`
`J. Agric. Food Chem. 2009, 57, 6014-6020
`DO|:10.1021/j1‘900903e
`
`JOURNALO
`
`AGRICULTURAI:/\_t">
`FOOD CHEMISTRY
`
`Investigation of Natural Phosphatidylcholine Sources:
`Separation and Identification by Liquid
`Chromatography—Electrospray |onization—Tandem Mass
`Spectrometry (LC—ES|—MS2) of Molecular Species
`
`JULIE LE GRANDoIs,l' ERIC MARcHIoN1,*’l‘ MINJIE ZHAo,l' FRANCESCA GIuFFR1DA,i3
`SAlD ENNAHAR,T AND FRANCOISE BINDLERT
`
`+Laboratoire de Chimie Analytique et Sciences de l”Aliment, lPHC—DSA, Universite de Strasbourg,
`CN RS, 74, route du Rhin, 67400 lllkirch, France, and ‘Nestle’ Research Center, Nestec Limited, Post Office
`Box 44, CH—l000 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—e|ectrospray ionization—tandem mass spectro-
`metry (LC- SI MS2). 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%), (1620-1822)
`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 (1620-1822)
`PC, (16:O—18:1)PC, (18:O—18:2)PC, or (18:0—18:1)PC. Finally, marine source (krill oil), which was
`particularly rich in (16:0—20:5)PC and (16:0—22:6) DC, appeared to be an interesting potential
`source for food supplementation with LC-PUFA—PLs, particularly eicosapentaenoic acid ( PA) and
`docosahexaenoic acid (DHA).
`
`KEYWORDS: Phosphatidylcholine; PUFA; supplementation; molecular species; lithium; LC—ES|—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 (1822) and d—linolenic acid (l8:3) 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 (1823 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
`l8:3 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.1narchioni@
`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 diet, 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 * 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
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`Published on Web 06/22/2009
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`©2009 American Chemical Society
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`animal source. Molecular species profiles were determined using
`liquid chromatography—electrospray ionization—tandem mass
`spectrometry (LC—ESl—MS2) 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, Molsheini, 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 al. (12), with minor modifications. A total of
`l g of the treated food matrix was suspended in 30 mL of a CHCl3/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%, w,°v). 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 CHCI3.
`Liquid Chromatography. Flash C'iirnnzatogi'cip/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 CHCl3, then 120 inL of CHCl3/CH3OH/H20 (35:65:4,
`vjvfv) mixture, and finally 120 m L ofethanol for a column wash. Fractions
`(10 mL) were collected and tested by thin—layer chromatography (TLC)
`using silica plates, CHCl3/‘CH3OH/H20 (35:65:4, v/v/v) as an eluent, and
`phosphomolybdic acid in ethanol (20:80, vfv) 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 7l7Plus 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 CHCI3;
`CH3OH (88:12, v/v) to CHCl3/CH3OH/l M formic acid adjusted to pH 3
`with triethylamine (28:60:l2, v,«v/V) (/3). 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 l0 mm, l0 ,LiI11 normal—phase Lichrospher
`column (lnterchim, Montlucon, France). A total ofz/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 [1111 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:flxio0
`.41
`
`where AFC is the PC peak area and AL is the total of peak areas on the
`chromatogram.
`Separation and Identification ofM0/eculai‘ 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 C18 reverse—phase 250 X 3 mm,
`3 pm Isis Nucleodur column (Macherey—Nagel) using an isocratic flow of
`CH3CNi°Cl-l3OH (40:60, v/v) 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 l 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 (MS2), 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/2))
`2 A(peaks)
`
`><l00
`
`where A(peak(m,°z)) refers to the peak area of the selected mfz and
`Z/t(peaks) refers to the sum of all peak areas. Analyses were performed
`in triplicate, with results expressed as mean 3: standard deviation (SD).
`Gas Clr'1r0mat0grap/iic 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 of Purified PC from Soybeans, Egg Yolk, Krill Oil, and Ox
`Livera
`
`FAs
`
`14:0
`15:0
`16:0
`16:1 (ri—7)
`17:0
`18:0
`
`18:1 (11-9)
`18:2 (H-6)
`18:3 (11-3)
`20:0
`20:1 (11-11)
`20:2 (n—6)
`20:3 (n—3)
`2014 (11-6)
`22:0
`22:1 (n—13)
`22:2 (n—6)
`24:0
`24:1 (n—9)
`20:5 (n—3)
`22:5 (113)
`Z saturated FAs
`2 monounsaturated
`FAs
`
`soy PC (%)"
`
`rid“
`rid
`19.9 :: 0.03
`rid
`0.2 :: 0.01
`4.8 :: 0.02
`
`9.4 :: 0.08
`57.6 :: 0.10
`5.5 :: 0.01
`rid
`|'iCi
`rid
`rid
`lid
`0.8 :: 0.01
`rid
`rid
`0.5 :: 0.01
`rid
`rid
`::
`26.1
`9.4
`
`egg yolk PC
`1°/ab
`
`krill oil PC
`mi"
`
`rid
`0.3 :: 0.01
`34.1 :: 0.2
`1.6 :: 0.03
`rid
`13.6 :: 0.2
`
`29.3 :: 0.4
`16.0 :: 0.2
`nd
`nd
`nd
`rid
`nd
`4.5 :: 0.02
`nd
`nd
`nd
`rid
`nd
`rid
`::
`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 :i: 0.03
`3.0 :: 0.02
`1.7 :i: 0.01
`0.1 3: 0.05
`0.6 :i: 0.01
`2.4 3: 0.01
`rid
`0.5 :i: 0.01
`rid
`1.3 3: 0.02
`0.8 3: 0.01
`rid
`0.5 3: 0.01
`34.1 :: 0.07
`::
`31.2
`9.9
`
`ox liver PC
`(%)"
`
`rid
`rid
`20.3 :: 0.6
`rid
`0.5 :: 0.4
`36.4 :: 0.02
`
`11.7 :i: 0.5
`21.2 :: 0.7
`|'i[i
`rid
`1.5 :i: 0.08
`rid
`2.5 3: 0.07
`5.8 :i: 0.2
`rid
`rid
`rid
`rid
`rid
`rid
`|'lCi
`57.2
`13.3
`
`Z PUFAs
`
`54.5
`
`21.
`
`59.0
`
`29.5
`
`“ Results in = 3) are expressed as mean :: SD. “Percentage of the total peak
`area of FAs. Ciid = not detected.
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`
`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 ,ML) were injected. Carrier gas was helium of high purity
`(99.9995%). 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 °C (rate of 15 °C/min).
`The temperature of 165 °C was held for 1 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—Fa1lavier, 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/2)) X 100
`Z A(peaks)
`
`where A(peak) refers to the area of each identified peak and ZA(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—po1ar 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 1ight—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 ox 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
`
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`
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`100 ~
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`75 ,
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`Retention time (min)
`
`25
`
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`Retention time (min)
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`
`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: CHECN/CH3OH (40:60) containing 0.1% NH4OH at a flow rate of 1 mL/min onto a reverse-phase column (250 >< 3 mm, 3,um). Letters are assigned
`to major identified peaks: a, 18:3—18:3-PC; b, 18:3—18:2-PC; c, 1822-1822-PC; d, 1520-1823-PC; e, 18:1—18:2-PC; f, 1620-1822-PC; g, 16:O—18:1-PC; h,
`1820-1822-PC +18:1—18:1-PC;i,18:0—18:1-PC;j,1820-2024-PC;k,18:0—20:3-PC;|,18:1—20:4-PC;m,18:2—20:3-PC;n, 1620-2025-PC;o,1520-1820-
`PC; p, 1620-2226-PC; q, 18:1—22:6-PC; r, 18:0—22:6-PC; s, 14:0—20:5-PC; t, 2025-2025-PC; u, 20:5—22:6-PC; v, 22:6—22:6-PC; w, 1821-2025-PC; X,
`1820-2025-PC; and y, 18:1—20:4-PC.
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`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 1). 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 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 source 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 -1- 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 l\/S2,
`which was previously shown to yield three main fragments:
`[M,4]: — FA]+ corresponding to the loss of a FA group, [MLi —
`FA 4]'.]+ corresponding to the loss of a lithium salt of an 7A
`group, and [MLi — TMA — FA]+ 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 -1 — (16:0) — TMA]t and [MLi — (l6:0)Li]+. The second FA
`was identified as 18:2 based on m/: 484, which corresponds to
`fragment [MLi— (l8:2)]+. 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
`[(l6:0—l8:2)PC + Liit.
`7
`Figure 2b shows the MS“ fragmentation spectrum of the
`molecular species m/: 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 — (22:6)—
`i]+ and [MLi — (l6:0)]+, showing WHA and 16:0 as the two
`«As of the molecule.
`
`A third example is given in Figure 2c with the MS3 spectrum of
`m/: 784 (RT = 6.0 min) containing three fragments that allowed
`
`600
`
`595
`
`
`
`600
`
`0%
`
`200
`
`400
`m/z
`
`Ions obtained in positive mode in ES|—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-l8:3)PC+ Li]+. Two of these
`fragments, m/2 506 and 447, were identified as [MLi—(l8:3)]+
`and [MLi — TMA — (l8:3)]+, which pointed to 18:3 as the sole
`VA. 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/2 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
`
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`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`449
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`(a) 50/2 764
`
`/,f*’<
`
`200
`
`400
`m/z
`
`600
`
`(17) m/Z872
`
`100%
`
`0
`750/
`
`Q)
`QC
`{U
`"CC:
`9
`3
`m 50%,
`Q)
`.2
`
`25%
`
`E E
`
`0%
`
`100%
`
`<1:
`8 75%
`C5
`'UC
`
`3 g
`
`03
`.24-3
`.903
`o:
`
`§
`g
`
`C:
`
`3 3
`
`500/
`°
`
`3
`L3
`E 25%
`
`0
`50%
`
`25%
`
`0%
`
`100%
`
`.,
`75%,
`
`200
`
`(C)/77/Z 784
`
`400
`mzz
`
`Y/f
`J
`
`1<
`
`505
`
`447
`
`
`
`000004
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`
`
`6018
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`J. Agric. Food Chem, Vol. 57, No. 14, 2009
`
`able 2. Molecular Species Profiles of Each Purified P03
`
`Le Grandois et al.
`
`m/2 [M + Li]
`
`structureb
`
`retention time (min)
`
`soy PC (%)”
`
`egg yolk PC (°/Q)“
`
`krill all PC (%)°
`
`ox liver PC (°/Q)“
`
`738.6
`740.6
`758.6
`
`762.6
`764.6
`
`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
`
`842.6
`858.6
`884.6
`
`(16:O—16:1)PC
`(16:O—16:O)PC
`(14:O—20:5)PC
`(14:1—20:4)PC
`(16:O—18:3)PC
`(16:0—18:2)PC
`(16:1—18:1)PC
`
`(16:0—18:0)PC
`(17:0—18:2)PC
`(17:0—18:1)PC
`(18:3—18:3)PC
`(14:0—22:6)PC
`(16:1—20:5)PC
`(18:2—18:3)PC
`(16:0—20:5)PC
`1812-1812030
`16:0—20:4)PC
`18I1—18I2)PC
`15I0—20I3)PC
`18:0—18:2)PC
`18:1—18:1)PC
`18:0—18:1)PC
`16:0—22:5)PC
`18:2—20:4)PC
`18:1 —20:5)PC
`18:0—20:5)PC
`18I1—20I41PC
`(18:2—20:3)PC
`18:0—20:4)PC
`(18:1—20:3)PC
`(18:0—20:3)PC
`(18:2—20:1)PC
`(20:5—20:5)PC
`(20:4—20:4)PC
`(18:1—22:6)PC
`(18:0—22:6)PC
`(20:3—20:3)PC
`(20:1—20:5)PC
`(20:4—20:1)PC
`(20:5—22:6)PC
`(22:6—22:6)PC
`
`11.5
`17.4
`6.5
`6.0
`10.4
`13.2
`13.6
`
`17.5
`15.5
`20.9
`6.0
`5.8
`6.5
`8.6
`8.1
`11.0
`8.7
`13.3
`12.5
`18.7
`17.8
`22.4
`8.6
`8.2
`7.5
`9.1
`9.7
`10.8
`15.5
`15.7
`16.0
`16.4
`4.4
`9.4
`8.9
`12.0
`13.2
`8.2
`15.2
`4.6
`5.0
`
`ndd
`0.9 :2 0.2
`nd
`nd
`2.8 22 0.4
`20.82210
`rid
`
`rid
`rid
`nd
`0.7 :2 0.2
`rid
`nd
`7.7 :2 1 2
`rid
`34.0 :i: 2.0
`rid
`15.3 :i: 0.2
`|'lC1
`6.3 :2 0.3
`3.2 22 0.4
`1.6 :2 0.2
`rid
`rid
`rid
`rid
`|'l[1
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`rid
`
`rid
`rid
`nd
`nd
`nd
`20.2 :2 0.7
`rid
`
`4 5220.1
`rid
`nd
`rid
`rid
`nd
`rid
`rid
`3.3 :i: 0 2
`rid
`3.1 :i: 0.3
`fld
`8.3 :2 0.7
`33:20.7
`8.9 :2 0.5
`2.8 :2 0.1
`rid
`rid
`rid
`|'lC1
`rid
`4 5 22 0.3
`rid
`rid
`rid
`rid
`rid
`0 3 :2 0.6
`1
`1 :2 0.3
`rid
`rid
`nd
`rid
`nd
`
`2.7 :2 0.1
`nd
`3.2 22 0.0
`nd
`nd
`2.6 220.0
`1.1 220.1
`
`rid
`rid
`rid
`rid
`1 4 :2 0.0
`1 3:: 0.3
`nc
`32.4 :2 0.3
`1.0 :i: 0.1
`4.1 :2 0.1
`1.0 :i: 0.1
`4.1 :i: 0.1
`6 6:20 9
`rid
`rid
`11.9 220.0
`rid
`7.4 22 0.4
`1.3 22 0.4
`1.9 :01
`rid
`rid
`rid
`nc
`rid
`1.7 :2 0.0
`rid
`rid
`1 :2 0.1
`rid
`rid
`rid
`2.3 :2 0.0
`0.8 :2 0.0
`
`1
`
`rid
`0.7 22 0.0
`nd
`0.2 22 0.0
`nd
`10.9220 2
`rid
`
`rid
`0.7 22 0 3
`0.9 22 0.0
`D 2 22 0.0
`rid
`rid
`rid
`0.3 22 0 0
`3.3 ::03
`rid
`3.5 :: 0.4
`2.8 :: 0.4
`34.32205
`rid
`15 92205
`rid
`0.4 2: 0 0
`rid
`rid
`1 5:115
`1.2 :: 0.7
`5.7 22 0 3
`
`5.5 22 0 2
`
`rid
`0.1 22 0 1
`rid
`rid
`0.8 22 0 0
`rid
`2.2122 0.2
`rid
`rid
`
`“ Results (n = 3) are expressed as mean 22 SD. “The position of fatty acids in the glycerol moiety has not been determined. “Percentages of all identified molecular species.
`dnd = not detected.
`
`(18:2— l8:2)PC (34.0%), followed by (16:0—18:2)PC (20.8%) and
`(l8:l—l8:2)PC (16.3%), while minor ones included (18:0—l8:2)-
`PC (3.2%), (16:0—18:3)PC (2.8%), (18:0—l8:l)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 1), 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
`(l6:0—l8:l)PC (39.7%), followed by (l6:0—18:2)PC (20.2%),
`(18:0—l8:2)PC (8.9%), and (1 820-1 8:l)PC (8.3%). Minor species
`included (l8:2—l8:2)PC (3.3%), (l8:l—l8:2)PC (3.1%), (16:0-
`22:6)PC (2.8%), and (l8:O—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 (l6:0—l8:l)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: 1.
`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—l8:2)PC (34.3%), (18:0—18:l)PC (15.9%),
`(16:0—l8:2)PC (10.9%), and (l6:0—l8:l)PC (9.1%). This
`matched the FA pattern, which showed 18:0 (36.4%), 18:2
`(21.2%), 16:0 (20.3%), 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%, (l8:0—2l: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—l8:2)PC, (18:0-
`l8:l)PC, and (l6:0—l8:l)PC being the main species and no
`(l8:0—2l:3)PC being detected (23). These remarks let us think
`that the identification performed by Bang et al.
`is probably
`
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`000005
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`Article
`
`wrong. Minor species found in ox liver included ( 18:0—20:3)—
`PC -1- (18:2—20:1)PC (5.5%), (18:0-20:4)PC -1- (18:l—20:3)PC
`(5.7%),
`(18:1—18:2)PC (3.5%),
`(l6:0—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 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 (Euphazr.siapac*zficia) (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
`(l6:0—22:6)PC (11.9%), (l8:1—20:5)PC (7.4%), (18:0—18:2)PC
`(6.6%), (l6:0—l8:l)PC (5.9%), and (l6:0—20:4)PC (4.1%), were
`less abundant. Once again, the patterns of molecular species and
`EA coincided, because both determinations indicated that EPA,
`)HA, and 16:0 were the predominant FAs. lnterestingly, minor
`molecular species of krill oil PC included two [(14:0-20:5)PC
`(3.2%) and (14:0—22:6)PC (l.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—PUFA—PLs. As an
`example, krill oil was shown as a potential source for food
`supplementation with *PA and DHA.
`
`ABBREVIATIONS USED
`
`‘PA or
`long—chain polyunsaturated fatty acid;
`LC—PUFA,
`20:5 (n—3), eicosapentaenoic acid; DHA or 22:6 (n—3), docosahex—
`aenoic acid; PL, phospholipid; PUFA—PL, PUFA—rich ?L; PC,
`ohosphatidylcholine; TAG, triacylglycerol; LC, liquid chroma-
`tography; *Sl, electrospray ionization; MS, mass spectrometry;
`‘LSD, eve porative light—scattering detector; GC, gas chroma-
`tography; TID, 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—1l), gadoleic
`acid; 20:3 (n—3), dihomo—y—linolenic acid; 20:4 (n—6), arachidonic
`acid; 22:0, behenic acid; 24:0, lignoceric acid.
`
`LITERATURE CITED
`
`(1) Shahidi, F. NZlfl‘aC€l4IiCa'l aria’ Specialty Lipids and Their C0—pr0a’iiers;
`Taylor and Francis: Oxford, U.K., 2006.
`(2) Goodnight, S. H.1r.; Harris, W. S.; Connor, W. E.; Illingworth, D.
`R. Polyunsaturated fatty acids, hyperlipidemia, and thrombosis.
`Arrerioselen, T/2romb., Vase. Biol. 1982, 2, 87-113.
`
`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, /3/, 1250-1255.
`(4) Heinemann, K. M.; Waldron, M. K.; Bigley, K. E.; Lees, G. E.;
`Bauer, 1. E. Long—chain (n—3) polyunsaturated fatty acids are more
`efficient than (1—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). P/iarmaea/. Res. 1999, 40, 21 1-225.
`(6) Auestad, N.; Montalto, M. B.; 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. Pediazr. 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,