`DOI 10.1007/s11745-010-3472-6
`
`O R I G I N A L A R T I C L E
`
`Elucidation of Phosphatidylcholine Composition in Krill Oil
`Extracted from Euphausia superba
`
`Bjørn Winther • Nils Hoem • Kjetil Berge •
`Le´on Reubsaet
`
`Received: 9 June 2010 / Accepted: 30 August 2010 / Published online: 17 September 2010
`Ó The Author(s) 2010. This article is published with open access at Springerlink.com
`
`Abstract High performance
`liquid chromatography-
`electrospray tandem mass spectrometry was used to
`elucidate the phospholipids in krill oil extracted from
`Euphausia superba, an emerging source for human nutri-
`tional supplements. The study was carried out in order to
`map the species of the choline-containing phospholipid
`classes: phosphatidylcholine and lyso-phosphatidylcholine.
`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-
`fication was obtained with class separation on a normal
`phase chromatography column. An Orbitrap system was
`used for the detection, and pulsed-Q dissociation frag-
`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-1 and sn-2.
`The phosphatidylcholine concentration was estimated to be
`34 ± 5 g/100 g oil (n = 5). These results confirm the
`complexity of the phospholipid composition of krill oil,
`and the presence of long chained, heavily unsaturated fatty
`acids.
`
`B. Winther L. Reubsaet (&)
`Department of Pharmaceutical Chemistry,
`School of Pharmacy, University of Oslo, Oslo, Norway
`e-mail: j.l.reubsaet@farmasi.uio.no
`N. Hoem K. Berge
`Aker BioMarine ASA, Fjordalle´en 16, Vika,
`P.O. Box 1423, 0115 Oslo, Norway
`
`Keywords Fish oil Krill oil Mass spectrometry
`Omega-3 Phosphatidylcholine Phospholipid
`
`Abbreviations
`Eicosapentaenoic acid
`EPA
`Docosahexaenoic acid
`DHA
`lyso-PtdCho Lyso-phosphatidylcholine
`NPLC
`Normal phase liquid chromatography
`PtdCho
`Phosphatidylcholine
`PtdEtn
`Phosphatidylethanolamine
`PtdIns
`Phosphatidylinositol
`PtdSer
`Phosphatidylserine
`PL
`Phospholipid
`RPLC
`Reverse phase liquid chromatography
`
`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 bound as ethyl esters (Omacor/Lovaza), krill oil contains
`a high proportion of omega-3 fatty acids bound to
`phospholipids.
`Krill oil has been investigated in several preclinical and
`clinical studies [1–4], and there is growing evidence that
`the molecular form of the omega-3 fatty acids (i.e. tri-
`glycerides,
`ethyl-esters, phospholipids) might be of
`importance for their biological effect as well as distribution
`of the omega-3 fatty acids in the body. In one animal study,
`
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`
`
`26
`
`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-
`sapentaenoic acid (EPA) ? docosahexaenoic acid (DHA),
`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
`that this difference may be linked to differences in the
`incorporation of omega-3 fatty acids into membranes, and
`consequently a reduction of inflammatory molecules and
`endocannabinoids, which might be relevant for the differ-
`ences observed between 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-
`tion, but not after fish oil administration, when compared to
`control animals [2]. Thus, omega-3 fatty acids linked to
`phospholipids may be differently distributed in the body
`compared to omega-3 fatty acids in other molecular forms.
`Moreover, in a clinical safety study, the presence of EPA
`and DHA in the blood plasma was determined after daily
`administration of 2 g krill oil or 2 g menhaden oil for
`4 weeks [3]. The authors concluded that EPA and DHA
`from krill oil are absorbed at least as well 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–MS(/MS),
`a technique which has lately played an important role in
`characterization of the lipidome in tissues and organisms
`[5]. An inherent limitation in the use of ESI for the ioni-
`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-
`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 MSn fragmentation with systems based on ion traps
`[10–13]. Normal phase liquid chromatography (NPLC) and
`reverse phase liquid chromatography (RPLC) are both
`frequently used for the separation of the components [10,
`14–17]. Of these two separation techniques, RPLC has
`been shown to be more suitable for species separation and
`characterization [8].
`Different ionization and fragmentation techniques can
`be used for the evaluation of phospholipids. Ionization of
`the phospholipids may be performed in negative- and
`positive-ionization mode.
`In general,
`fragmentation of
`phospholipids in the positive mode provides information
`about the phospholipid head group, while fragmentation in
`
`123
`
`the negative mode is the source of structural information.
`For phospholipids containing choline-headgroups,
`the
`choline-specific fragment m/z 184 has been used in pre-
`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-
`acterization. All phospholipid classes, except those con-
`taining choline, yield molecular ions [M-H]- 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 [M ? FA-H]- ions
`(m/z = M ? 45) [19, 20]. With fragmentation, this adduct
`dissociates with the loss of (HCOO ? CH3) into the frag-
`ion [M-CH3]-. This is in particularly useful
`ment
`in
`methods utilizing RPLC for separation. Although the
`chromatographic class information is lost in such setups,
`the class-specific fragments may be used in the character-
`ization of the species [11].
`Two ion activation techniques may be used for MS
`analysis utilizing ion traps: collision-induced dissociation
`(CID) and pulsed-Q dissociation (PQD) 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
`potential loss of low mass fragments [21, 22]. This dif-
`ference could be crucial in the fragmentation of larger
`molecules into low mass, specific fragments, as shown with
`detection of iTRAQ fragments with a linear ion trap [23].
`The fatty acid composition of phosphatidylcholine
`(PtdCho) from krill oil has previously been investigated by
`Le Grandois et al. [24]. This study was performed with a
`method based on the ESI operated in the positive mode
`with triple quadrupole 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-
`sented findings and offer new insights into the composition
`of krill oil. In addition; it shows the advantage of per-
`forming an additional fragmentation using an exclusion list
`in the identification of low prevalent species.
`
`Experimental Procedures
`
`Chemicals
`
`Phospholipid standards of lyso-phosphatidylcholine (lyso-
`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
`PtdIns source was glycine max and the PtdSer source was
`
`RIMFROST EXHIBIT 1107 Page 0002
`
`
`
`Lipids (2011) 46:25–36
`
`27
`
`Ò
`fish oil was donated by
`bovine brain. EPAX 6000 TG
`EPAX (A˚ lesund, Norway), and SuperbaTM krill oil was
`obtained from Aker BioMarine (Oslo, Norway). All other
`chemicals were of MS grade.
`
`Instrumentation
`
`The chromatography was carried out on a Dionex system
`consisting of an Ultimate 3000 pump, an Ultimate 3000 RS
`autosampler, and an Ultimate 3000 flow manager. Detec-
`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 DCMSLink 2.5 (all Instrument-Teknikk AS, Østera˚s,
`Norway).
`
`Mass Spectrometry
`
`The LTQ Orbitrap system was operated with a spray voltage
`of 5.00 kV, nitrogen as the sheath gas with flow rate set to
`30 arbitrary units, and helium as the collision gas. The
`quantification of the PtdCho class was performed with a
`scan from m/z = 400 to m/z = 1,000 operated in negative
`ionization mode. MSn experiments for identification of the
`choline-containing phospholipids were performed using
`data dependent PQD for the first 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 window
`from m/z = 400 to m/z = 1,000. The normalized collision
`energy was 200 and the isolation width 2.00 Da. Subse-
`quently, the most intense fragment ion detected was further
`fragmented using CID, with the normalized collision energy
`at 35 and an isolation width of 2.00 Da (MS3). The LTQ
`was utilized for the detection of the fragments and the
`m/z 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 m/z of the molecular ions, with an exclusion
`window from this mass-to-charge ratio, up to m/z ? 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 C18 column with particle diameter of 5 lm
`and the column dimensions were 150 9 2.1 mm i.d.
`
`The mobile phase A consisted of 90 parts 1% TEA and
`0.2% formic acid in water, and 10 parts mobile phase B (v/v).
`Mobile phase B consisted of 1% TEA and 0.2% formic acid
`in 60 parts methanol and 40 parts acetonitrile (v/v).
`A linear gradient was used for the separation. The sys-
`tem was first kept isocratic at 65% mobile phase B for
`5 min after injection of sample. The gradient was then run
`from 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 was set to 0.2 mL/min and the injection
`volume was 20 lL throughout the study.
`
`Chromatographic Conditions NPLC
`
`NPLC was performed on a HiCHROM LiChrospher 100
`DIOL column with a particle diameter of 5 lm and column
`dimensions of 250 9 2 mm i.d. Mobile phase C was 100%
`chloroform, and the mobile phase D consisted of 0.05%
`TEA, 0.05% ammonia and 0.1% formic acid in methanol
`(v/v). For the class separation of the phospholipids, a linear
`gradient was used. The gradient was run from 5 to 27.5%
`mobile phase D in 15 min, followed 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 lL
`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-
`form and methanol at a ratio of 2:1. These solutions were
`stored at -32°C and excessive heating cycles were avoi-
`ded. Samples were prepared by further dilution with sol-
`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
`Ò
`with samples of PL free fish oil (EPAX
`) spiked with a
`PtdCho standard purified from egg yolk to concentrations
`of 100 lg/mL. The spiking of PL free fish oil was per-
`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:MeOH 2:1.
`The concentration of krill oil and PtdCho standard was
`
`123
`RIMFROST EXHIBIT 1107 Page 0003
`
`
`
`28
`
`Lipids (2011) 46:25–36
`
`1 mg/mL and for PL free fish oil 10 mg/mL for these
`solutions. Respectively, 100 lL of PtdCho standards and of
`krill oil was added to 900 lL of the PL free fish oil, pro-
`ducing samples with concentrations of 100 lg/mL. For the
`calibration curve, the PtdCho standard samples were con-
`secutively diluted to the desired concentrations of 10.0,
`5.00, 2.50, 1.00, 0.50, 0.25, and 0.10 lg/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 lg/
`mL in order to measure the PtdCho content within the
`linear area of response of the calibration curve.
`
`Results
`
`Selection of MS-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-
`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. 1, is predominantly
`composed
`of
`choline-containing
`phospholipids,
`the
`emphasis of the work was focused on the elucidation of the
`species in the PtdCho and lyso-PtdCho classes. In NPLC,
`class separation of the phospholipids is achieved. A clear
`tendency of the elution order from the column was seen
`from high m/z to lower m/z. PtdCho class species eluted
`from 6.5 to 8 min, and the elution of the lyso-PtdCho class
`occurred between 10.5 and 12 min. Some species separation
`
`Fig. 1 Base peak
`chromatogram and three
`dimensional (3D) map of
`phospholipid class separation of
`krill oil, performed with a linear
`gradient NPLC/ESI–MS on a
`HiCHROM LiChrospher 100
`DIOL column (250 9 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 part
`of the species, the map show
`adduct ions in the form of
`[M ? FA–H]-
`
`123
`
`RIMFROST EXHIBIT 1107 Page 0004
`
`
`
`Lipids (2011) 46:25–36
`
`29
`
`was seen within the PtdCho class, however, this was not
`adequate for identification of the diverse species within the
`classes.
`As the identification of the components is performed
`with data dependent fragmentation, chromatographic sep-
`arations of the substances are critical for detection of the
`less prominent species. Hence, the separation for charac-
`terization of the species was performed utilizing a RPLC
`C18 column as described under ‘‘Experimental Proce-
`dures’’. This improved the chromatographic performance
`for species separation compared to NPLC (Fig. 2). As lyso-
`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 MS3 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-
`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 identification. The
`following criteria were applied for reliable interpretation of
`a choline-containing phospholipid: based on the mass-to-
`charge ratio of the molecular ion, it is likely to be a choline-
`containing phospholipid (i.e. m/z being an even number).
`Following the first CID with PQD, a daughter ion should be
`produced by the loss of (HCOO ? CH3) as 60 Da. Further
`fragmentation with CID of the resulting product should
`produce specific fragments revealing the nature of the fatty
`acyl groups in both the sn-1 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-1 and the sn-2 positions in
`addition to their corresponding fragments of the lyso-
`compound, ensuring identification of the species. The lyso-
`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 MS,
`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-
`PtdChoclasses. Chromatograms and fragmentation patterns
`
`are presented in Figs. 4 and 5 for the 10 foremost sub-
`stances characterized from the PtdCho class.
`As data dependent fragmentation methods are, by nat-
`ure, biased in the selection of the most prevalent sub-
`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 (Fig. 1). It was therefore attempted to quan-
`tify the absolute concentration of this class by use of class
`separation with NPLC. Quantification of the PtdCho class
`was 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 lg/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 lg/mL to 1.00 lg/mL, the calibration curve showed a
`linearity (r2 = 0.9995) with a linear
`high degree of
`regression curve (y = bx ? 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 spectrum 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 [24].
`As mentioned above, instrument response is affected by the
`acyl chain length of the PtdCho. These differences in chain
`lengths could therefore influence 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 g/100 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 quantifica-
`tion purposes. Due to the low mass cut-off limit at 28% of
`the molecular ion mass with CID fragmentation in ion
`
`123
`RIMFROST EXHIBIT 1107 Page 0005
`
`
`
`30
`
`Lipids (2011) 46:25–36
`
`traps, fragmentation utilizing PQD in the positive ioniza-
`tion mode was chosen as the first fragmentation step. In
`addition, utilizing PQD in the first fragmentation step of a
`MS3 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]-
`fragment formed after (HCOO ? CH3) loss from formate-
`molecular ion adducts. Consequently, performing a mode
`shift from 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-CH3]- frag-
`ments, without extensive fragmentation to secondary
`
`ions. Higher collision energies produced sec-
`fragment
`ondary fragments that could be used as a source of struc-
`tural information; however, this was better achieved with
`CID as a second fragmentation step. The CID was operated
`with a normalized collision energy of 35, for further
`fragmentation of the [M-CH3]- ion. This value was not
`optimized for the individual species, and additional struc-
`tural information could potentially have been achieved by
`specie specific optimization of this setting.
`With the described method for separation and frag-
`mentation of
`the phospholipids,
`typical
`fragmentation
`patterns were obtained, as shown in Fig. 3. The spectra
`were dominated by fatty acyl fragment ions originating
`
`RT:
`
`90
`
`10.00 - 35.00
`
`SM:
`
`9B
`
`20.37
`
`20.37
`
`1000
`
`950
`
`900
`
`850
`
`800
`
`750
`
`m/z
`
`700
`
`650
`
`600
`
`550
`
`500
`
`450
`
`400
`
`21.41
`
`23.33
`
`21.41
`
`23.33
`22.79
`22.79
`
`23.86
`23.86
`
`27.65
`
`27.65
`
`27.93
`
`27.93
`
`24.30
`24.30
`
`26.76
`26.76
`
`28.74
`28.74
`29.83
`29.83
`
`32.33
`
`31.68
`31.68 32.33
`
`32.99
`32.99
`
`13.96
`
`13.96
`
`17.05
`17.54
`17.05
`
`12.53
`12.53
`
`11.82
`11.82
`
`15.33
`15.33
`16.27
`16.27
`
`PtdCho
`
`17.54
`18.35
`18.35
`19.85
`19.85
`19.02
`19.02
`
`Lyso-
`PtdCho
`
`80
`100
`70
`80
`60
`50
`60
`40
`40
`30
`20
`20
`10
`0
`
`Relative Abundance
`
`Relative Abundance
`
`10
`
`12
`
`14
`
`16
`
`18
`
`20
`
`24
`22
`Time (min)
`
`26
`
`28
`
`30
`
`32
`
`34
`
`Fig. 2 Base peak chromatogram and three dimensional (3D) map of
`phospholipid specie separation of krill oil, performed with RPLC/
`ESI–MS on a ZORBAX Eclipse Plus C18 column (150 9 2.1 mm
`i.d., 5 lm). 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 ions in the form of
`[M ? FA–H]- is seen throughout the map
`
`123
`
`RIMFROST EXHIBIT 1107 Page 0006
`
`
`
`100
`
`301.2
`
`RT: 17.42-17.61 AV: 11 NL: 4.42E1
`
`31
`
`m/z
`
`Fragment identity
`
`836.3
`
`[M-CH3]
`
`-
`
`552.2
`
`[(LPC 22:6) - CH3]
`
`-
`
`534.3
`
`[(LPC 22:6) - CH3 - H2O]
`
`526.2
`
`[(LPC 20:5) - CH3]
`
`-
`
`508.3
`
`[(LPC 20:5) - CH3 - H2O]
`
`-
`
`-
`
`- -
`
`327.2
`
`[22:6]
`
`301.2
`
`[20:5]
`
`283.3
`
`257.3
`
`2O]-
`[20:5 - H
`Glycero phospho choline
`
`552.2
`
`526.2
`
`508.3
`
`500
`
`550
`
`600
`
`800
`
`850
`
`327.2
`
`283.3
`257.3
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Relative Abundance
`
`Lipids (2011) 46:25–36
`
`Fig. 3 MS3 product ion
`spectrum of 22:6–20:5 diacyl
`PtdCho obtained in negative
`ionization mode. The molecular
`ion of m/z 896.6 was selected
`for PQD fragmentation; this
`yielded a fragment ion of
`m/z 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
`
`650
`
`700
`
`750
`
`m/z
`
`Table 1 Identified lyso-phospholipid species with choline head
`group in krill oil
`
`Class
`
`Mass m/za Molecular specie Relative intensity
`
`Lyso-PtdCho
`
`493.4
`
`538.4
`
`16:1
`
`Lyso-PtdCho
`
`495.4
`
`540.4
`
`16:0
`
`Lyso-PtdCho
`
`509.4
`
`554.4
`
`17:0
`
`Lyso-PtdCho
`
`521.4
`
`566.4
`
`18:1
`
`Lyso-PtdCho
`
`541.4
`
`586.4
`
`20:5
`
`Lyso-PtdCho
`
`563.4
`
`608.6
`
`21:1
`
`Lyso-PtdCho
`
`567.4
`
`612.4
`
`22:6
`
`6.01
`
`32.42
`
`4.06
`
`23.21
`
`31.31
`
`16.70
`
`12.19
`
`Data were obtained with RPLC/ESI–MS3 operated in negative ioni-
`zation mode and with data dependent fragmentation without exclu-
`sion list
`a m/z for [M ? FA–H]- adduct
`
`from both sn-1 and sn-2, except in the fragmentation 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 were prevalent, con-
`firming the characterization of the species. The fragments
`associated with the lyso-PtdCho compounds were either in
`the form of [lyso-PtdCho–CH3]- or [lyso-PtdCho–CH3–
`H2O]-. Furthermore, fragments specific for phospholipids
`carrying a choline head group were often registered. These
`dissociation products were m/z 257, m/z 242 and m/z 223,
`representing [Glycero phospho choline]-, [Glycero phos-
`pho choline–CH3]- and [Glycero phospho choline–CH3–
`H2O]-, 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–H2O]- fragment ion with m/z 283 can often be
`detected. This ion can potentially be misinterpreted as the
`[18:0]-, but meeting the criteria
`fatty acyl
`fragment
`for reliable identification will rule out
`this erroneous
`conclusion.
`In some incidences, a molecular ion could be explained
`by either being a diacyl-, or an alkyl–acyl-compound. In
`these cases,
`the possible identification of alkyl–acyl
`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-
`tation of an ester bond in the opposite sn-position. An
`example of this is m/z 764.6 (spectrum shown in Fig. 5f)
`which could originate from both O16:0–20:5 alkyl–acyl
`PtdCho and 15:0–20:5 diacyl PtdCho. The presence
`m/z 466 [(lyso-PtdCho O16:0/15:0)–CH3]-, 448 [(lyso-
`PtdCho O16:0–15:0)–CH3–H2O]- and 301 [20:5]- indi-
`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-
`specie with an alkyl-acyl composition of O16:0–20:5.
`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 clarify the stereospec-
`ificity of the species. It is important to keep in mind that
`
`123
`RIMFROST EXHIBIT 1107 Page 0007
`
`
`
`32
`
`Lipids (2011) 46:25–36
`
`Table 2 Identified phospholipid species with choline head group in
`krill oil
`
`Class
`
`Mass
`
`m/za
`
`Molecular specie
`
`Relative intensity
`
`Table 2 continued
`
`Class
`
`Mass
`
`m/za
`
`Molecular specie
`
`Relative intensity
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`703.6
`
`717.7
`
`717.7
`
`731.6
`
`731.6
`
`737.6
`
`745.6
`
`748.6
`
`762.7
`
`762.7
`
`776.6
`
`776.6
`
`782.6
`
`790.6
`
`14:0–16:1
`
`15:0–16:1
`
`13:0–18:1
`
`14:0–18:1
`
`16:0–16:1
`
`13:0–20:5
`
`15:0–18:1
`
`5.33
`
`0.90
`
`21.08
`
`13.75
`
`27.48
`
`17.38
`
`6.71
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`861.7
`
`867.6
`
`875.7
`
`877.6
`
`906.7
`
`912.7
`
`920.7
`
`922.6
`
`20:5–22:1
`
`20:5–23:5
`
`20:5–23:1
`
`22:6–22:6
`
`20.80
`\0.01
`5.75
`
`7.49
`
`Data were obtained with RPLC/ESI-MS3 operated in negative ioni-
`zation mode and with data dependent fragmentation without exclu-
`sion list
`a m/z for [M ? FA–H]- adduct
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`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
`
`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
`
`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
`
`13:0–22:6
`
`O16:1–20:5
`
`O16:0–20:5
`
`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
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`PtdCho
`
`765.6
`
`777.6
`
`777.6
`
`777.6
`
`777.6
`
`777.6
`
`779.6
`
`779.6
`
`781.6
`
`783.7
`
`785.6
`
`789.6
`
`789.6
`
`791.7
`
`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
`
`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
`
`O16:1–22:6
`
`17:2–20:5
`
`17:1–20:5
`
`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
`
`stereoisomers would be difficult to separate and specifi-
`cally identify. Therefore, the relative intensity values pre-
`sented in Table 2 will 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-1 position, based on the fragment
`ratios in the fragmentation spectra (e.g. 22:6–18:1 diacyl
`PtdCho).
`In total, 58 species were characterized without the use of
`the exclusion list, whereof seven were from the lyso-
`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-
`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 n-3 groups in both the sn-1 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 the complexity of krill oil.
`Prior to analysis of the krill oil, the total fatty acid
`composition, wherein information on the concentration of
`individual fatty acids and their n-3 content was obtained
`and provided by Nofima, with the method AOCS Ce 1b-89
`(data not shown). The sum of polyunsaturated (n-3) fatty
`acids was reported to be 18.5 g/100 g oil. The assumed
`homologous distribution of the fatty acid composition
`between triacylglycerols, free fatty acids, and the lyso-
`PtdCho- and PtdCho-classes, combined with the described
`relative intensities of the known species (Tables 1, 2),
`makes it possible to estimate the prevalence of n-3 fatty
`acids in one or both sn positions of the PtdCho species. For
`the PtdCho class, approximately 58% of the components
`contained a single n-3 fatty acid, and 10% held an n-3 fatty
`acid in both sn-1 and sn-2 positions. Of the species in the
`lyso-PtdCho class, approximately 35% contained an n-3
`
`RIMFROST EXHIBIT 1107 Page 0008
`
`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
`
`O16:0–22:6
`
`O18:0–20:5
`
`18:4–20:5
`
`18:2–20:5
`
`18:1–20