PL class and can be divided into subclasses according to
`their polar head, with phosphatidylcholine (PC) being the pre—
`dominant one. In the human dict, TAGs are the major carriers of
`7As, with 50— 100 g/day for an adult, followed by PLs, with 2— 10
`g/day (9). Studies showed however that, when it comes to FA
`biodisponibility, PLs are much more efficient carriers than
`"AGs(3,10, II).
`Supplementation of food products with PUFA—rich phospho—
`lipids (PUFA—PLs) has recently emerged as an interesting way of
`increasing the assimilation and the health benefits of LC—PUFAs
`
`in the human body. The preparation of food supplements con—
`
`
`taining pure molecular species (rich in 18:2, 18:3, DHA, and a PA)
`is not only technically challenging but may turn out to be costly
`and industrially unrealistic. The solution may however lie in the
`exploration and tapping of natural PL sources rich in molecular
`species with PUFAs of nutritional interest.
`This study describes the development of an analytical method
`intended for the determination of molecular species from glycer—
`ophospholipids. PC was picked as the model and was extracted
`and purified from various food matrices: soy as a plant source,
`egg yolk and ox liver as animal sources, and krill oil as a marine
`
`pubs.acs.org/JAFC
`
`Published on Web 08/22/2009
`000001
`
`©2009 American Chemical Society
`
`AKER877ITCOO740544
`
`Petition for Inter Panes Review
`of Us, Patent 8,278,351
`Exhibit
`ENZYMOTEC -1013
`
`
`
`000001
`
`

`

`Article
`
`animal source. Molecular species profiles were determined using
`liquid chromatography—electrospray ionization—tandem mass
`spectrometry (LC—ESI—Msz) by fragmenting lithiated adducts
`of the 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 MET ODS
`
`
`
`Materials. All solvents used for high—performance liquid chromatog—
`raphy (HPLC) analyses were {PLC—grade. Methanol was purchased from
`VWR (Strasbourg, France), and acetonitrile was purchased from Sigma—
`Aldrich (Steinheim, Germany). Chloroform (Sigma—Aldrich, Steinheim,
`Germany), methanol, and n—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 ox liver (1.7% PC) were
`purchased from a local retailer. Krill oil (29.0% PC) has been kindly
`provided by Nestec SA (Lausanne, Switzerland)
`Sample Preparation and Lipid Extraction. Preparation was differ-
`ent for each sample depending upon its physical state. Both soy beans and
`ox 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
`1 g of the treated food matrix was suspended in 30 mL of a CHCi3/CH3OH
`(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%, wfv). The
`organic phase containing the lipid fraction was removed, evaporated
`under vacuum, and dried under a gentle stream of N2. The total lipid
`extract was weighed and dissolved in 1 mL of CHC13.
`Liquid Chromatography. Flash Chromatograp/iy. 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 sur la Sorgue, France) onto a 15 g silica cartridge precondi—
`tioned with n—heptane. Elution was performed at a flow rate of 10 mL/min
`using 60 mL of CHClg, then 120 mL of CHClg/CH30H/H20 (35:65:4,
`V/vj‘V) mixture, and finally 120m L ofethanol for a column wash. Fractions
`(10 mL) were collected and tested by thin—layer chromatography (TLC)
`using silica plates, CHClyCHgOH/HZO (3526524, v/v/v) as an eluent, and
`phosphomolybdic acid in ethanol (20:80, W) 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 detector, and a 717Plus autosampler (Waters, 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 of 45 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;
`CH30H (88:12, W) to CHClg/CH3OH/l M formic acid adjusted to pH 3
`with triethylamine (28:60:12, ViV/V) (13). This chromatographic system
`and this gradientwere both used for PC purification and to check purity of
`the isolated PC.
`ipidic
`To isolate PC from ox liver, a 100 mg aliquot of the ox liver
`extract was injected on a 250 x 10 mm, 10 ,er normal—phase Lichrospher
`column (Interchim, Montlucon, France). A total of2/3 ofthe mobile—phase
`flow, which was set at a rate of 2 mL/min, was diverted, ahead of the * LSD
`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 >< 3 mm. 3 pm Luna normal phase
`(Phenomenex, Le Pecq, France) using a flow rate of 0.5 mL/min. Purity (P)
`
`
`
`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`of the isolated PC was calculated as follows:
`
`6015
`
`P
`
`Arc
`:—><
`At
`
`100
`
`where AFC is the PC peak area and AL is the total of peak areas on the
`chromatogram.
`Separation and Identification ofMolecular Species by LC—
`ESI—iMS. Molecular species of PC were determined using a Prostar
`HPLC system made of two 210 solvent delivery modules, a 410 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 pm Isis Nucleodur column (Macherey—Nagel) using an isocratic flow of
`CH3CN1°CH3OH (40:60. Viv) containing 0.1% NH4OH at a rate of 1 mL/
`min. A split system however allowed the HPLC effluent to enter the mass
`spectrometer at a flow rate of0.2 mL/‘min. To help the identification ofthe
`molecular species, 3 mL of 10 mM lithium formate was added to 1 L of
`mobile phase. Major detected ions were [M+ Li]+ referring to the mo—
`lecular ion with a lithium adduct. Acquisition was performed in positive
`mode in a mass range between m/z 450 and 900. mi: ([M + Li]+) were
`fragmented in positive mode (M82), 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/:. 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:
`
`%
`
`_ A(peak(m/z))
`7 2A(peaks)
`
`X 100
`
`where A(peak(m,°z)) refers to the peak area of the selected mfz and
`ZA(peaks) refers to the sum of all peak areas. Analyses were performed
`
`in triplicate, with results expressed as mean a: standard deviation (SD).
`Gas Chronriaiagraphic 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
`
` able 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 (%)"
`W“
`(W
`cob
`
`14:0
`15:0
`16:0
`16:1 (n—7)
`17:0
`18:0
`
`ridC
`nd
`19.9 :: 0.03
`nd
`0.2 :: 0.01
`4.8 :: 0.02
`
`
`
`9.4 :: 0.08
`57.6 :: 0.10
`5.5 :: 0.01
`nd
`l'ld
`nd
`nd
`nd
`0.8 :: 0.01
`nd
`nd
`0.5 :: 0.01
`nd
`nd
`0.3 :: 0.01
`26.1
`9.4
`
`
`
`
`
`nd
`0.3 :: 0.01
`34.1 :: 0.2
`1.6 :: 0.03
`nd
`13.6 :: 0.2
`
`29.3 :: 0.4
`16.0 :: 0.2
`nd
`nd
`nd
`nd
`nd
`
`4.5 :: 0.02
`nd
`nd
`nd
`nd
`nd
`nd
`
`0.8 :: 0.7
`48.0
`30.9
`
`
`
`2.2 :: 0.09
`0.4 :: 0.02
`26.8 :: 0.1
`1.8 :: 0.08
`0.2 :: 0.06
`1.6 :: 0.05
`
`5.7 :r: 0.03
`3.0 :: 0.02
`1.7 :r: 0.01
`0.1 :1: 0.05
`0.6 :r: 0.01
`2.4 :1: 0.01
`nd
`0.6 :r: 0.01
`nd
`1.3 :1: 0.02
`0.8 a: 0.01
`nd
`0.5 a: 0.01
`34.1 :: 0.07
`15.4 :: 0.1
`81.2
`9.9
`
`
`
`nd
`I’ld
`20.3 :: 0.6
`nd
`0.5 :: 0.4
`36.4 :: 0.02
`
`11.7 :r: 0.5
`21.2 :: 0.7
`1'10
`nd
`1.5 :r: 0.08
`nd
`2.5 :1: 0.07
`5.8 :r: 0.2
`nd
`nd
`nd
`nd
`nd
`nd
`1’10
`57.2
`13.3
`
`18:1 (11'9)
`18:2 (rt—6)
`18:3(1’1'3)
`20:0
`20:1 (“'1 1)
`20:2 (n—6)
`20:3 (n—3)
`2024 (11-6)
`22:0
`22:1 (n—13)
`22:2 (n—6)
`24:0
`24:1 (n—9)
`20:5 (11-3)
`22:5 (n—3)
`2 saturated FAs
`E monounsaturated
`FAs
`Z PUFAs 29.5 54.5 21. 59.0
`
`
`
`
`
`
`
`
`
`
`“ Results (n = 3) are expressed as mean :: SD. 5Percentage of the total peak
`area of FAs. Crid = not detected.
`
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`6016
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`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 gm, Varian].
`Samples (1 ,LtL) were injected. Carrier gas was helium of high purity
`(99.999504). 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’C/min).
`The temperature of 165 0C was held for 1 min and then raised to 225 °C
`(rate of2 C’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, FAME were extracted using n—heptane.
`Gas chromatography (GC) data were normalized, and the percentage of
`each FA was calculated as follows:
`
`% :A(peak(m/z)) X 100
`Z A(peaks)
`
`where A(peak) refers to the area of each identified peak and 2,4(peaks)
`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
`
`Le Grandois et al.
`
`
`step, fractions containing PC were pooled and purity was checked
`
`
`by HPLC—evaporative light—scattering detector (ELS )). 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 ox 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 FA, as
`compared to PC from animal sources. For instance, 18:2
`(57.6%) 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, ox liver, and krill oil. Conversely, 20:4 was identified in egg
`yolk PC (4.5%) and 0x liver PC (5.8%), while it was not detected
`in soy PC.
`As far as egg yolk PC is concerned, 16:0 (34.1%) and 18:1
`(29.3%) were the two main FA identified, followed by 18:2
`(16.0%) and 18:0 (13.6%) (Table 1), which is in agreement with
`a previous report (14). The same :As 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 7A were 20:1 (1.5%) and 20:3
`(2.5%) in ox liver and 16:1 (1.6%) and DHA (0.8%) in egg yolk.
`
`Krill oil PC contained the largest variety of FAs in comparison
`
`
`to other matrices but with *PA (34.1%), 16:0 (26.8%), and DHA
`(16.4%) being the three major FAs. This result is in accordance
`
`
`
`MCounts
`
`MCounts
`
`150 d
`
`125 <
`
`100 ~
`
`75 ,
`
`MCou ts
`
`5
`
`10
`
`25
`
`20
`1 5
`Retention time (min)
`h
`
`MCounts
`
`5
`
`20
`is
`1b
`Retention time (min)
`
`11
`
`25
`
`(D)
`
`
`
`
`50‘
`
`
`
`
` 5
`
`20
`1‘5
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`Retention time (min)
`
`2’5
`
`U'H
`
`16
`
`£0
`-k
`Retention time (min)
`
`£5
`
`Figure 1. Chromatograms of purified PC: (A) soy, (B) ox liver, (C) egg yolk, and (D) krill oil using LC—ESl—MS. Separation was performed using isocratic
`conditions: CHSCN/CHSOH (40:60) containing 0.1% NH4OH at a flow rate of 1 mL/min onto a reverse-phase column (250 >< 3 mm, 3pm). Letters are assigned
`to major identified peaks: a, 18:3—18:3-PC; b, 18:3—18:2—PC; c, 18:2—18:2-PC; d, 15:0—18:3-PC; e, 18:1—18:2-PC; f, 16:0—18:2—PC; g, 16:0—18:1-PC; h,
`18:0—18:2-PC + 18:1—18:1-PC; i, 18:0—18:1-PC;j, 18:0—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, 15:0—18:0-
`PC; p, 16:0—22:6-PC; q, 18:1—22:6-PC; r, 18:0—22:6-PC; s, 14:0—20:5-PC; t, 20:5—20:5—PC; u, 20:5—22:6-PC; v, 22:6—22:6-PC; w, 18:1—20:5-PC; x,
`18:0—20:5-PC; and y, 18:1—20:4—PC.
`
`000003
`
`AKER877ITCOO740546
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`

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`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 FA composition varied according to the period of
`
`
`capture, its PC was always rich in *PA, DHA, and 16:0 (16).
`The relative rates of saturated, monounsaturated, and PUFAs
`were very different among food matrices (Table l). PUFAs were
`most abundant in soy PC (64.5%) 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 ox 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. Chromatographic Separation. Separa—
`tion was achieved using a 3 ,am 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 som‘ce ones: egg yolk and ox liver, where
`several peaks have the same retention times.
`Identification ofPC Jilolecular Species. Molecular species of
`PC were detected as m/: of lithium adducts [M + Li] T. 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 (I 7—I 9). To identify PC
`molecular species lithium adducts were fragmented in l\/'S2,
`which was previously shown to yield three main fragments:
`[Mei — FA]T corresponding to the loss of 21 FA group, [MLi —
`
`FA ai]T corresponding to the loss of a lithium salt of an :A
`group, and [MLi — TMA — FA]T corresponding to the loss of a
`trimethylamine 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 (17).
`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)— TMA]T and [MLi — (16:0)Li]T. The second FA
`was identified as 18:2 based on m/: 484, which corresponds to
`fragment [MLi— (18:2)]T. An additional fragment, m/: 575,
`matched the mass of the PC minus its phosphocholine head with
`a lithium adduct (17). Consequently, the molecule detected as a
`lithium adduct with m/z 764 (RT : 13.2 min) was identified as
`[(16:0—18:2)PC + L1]T.
`Figure 2b shows the MS2 fragmentation spectrum of the
`molecular species m/z 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 fact1espectively to fragments [M Li— (22:6)—
`
`
`
`
`i]T and [MLi — (16:0)]T, showing )HA and 16:0 as the two
`
`«As of the molecule.
`
`A third example is given in Figure 2c with the MS2 spectrum of
`m/: 784 (RT : 6.0 min) containing three fragments that allowed
`
`200
`
`400
`m/z
`
`600
`
`Ions obtained in positive mode in ESI—MS2 between 200 and
`Figure 2.
`600 from fragmentation of lithiated adducts: (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]T. Two of these
`fragments, m/z 506 and 447, were identified as [MLi—(18:3)]T
`and [MLi— TMA — (18. 3)]T, which pointed to 18. 3 as the sole
`3A. The third f1agrnent, m/z 595 corresponded as above fo1m/z
`575, to the loss of the phosphocholine head with a lithium adduct.
`"his 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
`
`AKER877ITCOO740547
`
`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`449
`
`6017
`
`(a) [39/2 764
`
`I
`”1K
`
`200
`
`400
`m/z
`
`600
`
`(b) m/z872
`
`
`
`
`
`200
`
`
`
`(cjm/Z 734
`
`600
`
`595
`
`400
`17712
`
`Yr
`1
`
`1<
`
`5‘76
`
`447
`
`000004
`
`

`

`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`6018
`
`
`
`able 2. Molecular Species Profiles of Each Purified PCa
`
`m/z [M + Li]
`structure"
`retention time (min)
`soy PC (%)C
`egg yolk PC (”/Q)C
`krill all PC (%)C
`ox liver PC (”/Q)C
`
`Le Grandois et al.
`
`738.6
`740.6
`758.6
`
`762.6
`764.6
`
`(16:0—16:1)PC
`(16:0—16:0)PC
`(14:0—20:5)PC
`(14:1 —20:4)PC
`(16:0—18:3)PC
`(16:0—18:2)PC
`(16:1—18:1)PC
`
`11.5
`17.4
`6.5
`6.0
`10.4
`13.2
`13.6
`
`ndd
`0.9 3: 0.2
`nd
`nd
`2.8 : 0.4
`20.8 : 10
`nd
`
`768.6
`778.6
`780.6
`784.6
`
`786.6
`
`788.6
`
`790.6
`
`792.6
`
`794.6
`812.6
`
`814.6
`
`816.6
`
`818.6
`
`832.6
`836.6
`838.6
`840.6
`
`
`
`
`
`
`nd
`nd
`nd
`nd
`nd
`20.2 : 0.7
`nd
`
`
`
`
`
`2.7 3: 0.1
`ml
`3.2 :: 0.0
`nd
`nd
`2.6 : 0.0
`1.1 :: 0.1
`
`
`
`
`
`
`1
`
`nd
`0.7 :: 0.0
`rid
`0.2 :: 0.0
`nd
`10.9 :0 2
`ml
`
`
`
`nd
`0.7 :: 0 3
`0.9 : 0.0
`0 2 :: 0.0
`nd
`nd
`nd
`0.3 :: 0 0
`3.3 2:03
`nd
`3.5 :: 0.4
`2.8 :: 0.4
`34.3 :05
`00
`15 9:05
`ml
`0.4 : 0 0
`nd
`nd
`1 5:: 0.5
`1.2 :: 0.7
`5.7 : 0 3
`
`
`
`nd
`4 5:: 0.1
`nd
`17.5
`(16:0—18:0)PC
`nd
`nd
`nd
`15.5
`(17:0—18:2)PC
`ml
`nd
`nd
`20.9
`(17:0—18:1)PC
`nd
`nd
`0.7 3: 0.2
`6.0
`(18:3—18:3)PC
`1 4 3: 0.0
`nd
`nd
`5.8
`(14:0—22:6)PC
`1 33: 0.3
`nd
`nd
`6.5
`(16:1—20:5)PC
`nc
`nd
`7.7 3: 1 2
`8.6
`(18:2—18:3)PC
`32.4 3: 0.3
`nd
`nd
`8.1
`(16:0—20:5)PC
`1.0 3: 0.1
`3.3 3: 0 2
`34.0 3: 2.0
`11.0
`18:2—18:2)PC
`4.1 3: 0.1
`nd
`nd
`8.7
`16:0—20:4)PC
`1.0 :i: 0.1
`3.1 :i: 0.3
`16.3102
`13.3
`18:1—18:2)PC
`4.1 3: 0.1
`nd
`nd
`12.6
`16:0—20:3)PC
`6 63:0 9
`8.3 3: 0.7
`6.3 3: 0.3
`18.7
`18:0—18:2)PC
`nd
`333: 0.7
`3.2:: 0.4
`17.8
`18:1—18:1)PC
`ml
`8.9 3: 0.5
`1.6 3: 0.2
`22.4
`18:0—18:1)PC
`11.9 : 0.0
`2.8 : 0.1
`ml
`8.6
`16:0—22:6)PC
`nd
`nd
`nd
`8.2
`18:2—20:4)PC
`7.4 : 0.4
`nd
`nd
`7.5
`18:1 —20:5)PC
`1.3 : 0.4
`nd
`nd
`9.1
`18:0—20:5)PC
`1.9 :01
`I'lCl
`I'lEl
`9.7
`18:1—20:41PC
`ml
`nd
`nd
`10.8
`(18:2—20:3)PC
`
`ml
`4 5 :: 0.3
`ml
`15.5
`18:0—20:4)PC
`nd
`nd
`nd
`15.7
`(18:1—20:3)PC
`nc
`nd
`nd
`16.0
`(18:0—20:3)PC
`nd
`nd
`nd
`16.4
`(18:2—20:1)PC
`nd
`1.7 3: 0.0
`nd
`nd
`4.4
`(20:5—20:5)PC
`0.1 :: 0 1
`nd
`nd
`nd
`9.4
`(20:4—20:4)PC
`nd
`nd
`0 3 3: 0.6
`nd
`8.9
`(18:1—22:6)PC
`
`nd
`1 3: 0.1
`1 3: 0.3
`1
`nd
`12.0
`(18:0—22:6)PC
`0.8 :: 0 0
`nd
`nd
`nd
`13.2
`(20:3—20:3)PC
`nd
`nd
`nd
`nd
`8.2
`(20:1 —20:5)PC
`2.21:: 0.2
`ml
`nd
`nd
`15.2
`(20:4—20:1)PC
`842.6
`nd
`2.3 3: 0.0
`nd
`nd
`4.6
`(20:5—22:6)PC
`858.6
`
`was
`(ac—acwc
`so
`m
`nd
`08:00
`m
`
`a Results (n = 3) are expressed as mean :: SD. bThe position of fatty acids in the glycerol moiety has not been determined. CPercentages of all identified molecular species.
`dnd = not detected.
`
`
`
`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—18:2)—
`PC (3.2%), (16:0—18:3)PC (2.8%), (18:0—18:1)PC (1.6%), and
`(18:3—18:3)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%),
`(18:0—18:2)PC (8.9%), and (1 820—1 8:1)PC (8.3%). Minor species
`included (l8:2—l8:2)PC (3.3%), (18:1—l8:2)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 Pacetti et al. (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 1). To a lesser
`extent, a similar observation was made for 18: l.
`The four main molecular species in ox liver were the same as in
`the other animal source food, egg yolk, albeit
`in different
`proportions: (18:0—18:2)PC (34.3%), (18:0—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 ox liver (Table 1). Bovine liver PC molecular species have been
`determined by Bang et al. (20) and Dobson et al. (23). With a
`content of21.59%, (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—18:2)PC, (18:0—
`18:1)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 al.
`is probably
`
`000005
`
`AKER877ITC00740548
`
`000005
`
`

`

`Article
`
`wrong. Minor species found in ox liver included (18:0—20z3)—
`PC + (18:2—20:1)PC (5.5%), (18:0—20:4)PC + (18:1—20:3)PC
`(57%),
`(18:1—18:2)PC (35%),
`(16:0—20:4)PC (3.3%), and
`(16:0—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 (Table 2).
`As far as kn'll 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 (l6:0—20:4)PC (4.1%), were
`less abundant. Once again, the patterns of molecular species and
`
`7A coincided, because both determinations indicated that EPA,
`
`)HA, and 16:0 were the predominant FAs. Interestingly, minor
`molecular species of krill oil PC included two [(14:0—20:5)PC
`(32%) and (14:0—22:6)PC (14%)] 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 4PA 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—PUFA—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 (n—3), eicosapentaenoic acid; DHA or 22:6 (n—3), docosahex—
`aenoic acid; PL, phospholipid; PUFA—PL, PUFA—rich PL; PC,
`
`ohosphatidylcholine; TAG, triacylglycerol; LC, liquid chroma—
`
`tography; *SI, electrospray ionization; MS, mass spectrometry;
`<LSD, eve porative light—scattering detector; GC, gas chroma—
`tography;
`:ID, flame ionization detector; 14:0, myristic acid;
`16:0, palmitic acid; 16:1 (n—7), palmitoleic acid; 17:0, heptadeca—
`noic acid; 18:0, stearic acid; 18:1 (n—9), oleic acid; 18:2 (n—6),
`linoleic acid; 18:3 (n—3), ot—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.
`
`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`
`6019
`
`(3) Amate, L.; Gil, A.; Ramirez, M. Feeding infant piglets formula with
`long—chain polyunsaturated fatty acids as triacylglycerols or phos—
`pholipids influences the distribution of these fatty acids in plasma
`lipoprotein fractions. J. Nutr. 2001, 13/, 1250—1255.
`(4) Heinemann, K. M.; Waldron, M. K.; Bigley, K. E.; Lees, G. E.;
`Bauer, J. E. Long—chain (n—3) polyunsaturated fatty acids are more
`efficient than d—linolenic acid in improving electroretinogram re—
`sponses ofpuppies exposed during gestation, lactation, and weaning.
`J. Nutr. 2005, 135, 1960—1966.
`(5) Horrocks, L. A.; Yeo Young, K. Health benefits of docosahexaenoic
`acid (DHA). Pliarmacal. Res. 1999, 40, 21 1—225.
`(6) Auestad, N.; Montalto, M. 3.; Hall, R. T.; Fitzgerald, K. M.; Wheeler,
`R. E; Connor, W. E; Neuringer, M.; Connor, S. L.; Taylor, J. A.;
`Hartmann, E. E. Visual acuity, erythrocyte fatty acid composition, and
`growth in term infants fed formulas with long chain polyunsaturated
`fatty acids for one year. Pediair. Res. 1997, 41, 1—10.
`(7) Innis, S. M.; Nelson, C. M.; Lwanga, D.; Rioux, F. M.; Waslen, P.
`Feeding formula without arachidonic acid and docosahexaenoic acid has
`no effect on preferential looking acuity or recognition memory in healthy
`full—term infants at 9 mo of age. Am. J. Clin. Nutr. 1996, 64, 40—46.
`(8) Makrides, M.; Simmer, K.; Goggin, M.; Gibson, R. A. Erythrocyte
`docosahexaenoic acid correlates with the visual response of healthy,
`term infants. Pediatr. Res. 1993, 33, 425—427.
`(9) Parmentier, M.; Sayed Mahmoud, C.; Linder, M.; Fanni, J. Polar
`lipids: n—3 PUFA carriers for membranes and brain: Nutritional
`interest and emerging processes. OCL 2007, [4, 224—229.
`(10) Lemaitre—Delaunay, D.; Pachiaudi, C.; Laville, M.; Pousin, J.;
`Armstrong, M.; Lagarde, M. B