Lipids (20ll) 46:25-36
`DOl 10.1007/s11745-010-3472-6
`
`Elucidation of Phosphatidykholine Composition in Krill Oil
`Extracted fron1 Euphausia superba
`
`Bj!'lrn Winther' Nils Hoem . Kjetil Berge'
`Leon Reuhsaet
`
`Received: 9 June 20101 Accepted: 30 Augmt 2010 1Published online: 17 September 2010
`(g Thc Authm(s) 2010. This mticlc is publishcd with open access at Springcrlink.com
`
`Abstract High
`chromatography(cid:173)
`liquid
`performance
`e1ectrospray tandem mass
`spectrometry was used to
`elucidate the phospholipids in krill oil extracted from
`Euphausia superba, an emerging source for human nutri(cid:173)
`tional supplements. Tbe study was carried out in order to
`map the species of the choline-containing phospholipid
`classes: phosphatidy1choEne and lyso·-phosphatidy1choline.
`In addition,
`the prevalent phosphatidylcholine class was
`quantified and the results compared with prior analysis.
`The qualification was performed with separation on a
`reverse phase chromatography column, while the quanti(cid:173)
`fication was obtained with class separation on a nonnal
`phase chromatography column. An Orbitrap system was
`used for the detection, and pulsed-Q dissociation frag(cid:173)
`mentation was utilized for the identification of the species.
`An asymmetrical exclusion list was applied for detection of
`phospholipid species of lower concentration, significantly
`improving the number of species observed. A total of 69
`choline-containing phospholipids were detected, whereof
`60 phosphatidylcholine substances, among others seven
`with probable omega-3 fatty acids in both sn-l and sn-2.
`The phosphatidylchoIine concentration was estimated to be
`(n = 5.L These results confirm the
`34 ± 5 g/lOO g oil
`complexity of the phospholipid composition of krill oil
`and the presence of long chained, heavily unsaturated fatty
`acids.
`
`B. Winl.'1er . L. Reubsaet (C;:<1)
`Department of Pharmaceutical Chemistry,
`School of Pharmacy, Univcrsity of Oslo, Oslo, Norway
`e-mail: j.J.reub;;aet@fatmasi.uio.no
`
`N. Hoem . K. Bcrge
`Aker BioMarine ASA, FjordaIJeen 16, Vika,
`P.O. Box 1423, 0115 Oslo, Norway
`
`Keywl)I'ds Fish oil . Krill oil . Mass spectrometry
`Omega..3 . Phosphatidylc:holine . Phospholipid
`
`Abbreviations
`EPA
`DBA
`lyso..PtdCho
`NPLC
`PtdCho
`PtdEtn
`Ptdlns
`PtdSer
`P10
`RP10C
`
`Eicosapentaenoic acid
`Docosahexaenoic acid
`Lyso-phosphatidylcholine
`Normal phase liquid chromatography
`Phosphatidylcholine
`Phosphatidylethanolamine
`Phosphatidylinositol
`Phosphatidylserine
`Phospholipid
`Reverse phase liquid chromatograpby
`
`Introduction
`
`Krill oil has emerged as an important source of omega-3
`fatty acids for human consumption during the last decade,
`and the amount sold on the world market
`is rapidly
`increasing. In contrast to traditional omega-3 supplements
`on today's market, which are based on omega-3 fatty acids
`bound to triglycerides (such as cod liver oil and fish oil)
`or bonnd as ethyl esters (Omacor/Lovaza), krin oil contains
`a high proportion of omega-3 fatty acids bonnd to
`phospholipids.
`Krill oil has been investigated in several preclinical and
`clinical studies II-4], and there is growing evidence that
`the molecular form of the omega··3 fatty acids (i.e.
`tri(cid:173)
`ethyl-esters,
`phospholipids) might
`be of
`glycerides,
`importance for their biological effect as weH as distribution
`of the omega··3 fatty acids in the body. In one animal study,
`
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`26
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`Lipids (2011) 46:25-36
`
`it was demonstrated that when krill oil and fish oil were
`administrated to Zucker rats with an equimolar dose eico(cid:173)
`sapentaenoic acid (EPA) +- docosahexaenoic acid iDHA),
`krill oil had stronger and in some instances different effects
`than fish oil on specific parameters related to the metabolic
`syndrome [1]. The lipid level in both heart and liver was
`significantly lower in rats treated with krill oil, when
`compared to rats fed the fish oil diet The authors suggest
`this difference may be linked to differences in the
`that
`incorporation of omega-3 fatty acids into membranes, and
`consequently a reduction of inflammatory molecules and
`endocannabinoids, which might be relevant for the differ(cid:173)
`ences observed betvveen fish oil and krill oiL Further, in the
`same study, it was demonstrated that the level of DHA in
`the brain increased significantly after krill oil administra(cid:173)
`tion, but not after fish oil administration, when compared to
`control animals 12l Thus, omega-3 fatty acids linked to
`phospholipids may be ditTerently distributed in the body
`compared to omega-3 fatty acids in other molecular fOlTI1S"
`Moreover, in a clinical safety study, the presence of EPA
`and DHA in the blood plasma >,vas determined after daily
`administration of 2 g krill oil or 2 g menhaden oil for
`4 weeks [3]. The authors concluded thatEPA. and DHA
`from krill oil are absorbed at least as wen as that from
`menhaden oil.
`The aim of the current study was to characterize the
`phospholipids in krill oil in more detail
`to evaluate the
`composition of the fatty acids present in the phospholipids.
`The composition was determined using LC!ESI--?vlS(lJYlS),
`a technique which has lately played an important role in
`characterization of the lipidome in tissues and organisms
`[5J. An inherent limitation in the use of ESI for the ioni(cid:173)
`zation of long chained fatty acids has been described by
`Koivusalo et al. [6]. The study showed that the instrument
`response is affected by the acyl chain length. This is a
`consideration which is important particularly in the quan(cid:173)
`tification of the lipids.
`The elucidation of the phospholipid species is often
`performed either by doing a precursor ion scan or a neutral
`loss scan with triple quadrupole instrumentation [7-9], or
`with MS n ti'agmentation with systems based on ion traps
`[10-l3]. Nonnal phase liquid chromatography (NPLC) and
`reverse phase liquid chromatography (RPLC) are both
`frequently used for the separation of the components [IO,
`l4-17]. Of these two separation techniques, RPLC has
`been shown to be more suitable for species separation and
`characterization 18l
`Different ionization and fragmentation techniques can
`be used for the evaluation of phospholipids. Ionization of
`the phospholipids may be performed in negative- and
`fragmentation of
`positive-ionization mode" In general,
`phospholipids in the positive mode provides information
`about the phospholipid head group, while fragmentation in
`
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`the negative mode is the source of stmctural information.
`For phospholipids
`containing choline-headgroups,
`the
`choline-specific fragment mlz 184 has been used in pre(cid:173)
`cursor ion scanning operating in the positive ionization
`mode for class determination [7, 18]. Also in the negative
`mode, class-specific fragments may be used in the char(cid:173)
`acterization. All phospholipid classes, except those con(cid:173)
`taining choline, yield molecular ions [M-HJ- when a
`formate-based mobile phase is used. On the other hand, the
`choline-containing classes form stable adducts with formic
`acid in the mobile phase, yielding 1M +- FA---Hr ions
`(mIz = M -1- 45) [19, 20]. With fragmentation, this adduct
`dissociates with the loss of (HCOO +- CH3) into the frag(cid:173)
`ion IM---O-l3r-" This is in particularly useful
`in
`ment
`separation. Although the
`methods utilizing RPLC for
`chromatographic class information is lost in such setups,
`the class-specific fragments may be used in the character(cid:173)
`ization of the species I] l J.
`Two ion activation techniques may be used for ]VIS
`analysis utilizing ion traps: collision·induced dissociation
`(CID) and pulsed··Q dissociation (l'QD) techniques. While
`CID has a low mass cut off below 28% of the m!z for the
`precursor ion,
`the novel PQD technique eliminates the
`,n]. This dif·
`potential
`loss of low mass fragments
`ference could be crucial
`in the fragmentation of larger
`molecules into low mass, specific fragments, as shown with
`detection of iTHAQ fragments with a linear ion trap [23].
`The fatty acid composition of phosphatidyIcholine
`(PtdCho) from krill oil has previously been investigated by
`Le Grandois et al.
`[2.t]. This study was pelforrned with a
`method based on the ESI operated in the positive mode
`with triple quadmpole detection of lithium adduct ions, and
`showed the presence of a higher number of PtdCho species
`with long chained unsaturated fatty acids, than seen in egg
`yolk, ox liver and soy.
`We believe the current study verifies previously pre(cid:173)
`sented findings and offer new insights into the composition
`of krill oi1. In addition;
`it shows the advantage of per(cid:173)
`forming an additional fragmentation using an exclusion list
`in the identification of low prevalent species.
`
`Experimental Prucedures
`
`Chemicals
`
`Phospholipid standards of lyso-phosphatidylcholine (1yso(cid:173)
`PtdCho), PtdCho,
`phosphatidylethanolamine
`(PtdEtn),
`phosphatidylinositol
`(PtdIns)
`and
`phosphatidylserine
`(PtdSer) were purchased from Sigma-Aldrich (St. Louis,
`MO, USA). Lyso-PtdCho, PtdCho and PtdEtn were
`lyophilized powders obtained from egg yolk, whereas the
`Ptdlns source was glycine max; and the PtdSer source \vas
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`Lipids (2011) 46:25-36
`
`27
`
`bovine brain. EPAX 6000 1'0'8 fish oil was donated by
`EPAX (Alesund, Norway), and Superba™ bill oil was
`obtained from Aker BioMarine (Oslo, Norway). All other
`chemicals were of MS grade.
`
`Instmmentation
`
`The chromatography was canied out on a Dionex system
`consisting of an Ultimate 3000 pump, an Ultimate 3000 RS
`autosampler, and an Ultimate 3000 flow manager. Detec(cid:173)
`tion was obtained using a linear ion trap LTQ XL coupled
`to an Orbitrap Discovery, LC-operation, data acquisition
`and processing were carried out using Chromelion SDK
`6.80 SP2 Build 2327 and Xcalibur version 2.0.7 coupled
`with DCJ'viSLiflk 2.5 (all Instmment-Teknikk AS, 0steras,
`Norway).
`
`J'vlass Spectrometry
`
`The LTQ Orbitrap system was operated with a spray voltage
`of 5.00 kV, nitrogen as the sheath gas \vith flow rate set to
`30 arbitrary units, and helium as the collision gas. The
`quanti5cation of the PtdCho class was performed with a
`scan from m/z. co:: 400 to mlz. co:: 1,000 operated in negative
`ionization mode.MS n experiments for identitlcation of the
`choline-containing phospholipids were performed using
`data dependent PQD for the5rst fragmentation step. The
`molecular ion selected for each fragmentation in this step,
`was the most intense ion detected by the Orbitrap analyzer
`with target mass resolution of 30,000 and a scan windmv
`from mlz = 400 to In!z = 1,000. The normalized collision
`energy was 200 and the isolation width 2.00 Da. Subse(cid:173)
`quently, the most intense fragment ion detected was further
`fragmented using OD, with the normalized collision energy
`at 35 and an isolation width of 2.00 Da (MS'). The LTQ
`was utilized for the detection of the fragments and the
`ml? range was relative to the m!z of the molecular ion. An
`alternative method was used in order to be able to observe
`species that were not selected for fragmentation in this way.
`The overall setup of this method was as described above,
`with the distinction of adding an asymmetric exclusion list.
`The exclusion list was generated with the purpose of the
`LTQ to ignore already identified substances. The list was
`based on the mlz of the molecular ions, with an exclusion
`window from this mass-to-charge ratio, up to mlz + 1. The
`width of the exclusion window was selected in order to
`diminish the detection of isotopes of the molecular ions.
`
`Chromatographic Conditions RPLC
`
`Chromatographic separation was performed on a ZORBAX
`Eclipse Plus C 18 column with particle diameter of 5 flm
`and the colmnn dimensions were 150 x 2.1 mm i.d.
`
`The mobile phase A consisted of 90 parts j % TEA and
`0.2% formic acid in water, and 10 parts mobile phase B (1'/1').
`Mobile phase B consisted of 1% TEA and 0.2% formic acid
`in 60 parts methanol and 40 parts acetonitrile (vlv).
`A linear gradient was used for the separation. The sys(cid:173)
`tem was first kept isocratic at 65% mobile phase B for
`5 min after injection of sample. The gradient was then run
`ii-om 65 to 100% mobile phase B in 5 min and was kept
`isocratic at 100% mobile phase B for 20 min, before it was
`returned to the initial condition in 0.1 min. The column
`was regenerated with 65% mobile phase B for 16 min. The
`mobile phase flow \vas set to 0.2 mLimin and the injection
`volume \vas 20 pL throughout the study.
`
`Chromatographic Conditions NPLC
`
`NPLC \vas performed on a HiCHROM LiChrospher 100
`DIOL column with a particle diameter of 5 ~!m and column
`dimensions of 250 x 2 mm i.d. Mobile phase C \vas 100%
`chlorofmm, and the mobile phase D consisted of 0.05%
`TEA, 0.05°"j! ammonia and 0.1 (;~ formic acid in methanol
`(vlv). For the class separation of the phospbolipids, a linear
`gradient \vas used. The gradient \vas run from 5 to 27.5(fo
`mobile phase D in is min, fonowed by a rise to 80% in
`2 min to flush the column. This concentration was kept
`isocratic for 4 min, before it was returned to the initial
`condition in 2 min. The column was regenerated with 5(;;:)
`mobile phase D for 12 min. The mobile phase flow was set
`to 0.3 mL/min, and the injection volume was 20 pL
`throughout the study.
`
`Sample Preparation
`
`Samples of krill oil and stock solutions of standards were
`prepared by dissolving the lipids in a mixture of chloro(cid:173)
`form and methanol at a ratio of 2: 1. These solutions were
`stored at -32"C and excessive heating cycles were avoi(cid:173)
`ded. Samples were prepared by further dilution with sol(cid:173)
`vents compatible with the mobile phases used. For NPLC,
`this was achieved with chloroform:MeOH 95:5, while it
`was attained by dilution in mobile phase A for RPLC
`
`Calibration Curve
`
`For the quantification, a calibration curve was established
`with samples of PL free fish oil (EPAX''')) spiked with a
`PtdCho standard purified from egg yolk to concentrations
`of 100 flg/mL. The spiking of PL free fish oil was per(cid:173)
`formed in order to produce comparable matrixes in the
`standards and the krill oil samples. Stock solutions were
`made by dissolving PtdCho standard, PL free fish oil and
`krill oil separately in mixtures of chloroform:J'vleOH 2: 1.
`The concentration of krill oil and PtdCho standard \vas
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`28
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`Lipids (2011) 46:25-36
`
`l mg/mL and for PL free fish oil 10 mg/rnL for these
`solutions. Respectively, 100 ~iL of PtdCho standards and of
`krill oil was added to 900 ~lL of the PL free fish oil, pro(cid:173)
`ducing samples with concentrations of I 00 ~lg/mL. For the
`calibration curve, the PtdCho standard samples were con(cid:173)
`secutively diluted to the desired concentrations of 10.0,
`5.00, 2.50, 1.00, 0.50, 0.25. and 0.10 fig!mL (n = 5) with a
`mixture of chloroform:MeOH 95:5. The krill oil samples
`were diluted in the same way to a concentration of 1.00 fig!
`mL in order to measure the PtdCho content within the
`linear area of response of the calibration curve.
`
`Results
`
`Selection of l\iIS-Mode for PtdCho-Classification
`
`Initial experiments with standards of PtdEtn, PtdIns.
`PtdSer, PtdCho and lyso-PtdCho, were performed in both
`
`positive- and negative- ion mode using the LTQ Orbitrap
`as a detector. Separation of these substances on a NPLC
`column yielded class-specific baseline separation (data not
`shown). The results of these tests indicated a minor dif(cid:173)
`ference in the signal intensities obtained between the two
`settings, with a slightly higher response in negative mode.
`
`Identification of Choline-Containing Phospholipids
`in Krill Oil
`
`Since krill oiL as established in Fig. I, is predominantly
`of
`choline-containing
`phospholipids,
`the
`composed
`emphasis of the work was focused on the elucidation of the
`species in the PtdCho and lyso-PtdCho classes. In NPLC,
`dass separation of the phospholipids is achieved. A dear
`tendency of the elution order from the column was seen
`from high mlz to lower mh. PtdCho class species eluted
`from 6.5 to 8 min, and the elution of the lyso-PtdCho class
`occurred between lO.5 and 12 min. Some species separation
`
`RT: 5.00"'1~).OO
`
`SM:
`
`96
`
`Fig. 1 Base pea.];:
`chromatogram and three
`dimensional (3D) map of
`phospholipid class separation of
`krill oil, performed with a linear
`gradient NPLClESI-MS on a
`HiCHROM LiChrospher 100
`DIOL column (250 x 2 mm
`i.d., 5 ~lm). MS was operated in
`the negative ionization mode
`and set to scan m/z 400-1,000.
`Only the relevant part of the
`chromatogram is shown
`(5-15 min). For the major pmt
`of l.'1e ;;peeie;;, the map show
`adduct ions in the form of
`[M +FA--Hr
`
`10.03
`
`~ L83
`
`13.85
`13.02
`1L20
`I
`I 18.1e
`I li~7 /
`14.40
`Ino
`\;0.74
`"""-~-~~
`
`I
`
`10GO
`
`t
`
`5
`
`7
`
`10
`Time (min)
`
`11
`
`12
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`Lipids (2011) 46:25-36
`
`29
`
`was seen within the PtdCho class, however, this \vas not
`adequate for identification of the diverse species within the
`classes.
`As the identification of the components is perfol1lled
`with data dependent fragmentation, chromatographic sep(cid:173)
`aJ-ations of the substances are critical for detection of the
`less prominent species. Hence, the separation for charac(cid:173)
`terization of the species was performed utilizing a RPLC
`(~18 colun1]} as described under ~~[:xperirnental Proce-
`dure"". This improved the chromatographic performance
`for species separation compared to NPLC (Fig. 2). As lyso(cid:173)
`PtdCho only carry one fatty acyl group, these components
`elute earlier in the chromatogram than the PtdCho species.
`Lyso-PtdCho dominate the region between 12.0 min and
`14.5 min in the chromatogram, while the heavier PtdCho
`components dominate the chromatogram after 14.5 min.
`The identification of the species was performed utilizing
`a MS 3 data dependent fragmentation method, with an initial
`PQD fragmentation resulting in the loss of methyl formate,
`followed by a CID fragmentation. Analysis of the frag(cid:173)
`mentation spectra obtained typically revealed the identity of
`the substances without ambiguity. However, co-elution of
`isobaric compounds could potentially complicate the
`interpretation of the spectra. This challenge is minimized by
`applying a set of criteria for reliable identitlcation. The
`following criteria were applied for reliable interpretation of
`a choline-containing pbospholipid: based on the mass··w(cid:173)
`charge ratio of the molecular ion. it is likely to be a choline(cid:173)
`containing phospholipid (i.e. mh being an even number).
`Following the first CID with PQD, a daughter ion should be
`produced by the loss of (HCaO + CH3) as 60 Da. Fmther
`fragmentation with CID of the resulting product should
`produce specific fragments revealing the nature of the fatty
`acyl groups in both the sn-l and the sn-2 position, either by
`the occurrence of the fragment for the fatty acyl group itself,
`or by the presence of the corresponding fragments of the
`lyso-compound. The sum of the fatty acyl groups elucidated
`in this matter should yield a mass matching the initial
`molecular mass. This is illustrated in Fig. 3, showing the
`elucidated fragment identity for the fragmentation of the
`20:5-22:6 diacyl PtdCho.
`The spectra were generally dominated by fatty acyl
`fragments from both the sn-l and the sn-2 positions in
`addition to their cOlTesponding fragments of the lyso(cid:173)
`compound, ensuring identification of the species. The lyso(cid:173)
`PtdChoand PtdCho substances identified by applying this
`method are presented in Tables 1 and 2. The relative
`intensity of the molecular ions is also presented.
`As described earlier, the use of signal intensities in ]\,IS,
`for concentration comparison of the different substances, is
`only semi-quantitative. However,
`it provides a valuable
`indication of the composition of the PtdCho and lyso(cid:173)
`PtdChoclasses. Chromatograms and fragmentation patlems
`
`are presented in Figs. 4 and 5 t~")r the 10 foremost sub(cid:173)
`stances characterized from the PtdCho class.
`As data dependent fragmentation methods are, by nat(cid:173)
`ure, biased in the selection of the most prevalent sub(cid:173)
`stances, the experiments where repeated with the use of an
`asymmetric exclusion list added to the MS-method. The
`exclusion list was based on the data sets obtained with the
`initial settings (i.e. Tables 1, 2). This method allowed
`the detection and identification of the additional substances
`presented in Table 3.
`
`Quantification of PtdCho-Class in Krill Oil
`
`Krill oil predominantly contains phospholipids from the
`PtdCho class (l:ig. )). It \vas therefore attempted to quan(cid:173)
`tify the absolute concentration of this class by use of class
`separation with NPLC. Quantification of the PtdCho class
`\vas performed with a method developed "in-house" with a
`LTQ Orbitrap mass spectrometer for the detection.
`In the construction of the calibration curve, PtdCho
`concentrations above 1.00 llg/mL resulted in a relative
`decrease in the MS signal response, yielding a quadratic
`polynomial curve (y ;::: ax2 -+ bx: -+- c) where a;::: ---9,766,
`b :::; 330.360 and c ;::: 4763.8 with r2
`;::: 0.9995. From
`0.10 ~!g/mL to 1.00 llg/mL, the calibration cmve showed a
`linearity (r2
`:::: 0.9995) witb a linear
`high degree of
`regression curve (y ;::: 17x +- c) where b :::; 368,737 and
`c·::: ----16,547.
`The latter area was chosen for quantification purpose.
`From this, the PtdCho content of the undiluted krill oil was
`determined to be 34 ± 5% (w/w) (n = 5). Comparisons of
`the mass spectmm of the sample with the spectrum of the
`PtdCho standard indicated a difference in PtdCho class
`composition (Fig. 6). From the results,
`the average acyl
`chain lengths appear to be higher in krill oil than in egg
`yolk. This has also previously been shown by others [2.t].
`As mentioned above, instrument response is affected by the
`acyl chain length of the PtdCho. These differences in chain
`lengths could therefore infl.uence the quantification of the
`PtdCho class as discussed later. The quantitative results
`were compared with an earlier analysis of the krill oiL
`performed by the accredited analytical company Nofima
`(Bergen, Norway). They reported the PtdCho concentration
`in the krill oil sample to be 35 gil 00 g oiL
`
`DiSCUSSIon
`
`The fact that fatty acyl chain lengths of the PtdCho species
`are relatively long, affects both the choice of fragmentation
`technique and the effect of standards used for qmmtifica(cid:173)
`tion purposes. Due to the low mass cut-off limit at 28% of
`the molecular ion mass with CfD fragmentation in ion
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`30
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`Lipids (2011) 46:25-36
`
`traps, fragmentation utilizing PQD in the positive ioniza(cid:173)
`tion mode was chosen as the first fragmentation step. In
`addition, utilizing PQD in the first fragmentation step of a
`MS 3 method, operating with negative mode ionization, also
`yields class elucidation of phospholipids with choline head
`groups. This is achieved by the detection of the [M-CH3](cid:173)
`fragment formed after (HCOO + CH3) loss ii'om formate(cid:173)
`molecular ion adducts. Consequently, perfoDning a mode
`shift hom the positive to the negative ionization mode is
`not necessary for the overall identification of lyso-PtdCho
`and PtdCho class phospholipids. PQD with a normalized
`collision energy at 200 produced the [M---CH3r
`frag(cid:173)
`ments, without
`extensive fragmentation to secondary
`
`ions. Higher collision energies produced sec(cid:173)
`fragment
`ondary fragments that could be used as a source of struc(cid:173)
`tural information; however, this was better achieved with
`CID as a second fragmentation step. The elD was operated
`with a normalized collision energy of 35.
`for
`fruther
`ii'agmentation of the [M-CH3r ion. This value was not
`optimized for the individual species, and additional struc(cid:173)
`tural information could potentially have been achieved by
`specie specific optimization of this setting.
`With the described method for separation and hag(cid:173)
`mentation of the phospholipids,
`typical
`fragmentation
`patterns vvere obtained, as shown in Fig. 3. The spectra
`were dominated by fatty acyl fragment
`ions originating
`
`RT'
`
`10.00 - 35.00
`
`SM:
`
`9B
`
`"
`
`1000
`
`950
`
`900
`
`850
`
`800
`
`750
`
`..
`
`700
`
`!::'
`E
`
`'*.~.. ~..
`.......................•.'"....,.;,,,..,',.".",..
`
`•
`
`~, · l l . "
`·
`·..·
`· · · ···•
`·"""' ~·t··
`............,.L[\"'
`·..······
`f~Jl·',··········
`.jI:
`tJ,., J~' ....Er,"·llM1f"v,h,lv"'···........ ··· .. ·
`~ L'!so-
`J7
`
`i
`
`·····..· , ··..·..·
`
`,
`
`,
`
`..
`
`, ,.
`
`•
`
`650
`
`600
`
`550
`
`500
`
`450
`
`400
`
`10
`
`12
`
`•
`
`14
`
`16
`
`18
`
`20
`
`22
`24
`Time (min)
`
`26
`
`28
`
`30
`
`32
`
`34
`
`Fig. 2 Base peak chwmatogram and three dImensional (3D) map of
`phospholipid specie separation of krill oiL performed with RPLCf
`ESI-IvrS on a ZORBAX Eclipse Plus e18 column U50 x 2.1 mm
`i.d., 5 pm). MS was run in negative the ionization mode and set to
`
`scan from m/z 400 to m/z 1,000. Only the most relevant part of the
`chromatogram is shown (10--35 min). Adduct iOlls in the form of
`[Tv1 +- FA-H]' is seen throughout the map
`
`~ Springer AC:X:S ~
`
`000006
`
`

`
`31
`
`Fragrnent identity
`
`rn/z
`
`836.3
`
`552.2
`
`[(LPC 22:6) - CH3r
`
`534.3
`
`[(LPC 22:6) - CH3 - H20r
`
`526.2
`
`[(LPC 20:5) - CH3r
`
`508.3
`
`327.2
`
`[(LPC 20:5) - CH3 - H~~Or
`
`[22:6] -
`
`301,2
`
`[20.5J -
`
`283.3
`
`[20:5 - Hpr
`
`257.3
`
`G!ycero phospho choline
`
`RT'17.42-17.61 AV: 11 NL:4.42E1
`
`3012
`
`100
`
`90
`
`80
`
`70
`
`C.l
`CJ
`~ 60
`'0c
`::l
`Ll
`
`50
`
`~ C
`
`~'83.3
`
`40
`
`30
`
`20
`
`10
`
`0
`
`)>
`
`:;:;
`
`'"Q)
`a::
`
`Lipids (2011) 46:25-36
`
`Fig. 3 1\15 3 product ion
`spectlllm of 22:6--20:5 diacyl
`PtdCho obtmned in negative
`ionization mode. The molecular
`ion of rn/z 896,6 \vas selected
`for PQD fragmentation~ Gns
`yielded a fragment ion of
`mI;. 836.3 which was further
`fragmented with CID resulting
`in the presented spectrum.
`Fragment identity is explained
`in the table on the right hand
`side, indicating the high level of
`certainty in the characterization
`
`250
`
`300
`
`350
`
`400
`
`450
`
`500
`
`550
`
`600
`
`650
`
`700
`
`750
`
`800
`
`850
`
`m/z
`
`Table 1 Idemified lyso-phospholipid species with choline head
`group in krill oil
`
`Class
`
`Mass miz" Moleculat specie Relative intem;ity
`
`Lyso-PtdCho
`Lyso- PtdCho
`Lyso-PtdCho
`Lyso-PtdCho
`Lyso- PtdCho
`Lyso-PtdCho
`Lyso-PtdCho
`
`16:1
`493.4
`538A
`495A 540A 16:0
`554,4
`17:0
`509.4
`521.4
`18:1
`566A
`541.4
`586A 20:5
`563.4
`608.6
`21:1
`6124
`22:6
`567.4
`
`6.01
`32.42
`4.06
`23.21
`31.31
`16.70
`12.19
`
`Data were obtained with RPLCiESI---MS 3 operated in negative ioni(cid:173)
`zation mode and with data dependent fragmentation without exclu(cid:173)
`sion list
`a mil. for [M + FA---I-Ij- adduct
`
`from both sn--l and sn--2, except in the f-ragmentation of
`alkyl--acyl species, where only a single fatty acyl fragment
`ion was observed. In addition, the corresponding fragment
`ions of the lyso-PtdCho compound \vere prevalent, con(cid:173)
`firming the characterization of the species. The fragments
`associated with the lyso-PtdCho compounds were either in
`the form of [lyso-PtcK---':ho--CH3J"" or [lyso--PtdCho---CH3--(cid:173)
`H20]-. Furthermore, fragments specific for phospholipids
`carrying a choline head group were often registered. These
`dissociation products were mlz 257, mil 242 and mil 223,
`representing [Glycero phospho cholineJ-, [Glycero phos(cid:173)
`and [Glycero phospho choline-CHr
`pho choline-CH3r
`H20r, respectively. The identification of the phospholipid
`class was made based on the loss of 60 Da in the
`PQD fragmentation step. However, fragments specific for
`
`phospholipids carrying a choline head group affirms this
`interpretation. In spectra obtained from the dissociation of
`species carrying a 20:5 fatty acyl group, an ambiguous
`[20:5-H20]--- fragment
`ion with mlz 283 can often be
`detected, This ion can potentially be misinterpreted as the
`fatty acyl
`fragment
`[18:0]'--, but meeting the criteria
`for
`reliable identification will
`rule out
`this etToneous
`conclusion.
`In some incidences, a molecular ion could be explained
`by either being a diacyI-, or a.n alkyI--acyl--compound. In
`the possible identification of alkyl--acyl
`these cases,
`PtdCho species was based on the presence of a relatively
`high MS--signal for a single fatty acyl fragment ion and the
`corresponding lyso-PtdCho compound. In addition,
`there
`should be a total absence of signals (i. e. both fatty acyl--,
`and lyso--PtdCho-ions) potentially explained by fragmen(cid:173)
`tation of an ester bond in the opposite sn--position. An
`example of this is In!z 764.6 (spectrum shown in Fig. 5f)
`which could originate from both 016:0--20:5 alkyl--acyl
`PtdCho and 15:0--20:5 diacyl PtdCho. The presence
`mh 466 [(lyso--PtdCho 016:0/15:0)--CH3]-, 448 [(lyso-(cid:173)
`PtdCho 016:0--15:0)---CHr H20]- and 301 [20:5]- indi(cid:173)
`cate a fatty acyl group of 20:5, while there is no fragment
`indicating a fatty acyl group of 15:0 in the opposite
`sn-position. This is therefore assumed to be a PtdCho(cid:173)
`specie with an alkyl-acyl composition of 016:0-20:5.
`Altogether, seven different potential alkyl-acyl PtdCho
`species were characterized. :For all these species, the fatty
`alkyl chains were either hexadecanoic or octadecanoic, and
`either saturated or with a single double bond.
`No further attempt was made to clmify the stereospec(cid:173)
`ificity of the species. It is important to keep in mind that
`
`if) Springer Aa::s ~
`
`000007
`
`

`
`32
`
`Lipids (2011) 46:25-36
`
`Tllble 2 Identified phospholipid species with choline head group in
`krill oil
`
`Class
`
`Mass
`
`Molecular specie
`
`Relative intensity
`
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PcdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PcdCho
`PtdCho
`PtdCho
`PcdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PcdCho
`PtdCho
`PtdCho
`PcdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PtdCho
`PcdCho
`PtdCho
`PtdCho
`PcdCho
`
`703,6
`717.7
`717.7
`731,6
`731.6
`737.6
`74.5.6
`745.6
`749.6
`751.6
`753.6
`753.6
`755.6
`757.6
`757,6
`759.7
`759.7
`761.7
`763.6
`763.6
`765.6
`'1'77.6
`
`T/7.6
`777.6
`777.6
`
`T/7.6
`779.6
`779.6
`781.6
`783.7
`785.6
`789.6
`789.6
`791.7
`791.7
`793.6
`799.6
`803.6
`805.6
`805.6
`807.6
`825.6
`825.6
`827.6
`831.7
`833.7
`851.6
`
`748.6
`762.7
`762.7
`776,6
`776.6
`782.6
`790.6
`790.6
`794.6
`796.6
`798.6
`798.6
`800.6
`802.7
`802,7
`804.7
`804.7
`806.7
`808.6
`808.6
`810.7
`822.6
`822.6
`822.6
`822.6
`822.6
`824.6
`824.6
`826.6
`828.7
`830.7
`834.7
`834.7
`836.7
`836.7
`838.6
`844.6
`848.7
`850.6
`850.6
`852.6
`870.6
`870.6
`872.6
`876.7
`878.7
`896.6
`
`14:0-16:1
`15:0--16:1
`13:0--18:1
`14:0-18:1
`16:0-16:1
`l3:0--20:5
`15:0--18:1
`16:0-17:1
`14:1-20:5
`14:0--20:5
`14:0-20:4
`16:0-18:4
`16:0--18:3
`16:1--18:1
`16:0-18:2
`16:1-18:0
`16:0--18:1
`16:0-18:0
`J3:0-22:6
`016:1--20:5
`016:0--20:5
`18:3-18:3
`12:4-24:2
`18:1--18:5
`16:1--20:5
`14:0-22:6
`18:1--18:4
`16:0--20:5
`16:0-20:4
`18:1-18:2
`18:1--18:1
`016:1-22:6
`17:2-20:5
`17:1--20:5
`016:0--22:6
`018:0-20:5
`18:4-20:5
`18:2--20:5
`18:1--20:5
`16:0-22:6
`18:0--20:5
`18:4--22:6
`20:5-20:5
`20:4-20:5
`18:1--22:6
`20:1--22:6
`20:5-22:6
`
`~ Springer AC:X:S ~
`
`5.33
`0.90
`21.08
`13,75
`27.48
`17.38
`6.71
`6.80
`6.94
`17.62
`0.26
`14.88
`13.30
`1.41
`32,42
`0.10
`100.00
`15.83
`0.30
`20.17
`28.24
`<0.01
`0.03
`0.07
`4.85
`12.25
`0.10
`96.57
`24.23
`7.69
`14.69
`3.50
`3.85
`15.64
`22.97
`15.01
`g,17
`
`9.28
`3.51
`76.34
`23.92
`0.15
`30.31
`16.18
`17.10
`<0.01
`24.80
`
`Table 2 continued
`
`Class
`
`Mass
`
`Moleculm- specie
`
`Relative intensity
`
`PtdCho
`PldCho
`PtdCho
`PtdCho
`
`86L7
`867.6
`875_7
`87'1.6
`
`90fi7
`912.7
`920_7
`9226
`
`20:5-22:1
`20:5--23:5
`20:5---23:1
`22:6-22:6
`
`2(180
`<0.01
`5.75
`749
`
`Data were obtained with RPLCt1---:SI--MS 3 operated in negative ioni(cid:173)
`zation mode and with data dependent fragmentation without exclu(cid:173)
`sion list
`a m!z lor [jVI + FA--HJ- adduct
`
`srereoisomers would be difficult to separate and specifi(cid:173)
`cally identify. Therefore, the relative intensity values pre(cid:173)
`sented in Table 2 win in many cases be the sum of the
`signal
`intensities of the two stereoisomers. The ratio
`between the stereoisomers will vary among the different
`species. However, an interesting observation was that
`several of the n-3 acylated species appear to carry the n-3
`fatty acyl group in the sn-l position, based on the fragment
`ratios in the hagmentation spectra (e.g. 22:6-18:1 diacyl
`PtdCho).
`In total, 58 species were characterized without the use of
`the exclusion list whereof seven vvere from the lyso(cid:173)
`PtdCho class and 51 from the PtdCho class. An additional
`11 species were detected by applying the asymmetric
`exclusion list Of these latter, two were identified as lyso(cid:173)
`PtdCho and nine as PtdCho compounds, yielding an overall
`total of nine lyso-PtdCho class species and 60 PtdCho class
`species elucidated. Seven species yielded signals for highly
`probable fatty acyl 17-3 groups in both the sn-l and sn-2
`positions
`(i.e.
`the diacyl PtdCho species 18:4-20:5.
`18:4--22:6, 20:5--20:5, 20:5---22:6, 20:5--23:5, 22:6--22:6
`and 20:5--22:5), Those species and the detection of more
`exotic species such as 22:6--23:5 and 20:5--26:4 diacyl
`PtdCho, show tbe complexity of krill oiL
`Prior to analysis of the krill oil,
`the total fatty acid
`composition, wherein information on tbe concentration of
`individual fatty acids and their n--3 c