Individual Phosphatidylcholine Species Analysis by RP-HPLC-ELSD

Apr 3, 2015 - Food Chem. , 2015, 63 (15), pp 3851–3858 ... v/v/v) at a flow rate of 1 mL/min, and ELSD detection was performed using 80 °C for the dri...
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Individual Phosphatidylcholine Species Analysis by RP-HPLC-ELSD for Determination of Polyenylphosphatidylcholine in Lecithins Wei-Ju Lee,† Shun-Hsiang Weng,‡ and Nan-Wei Su*,† †

Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan Department of Food Science and Nutrition, Meiho University, Pingtung County 91202, Taiwan

‡

S Supporting Information *

ABSTRACT: Polyenylphosphatidylcholine (PPC), a subgroup of the bioactive agents in phosphatidylcholine (PC), has been indicated to possess liver-protective effects. This study aimed to investigate a promising and feasible method to determine PC molecular species with a reverse phase (RP) high-performance liquid chromatograph (HPLC) equipped with an evaporative light scattering detector (ELSD). Chromatography was achieved using a C30 column and an isocratic mobile phase consisting of acetonitrile/methanol/triethylamine (40/58/2, v/v/v) at a flow rate of 1 mL/min, and ELSD detection was performed using 80 °C for the drift tube and an air flow rate of 1.8 L/min. To identify individual peaks on the chromatogram, MALDI-TOF-MS was employed for initial detection, and then the results were used to investigate the relationship between the retention time and fatty acyl chains of each PC molecule. A linear correlation was observed between the retention time and theoretical carbon number (TCN) of individual PC species. The compositions of PC molecular species in soybean and sunflower lecithins were similar to each other, and the major PC molecular species were 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LLPC), 1-oleoyl-2-linoleoylsn-glycero-3-phosphocholine (OLPC), and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC). The contents of LLPC in soybean PC and sunflower PC were 40.6% and 64.3%, respectively. KEYWORDS: polyenylphosphatidylcholine, phosphatidylcholine molecular species, RP-HPLC-ELSD, lecithin, MALDI-TOF-MS, theoretical carbon number

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in a structured environment.9 Therefore, to characterize PCs, it is necessary to determine the individual PC molecular species. Reverse phase (RP) high-performance liquid chromatography (HPLC) is a preferred tool for separating PC molecular species with various aliphatic chains.10 Brouwers et al. reported the use of RP-HPLC to separate PC11 and phosphatidylethanolamine (PE)12 molecular species from animal origins; the corresponding eluates were detected using an evaporative light scattering detector (ELSD). To determine the molecular species of a given peak, the eluate was collected manually, subjected to derivatization, and then analyzed by HPLCultraviolet (UV) analysis or a gas chromatograph (GC) with a flame ionization detector (FID).11−13 Currently, RP-HPLC instruments equipped with mass spectrometers have become the most popular tools in identifying PC molecular species.14−19 However, the high cost of mass spectrometry instruments and their maintenance has exceeded the means of general analytical chemistry laboratories, and an alternative for determining PC species is still desirable. RP-HPLC equipped with ELSD appears to be a better choice. Nevertheless, in view of the highly complicated composition of PC molecular species and the lack of sufficient commercial standards, the identification of each peak is a nontrivial task. In general, PCs can be obtained from a variety of readily available sources, such as egg yolk and plant lecithins.

INTRODUCTION Phosphatidylcholines (PCs), a class of phospholipids, are a major component of biological membranes. The molecular structure of PCs incorporates choline as a polar headgroup at the sn-3 position in the glycerol backbone and two hydrophobic fatty acyl chains at the sn-1 and sn-2 positions. As an amphipathic component with surface-active properties, PCs can be used as emulsifiers and promote oil-in-water emulsion stability.1 The health effects of PCs have been extensively studied, and these compounds have been used in pharmaceuticals, foods, and food supplements. Previous studies have shown that PCs have numerous functions, including as a choline supplement,2 a possible memory booster,3 and a liver function repairer.4,5 The physical properties, nutritional aspects, and oxidative stability of PCs are to a large extent determined by the aliphatic groups. For example, the supplementation of polyenylphosphatidylcholines (PPCs), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LLPC) in particular, has been considered to increase health benefits for liver disease patients.4,5 For a long time, enzymatic interesterification of PCs has been an efficient tool to adjust for specific fatty acid compositions;6 in most cases, n-3 polyunsaturated fatty acids, including αlinolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, were incorporated to synthesize modified PCs to meet particular functional requirements.6,7 Moreover, Vikbjerg et al. reported that caprylic acid rich structured PCs with altered emulsifying and dispersing properties extended the application range of PCs.8 The oxidative capability of PCs in bilayers was substantially influenced by the number of intramolecular oxidizable acyl chains and the content of bis-allylic hydrogen © 2015 American Chemical Society

Received: Revised: Accepted: Published: 3851

February 25, 2015 April 2, 2015 April 3, 2015 April 3, 2015 DOI: 10.1021/acs.jafc.5b01022 J. Agric. Food Chem. 2015, 63, 3851−3858

Article

Journal of Agricultural and Food Chemistry

PC Analysis by RP-HPLC-ELSD. The purity of commercially available L-α-PC was examined by TLC. Briefly, a silica gel 60 TLC plate and a mobile phase consisting of chloroform/methanol/acetic acid/water (75/40/8/3, v/v/v/v) were used for the separation of phospholipids. L-α-PC standard solution was prepared by dissolving 5 mg of L-α-PC in 1 mL of methanol, and then 10 μL of sample was applied by a microsyringe to the TLC plate prior to developing. The spot was visualized using iodine vapor and exposed in an oven set at 60 °C for 5 min. For HPLC analysis, a 20 μL aliquot of the PC sample was analyzed using an HPLC system (P1000, Thermo Fisher Scientific, Barrington, IL, USA) equipped with an ELSD (Alltech 2000, Deerfield, IL, USA) and a C30 analytical column (Develosil C30-UG-5, 4.6 × 250 mm, 5 μm; Nomura Chemical Co., Anada-cho Seto, Japan). The mobile phase was composed of acetonitrile, methanol, and triethylamine in a 40/58/2 ratio (v/v/v) at a flow rate of 1 mL/min. For the ELSD, the temperature of the drift tube was 80 °C and the air flow rate was 1.8 L/min. Each run lasted 40 min and required another 10 min to reach equilibrium. In addition, to quantitate the individual PC species, five species of pure PC molecules, namely LLPC, PPPC, OOPC, OPPC, and POPC, were analyzed at concentrations of 0.25, 0.5, 1, 2, and 5 g/L to prepare calibration curves. Identification of PC Molecular Species by MALDI-TOF-MS. To identify the fatty acyl chains at the sn-1 and sn-2 positions, each PC molecular species eluted from HPLC was collected manually by using a flow splitter according to retention time and then analyzed by MALDI-TOF-MS. Aliquots of the PC samples were directly mixed (1/ 1, v/v) with the 2,5-DHB matrix solution (1 mL of 20 mg/mL 2,5DHB in 70% acetonitrile with 0.1% trifluoroacetic acid). A 1 μL portion of the PC sample solution was used for MALDI-TOF-MS analysis. The sample/matrix solution was deposited onto the sample plate and vacuum-dried before being transferred into the ion source of a MALDI-TOF-MS (Autoflex III, Bruker Daltonics, Bremen, Germany). The sample spot was irradiated with a neodymiumdoped yttrium aluminum garnet (Nd:YAG) laser (emitting at 355 nm; pulse duration, 3 ns; 100 Hz) for desorption and ionization. The extraction voltage was set to 2.5 kV, and gated matrix suppression was applied to prevent detector saturation. Positive ion mass spectra in the range m/z 400−1000 were acquired in reflectron mode under delayed extraction. In total, 300 laser shots were averaged for each sample analysis to obtain representative mass spectra, and three mass spectra were recorded from each sample. Isolation of PC from Lecithins. The crude lecithins from soybean and sunflower seeds were fractionated by acetone prior to silica gel column chromatography. Briefly, 10 g of the crude lecithin was suspended with acetone (1/5, w/v), and then the insoluble fraction was separated from the solvent. This step was performed two or three times until the supernatant was colorless. The acetone-insoluble fraction was resuspended with 95% ethanol (1/4, w/v). The alcohol fraction was concentrated to approximately 20 mL under vacuum and then mixed with 10 g of Celite to obtain the preadsorbed sample for further silica gel chromatography. After the solvent was removed using a rotary evaporator, the dry powder was transferred to the top of the silica gel column (containing 40 g of silica gel, 2.2 cm i.d. × 28 cm) that had been previously equilibrated with ethyl acetate. Chromatography was performed by eluting the column with 180 mL of ethyl acetate to remove neutral lipids and impurities, and then eluting with 180 mL of ethanol, followed by 300 mL of methanol. The eluate was collected in 30 mL fractions and was monitored by TLC according to retention value (Rf). L-α-PE and L-α-PC standard solutions were used in TLC analysis as references. The fractions dominated by PC were combined, and the solvent was removed under reduced pressure for further analyses of PC content and molecular species. The PC content of the sample was measured using a normal phase HPLC system equipped with a ConstraMetric 3200 solvent delivery pump and a SpectroMonitor 3200 UV−visible detector (Thermo Fisher Scientific, Barrington, IL, USA). Analysis was performed according to the American Oil Chemists’ Society (AOCS) official method Ja 7b-91. The analytical column was a 250 × 4.6 mm i.d. 5 μm Reprosil 70 Si column (Dr. Maisch GmbH, Ammerbuch, Germany).

Depending on the amount and availability of phosphatides, only parts of oilseed gums have the potential to be processed as lecithins. Common lecithin sources include cottonseed, corn, sunflower seed, and rapeseed. Due to the mass production of soybean oil, soybean gum obtained as a byproduct of the oilrefining process is the primary source of commercial lecithin. Recently, the demand for sunflower lecithin has grown because this lecithin is considered the product of a non genetically modified organism.1,20−22 Moreover, the rheological and emulsifying properties of sunflower lecithin indicate that it is a desirable alternative to soybean lecithin.20−22 However, little or no information is available regarding the composition of PC molecular species derived from sunflower lecithin. A previous study only focused on the fatty acid composition of lecithin, which was considered to be largely equivalent to that of the oil source.1 Thus, analyzing PC species in conjunction with triacylglycerol (TAG) composition in crude lecithins may provide a more complete description of the deposition of fatty acids in plant lecithins. In our previous study, TAG molecular species were successfully determined through HPLC-ELSD analysis by their retention times and theoretical carbon numbers (TCNs).23 The retention times were found to correspond with their TCNs, which were calculated on the basis of the total carbon number, unsaturation degree, and types of unsaturated fatty acid. Nevertheless, these attempts to establish a correlation between the HPLC elution sequence of the PC molecular species and TCN with regard to the physical conformation of acyl chains remained inconclusive. In this study, an RP-HPLC-ELSD method intended for the determination of the PC molecular species was introduced. L-αPC from soybean origin was used to evaluate the chromatographic characteristics. Furthermore, the correlation between the TCNs of PC molecular species and their respective HPLC retention times was investigated. We revealed that, as for TAG, profiling could be achieved by comparison with TAG standards with reference to retention times to make a regression curve; by calculation of the TCN of respective peaks with reference to the regression analysis, PC peak profiling in the HPLC-ELSD chromatogram could be achieved by the regression curve that was made from the TCNs of known peaks with reference to retention times.

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MATERIALS AND METHODS

Chemicals. L-α-PC (≥99%), L-α-PE (≥98%), five molecular species of PC, including LLPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (OOPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), and 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 2,5dihydroxybenzoic acid (2,5-DHB) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All chromatographic-grade reagents used in HPLC and other analytical-grade solvents, as well as thin-layer chromatography (TLC) plates silica gel 60 (No. 5553), were obtained from Merck (Darmstadt, Germany). Silica gel (SiliaFlash G60, 70−230 mesh (60−200 μm), average pore size 25 Å) for column chromatography was obtained from SiliCycle (Quebec, Canada). Silica solid-phase extraction cartridges were obtained from J. T. Baker (Phillipsburg, NJ, USA). Materials. The crude soybean lecithin was obtained as industrial soybean oil degummed residue from the Taiwan Sugar Corporation (Kaohsiung, Taiwan). Sunflower liquid lecithin was bought from Now Foods Company (Bloomingdale, IL, USA). The acetone-soluble fractions of crude soybean lecithin and sunflower liquid lecithin accounted for 43.6% and 61.7%, respectively. 3852

DOI: 10.1021/acs.jafc.5b01022 J. Agric. Food Chem. 2015, 63, 3851−3858

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chromatogram of L-α-PC by RP-HPLC-ELSD. Separation was performed using isocratic conditions: acetonitrile/methanol/triethylamine (40/58/2, v/v/v) at a flow rate of 1 mL/min on a C30 column (250 × 4.6 mm, 5 μm). Numbers were assigned to major identified peaks by MALDITOF-MS: (1) LLnPC; (2) LLPC; (3) OLnPC; (4) PLnPC; (5) OLPC; (6) PLPC; (7) SLPC; (8) PPPC; (9) SOPC. The HPLC mobile phase was composed of n-hexane, isopropyl alcohol, and 0.2 M acetate buffer (pH 4.2) in a ratio of 8/8/1 (v/v/v) at a flow rate of 2 mL/min. The detection wavelength was set at 206 nm. The retention time of L-α-PC was used as a reference to determine the PC peak in lecithin profiles. The PC content was calculated from a linear calibrated curve derived from the corresponding peak areas of a serial dilution of authentic L-α-PC standard. TAG Species Analysis by RP-HPLC-ELSD. The acetone-soluble fractions of crude lecithins were combined, and the solvent was removed under vacuum to obtain the oil. The TAG species of oils were analyzed on the basis of a previous study with some modifications.23 Briefly, oil samples were dissolved in a small amount of n-hexane and pretreated using a silica solid-phase extraction cartridge to remove the potent polar constituents prior to HPLC analysis. The HPLC mobile phase was composed of acetonitrile (solvent A) and isopropyl alcohol (solvent B); the gradient elution started with 85% A, decreased to 30% over 55 min, was held at 30% for 10 min, and finally increased to 85% A over the next 5 min. The flow rate of the mobile phase was 1.2 mL/min. ELSD settings were 70 °C for the drift tube and a 2.0 L/min air flow rate. The peaks were determined by calculating the TCN and referencing the results with our previous study.23 The percentage of TAG species was calculated using eq 1:

% of TAG =

increased, and the optimized solvent system varied with the different PC molecular species patterns of biological samples from animal origin.11 We considered that triethylamine acted as an ion-pair reagent to reduce extensive peak tailing, because amines are known to prevent interactions between the stationary phase and the phospholipid headgroup moiety. Moreover, because of the repulsion between the positively charged choline of PC and triethylamine, the chromatographic time could be reduced by the addition of triethylamine. We obtained results similar to those of Brouwers et al.;11 the content of triethylamine in the mobile phase affected the efficiency of separation greatly. Figure 1 revealed that nine molecular species of PC in L-α-PC were separated successfully through an isocratic mobile phase that was composed of acetonitrile/methanol/triethylamine (40/58/2, v/v/v); moreover, only one C30 column was used in the HPLC system, which could reduce excessive back pressure in the HPLC system. Identification of PC Molecular Species by MALDI-TOFMS. To identify the PC species corresponding with the individual peaks in the HPLC chromatogram, L-α-PC was analyzed using MALDI-TOF-MS in positive-ion mode. The identification of fatty acyl chains and the regiodistribution of individual PC species in these chains was achieved by verifying the fragment ions from the spectra. Parts A−E of Figure 2 shows the fragmentation patterns of the PC molecules associated with peak nos. 1, 2, 4, 6, and 7 in Figure 1. The mass signal at m/z 520.3 in Figure 2A refers to the molecular ion [M + H]+ of lysophosphatidylcholine (LPC) esterified with linoleic acid. Furthermore, m/z 780.5 was identified as the molecular ion [M + H]+ of linoleoyl-linolenoyl-glycerophosphocholine, whereas m/z 802.5 corresponded to the molecular ion of [M + Na]+. Previous studies have reported that all PC molecular species normally occur in LPCs and fatty acids.24 In addition, the sn-2 position of phospholipids contains unsaturated fatty acid, and the saturated fatty acids are most abundant at the sn-1 position.25 Fuchs et al. reported that the resulting LPCs from diacyl-PCs possess a saturated fatty acid residue at the sn-1 position.26 Therefore, the PC molecular species of peak no. 1 was identified as 1-linoleoyl-2-linolenoyl-sn-glycero-3phosphocholine (LLnPC). Similar results can be observed in

A(peak area of individual TAG species) × 100 ∑ A(total peak areas of TAG) (1)

Data Analysis. The HPLC chromatograms were recorded and analyzed using SISC Chromatography Data Station v3.0 (SISC Inc., Taipei, Taiwan), and the results were calculated using Windows Excel 2010. The peak picking software used with MALDI-TOF-MS was FlexAnalysis 3.3 (Bruker Daltonics).

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RESULTS AND DISCUSSION PC Molecular Species Analysis by RP-HPLC-ELSD. L-αPC was used as the reference to evaluate the separation of various species. L-α-PC was examined by TLC and presented as a single spot (Rf 0.41) on the silica-TLC plate, indicating that it was an assemblage of PC molecular species. Brouwers et al. reported that the separation was performed by connecting two C18 columns in series; the retention of PC molecular species decreased as the amount of triethylamine in the mobile phase 3853

DOI: 10.1021/acs.jafc.5b01022 J. Agric. Food Chem. 2015, 63, 3851−3858

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Journal of Agricultural and Food Chemistry

Figure 2. Ions obtained by MALDI-TOF-MS in positive-ion mode from m/z 400 to 1000 and corresponding structures of product nos. 1 (A), 2 (B), 4 (C), 6 (D), and 7 (E) in the L-α-PC profile. C16:0 refers to palmitic acid, C18:0 refers to stearic acid, C18:1 refers to oleic acid, C18:2 refers to linoleic acid, and C18:3 refers to linolenic acid.

PC Profiling: Relation between Retention Time and TCN. The TCN of PC (TCNPC) was calculated according to the TCN of TAG (TCNTAG) with some modification. The equivalent carbon numbers (ECNs), TCNs, and retention times of L-α-PC and soy TAG molecular species are shown in Table 1. According to El-Hamdy and Perkins, TAGs with the same ECN act as critical pairs, which means that these TAGs behaved similarly in response to RP chromatography in spite of the difference in chain lengths, number of double bonds, geometrical configuration, and their ability to be further

Figure 2B−E. As far as L-α-PC was concerned, the major species were LLPC, 1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine (OLPC), and 1-palmitoyl-2-linoleoyl-sn-glycero-3phosphocholine (PLPC), with LLPC being the predominant species, whereas minor components included LLnPC, 1-oleoyl2-linolenoyl-sn-glycero-3-phosphocholine (OLnPC), 1-palmitoyl-2-linolenoyl-sn-glycero-3-phosphocholine (PLnPC), 1stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC), PPPC, and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). The PC composition was in good agreement with that in previous studies.14,15 3854

DOI: 10.1021/acs.jafc.5b01022 J. Agric. Food Chem. 2015, 63, 3851−3858

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Journal of Agricultural and Food Chemistry Table 1. ECNs, TCNs, and Retention Times of PC Molecular Species of L-α-PC and TAG Species of Soybean Oil

a

PC species

ECNPCa

TCNPCa

retention time

TAG species

ECNTAGa

TCNTAGa

retention time

LLnPC LLPC OLnPC PLnPC OLPC PLPC SLPC PPPC SOPC

26 28 28 28 30 30 32 32 34

24.42 26.50 26.55 27.17 28.63 29.25 31.25 32.00 33.38

11.89 14.85 15.98 17.58 20.28 22.26 28.13 31.20 35.07

LLnL LLL OLnL PLnL OLL OLnO PLL PLnO PLnP OLO PLO PLP OOO POO POP SOO SOP

40 42 42 42 44 44 44 44 44 46 46 46 48 48 48 50 50

37.67 39.75 39.80 40.42 41.88 41.92 42.50 42.55 43.17 44.00 44.63 45.25 46.13 46.75 47.38 48.75 49.38

33.57 37.03 37.88 39.69 41.35 42.16 43.18 44.08 44.83 45.64 47.50 48.18 49.78 51.71 52.34 54.82 56.41

ECNTAG, TCNTAG, ECNPC, and TCNPC were calculated based on the basis of eqs 2, 3, 4. and 5, respectively.

curve. The parameters appeared to fit a linear regression model, and the regression curve between the retention time and TCNPC values of PC species showed good linearity (Figure S1A, Supporting Information). For the retention time and TCNTAG, the linearity of the regression curve was also examined (Figure S1B, Supporting Information). The results suggested that, similar to the case for TAG species, L-α-PC could be separated into molecular species by a highly efficient C30 column, and the elution order paralleled TCN correspondingly. Therefore, we considered that linear regression curves from the TCN and retention time of individual molecular species can be used for the determination of molecular species in both PC and TAG. Brouwers et al. suggested that a mathematical relationship exists between the retention time and ECN of each PE molecule, which was defined as ECN = CN − 1.505n (where CN is the total carbon number of fatty acid residues per PE molecule and n is the number of double bonds in the fatty acid residue); this relationship has been used to assist in the identification of unknown PE species when mass spectrometry is not available.12 The retention time of the PC molecule increases proportionally to the ECN coefficient (ECN = CN − 2n), which is a discriminating factor that can provide valuable information for PC separation.15,28 Choi et al. developed a retention time index for PC profiling, which showed that the fatty acid chain length and the degree of unsaturation were linked, achieving linear curves between the relative retention time and mass over charge (m/z) of PC.29 However, the relation between TCN and retention time of phospholipids has remained unclear to date. For those PC species with the same ECNPC values, such as LLPC, OLnPC, and PLnPC, their TCNPC values showed slight differences, enabling further discrimination (Table 1). Elhamdy and Perkins concluded the separate mechanism of TAGs was likely that the affinity between the stationary phase and fatty acids becomes stronger when the methylene groups in the chain increase or double bonds decrease, causing molecules to travel more slowly in a column, resulting in longer retention times.27 Moreover, the TCNPC and retention time of PC species were easily regressed as a linear curve (R2 > 0.98) (Figure S1A, Supporting Information). Since TCNTG has been widely used to predict

separated in sequence with TCN.27 The ECN of TAG (ECNTAG) and TCNTAG were calculated using eqs 2 and 3: ECNTAG = CN − 2n

(2) 3

TCNTAG = ECNTAG −

∑ Ui 1

(3)

where CN is the total carbon number of the fatty acid residue per TAG molecule and n is the number of double bonds in the fatty acid residue per TAG molecule. The Ui value of unsaturated fatty acid was calculated from experimental data for known TAG species according to Elhamdy and Perkins.27 In brief, a plot of retention times versus carbon numbers of several saturated TAG standards was used as a calibration curve. For any saturated TAG, the TCN value is equal to the actual carbon number of fatty acid residues while the Ui of unsaturated fatty acid was calculated from experimental data for the known fatty acyl chains of TAGs. For example, the triolein peak has ECN and TCN (which is calculated from the calibration curve) values of 48 and 46.125, respectively; then, ∑31Ui = 48 − 46.125 = 1.875. Consequently, the Ui factor for oleoyl is 0.625. The other Ui factors were obtained in the same way and are as follows: 0.75 for linoleyl, 0.83 for linolenyl groups, and 0.0 for saturated acyl groups. As regards PC, only two fatty acids were taken into consideration to calculate their TCNPC values. The ECN of PC (ECNPC) and TCNPC were calculated on the basis of eqs 4 and 5, respectively: ECNPC = CN − 2n

(4)

TCNPC = ECNPC − (U1 + U2)

(5)

where CN is the total carbon number of the fatty acid residue per PC molecule and n is the number of double bonds in the fatty acid residue per PC molecule. U1 and U2 are factors adapted from TAG for fatty acyl chains at the sn-1 and sn-2 positions, respectively. As shown in Table 1, as the value of TCN increased, the relative retention time increased correspondingly. In addition, the TCNs and retention times of nine PC molecular species in the L-α-PC profile (Figure 1) were selected to construct a 3855

DOI: 10.1021/acs.jafc.5b01022 J. Agric. Food Chem. 2015, 63, 3851−3858

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Journal of Agricultural and Food Chemistry the unknown TG species in previous studies,27,30 using the plot of the TCNPC and retention time of known PC species to predict the fatty acid components of unknown PC peaks should be a promising alternative to specify the PC molecular species in HPLC-ELSD analysis. In addition, on the basis of the relation of TCN to retention time, we considered not only for TAG and PC species determination but also the molecular species of other phospholipids could be determined. Furthermore, we observed a disadvantage that the sn positional isomers, i.e., molecular species such as OPPC and POPC, have identical retention times and cannot be separated under the current HPLC separation conditions. The same situation also occurred in TAG analysis if their TCNTAG values were near or identical. For instance, the TAG molecular species identified as POL represented a mixture of POL, PLO, and OPL. The detailed positional assignment of individual fatty acid residues is much more difficult or even impossible for TAG. Quantitation of Individual PC Molecular Species. To prepare quantitative curves with this RP-HPLC-ELSD system, five pure PC species, including OOPC, POPC, OPPC, PPPC, and LLPC, were analyzed individually to determine their respective relationships between concentration and peak area. Regression analyses of these linear plots revealed that the correlations between ELSD responses to all analytes were almost identical (Figure S2, Supporting Information). The list of relative response ratio of their interception values showed that the relative response factor values were highly similar to one another (Figure S2, Supporting Information). Therefore, the contents of corresponding HPLC fractions could be determined on the basis of peak area percentage. The percentage of individual PC species was calculated by using eq 6. % of PC species =

Table 2. Distribution of the PC Molecular Species from Soybean Lecithin and Sunflower Lecithin area (%)a PC species LLnPC LLPC OLnPC PLnPC OLPC PLPC SLPC PPPC SOPC

PC soybean 3.77 40.64 0.97 1.28 15.18 26.83 2.42 3.63 5.29

± ± ± ± ± ± ± ± ±

PC sunflower

0.25 0.28 0.01 0.01 0.11 0.19 0.01 0.01 0.02

0.00 64.25 0.00 0.00 16.85 13.02 3.19 2.32 0.36

± ± ± ± ± ± ± ± ±

0.00 0.75 0.00 0.00 0.37 0.42 0.25 0.16 0.19

Data are mean ± SD of triplicate determinations. The contents of PC molecular species were determined according to eq 6.

a

contains 63% linoleic acid, 18% oleic acid, 11% palmitic acid, and 4% stearic acid but no linolenic acid.1 Our results were comparable with these data, and no linolenic acid was observed in isolated sunflower PC. In other words, sunflower PC lacks the linolenic acid-related PC molecular species, which significantly differentiates it from soybean PC. Soybean oil and sunflower oil from their respective acetonesoluble fractions were analyzed by HPLC-ELSD (Figure S3C,D, Supporting Information). The TAG species were assigned by calculating the TCNTAG value and comparing the results with TAG profiles of known TAG components.23 The TAG compositions of soybean oil and sunflower oil samples are given in Table 3. The TAG species in soybean oil and Table 3. TAG Compositions of Soybean Oil and Sunflower Oil

A(peak area of individual PC species) × 100 ∑ A(total peak areas of PC species) (6)

area (%)a TAG species LLnL LLL OLnL PLnL OLL OLnO PLL PLnO PLnP OLO PLO PLP OOO POO POP SOO SOP

Determination of PC Contents in Lecithins and PC Profiling. The contents of PC in crude lecithins were determined according to AOCS method Ja 7b-91 to be 12.3 ± 2.6% (w/w) for crude soybean lecithin and 19.4 ± 1.5% (w/ w) for sunflower liquid lecithin. The PC content in lecithin was quantitated by normal-phase HPLC-UV on the basis of the absorbance of the PC at 206 nm being proportional to the number of double bonds in PC from the same origin. Therefore, quantitation by normal-phase HPLC-UV is not appropriate when the PC molecular species of lecithin samples contain diverse fatty acyl chains. PC profiles of isolated soybean PC and sunflower PC were analyzed by HPLC-ELSD (Figure S3A,B, Supporting Information). The compositions of isolated PC from soybean and sunflower lecithins are given in Table 2. Like the species L-αPC, the isolated soybean PC was composed of nine PC species. The most abundant PC species was LLPC, accounting for 40.6%. The total amount of PC species with two polyunsaturated fatty acyl chains at the sn-1 and sn-2 positions, such as LLPC and LLnPC, were 44.4%. The major PC species in the sunflower PC profile were very similar to those in the soybean PC profile. The LLPC level was quite high (64.3%) in sunflower PC, but LLnPC, OLnPC, and PLnPC peaks were absent. Regarding of the fatty acid composition of lecithins, Nieuwenhuyzen and Tomas reported that soy lecithin contains 55% linoleic acid, 17% oleic acid, 16% palmitic acid, 7% linolenic acid, and 4% stearic acid and sunflower lecithin

soybean oil 3.76 23.93 2.59 0.83 25.41 0.47 13.95 0.30 0.18 9.91 8.34 2.37 4.20 1.32 1.76 0.42 0.27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

sunflower oil

0.11 0.11 0.12 0.19 0.11 0.03 0.25 0.02 0.01 0.19 0.04 0.06 0.03 0.15 0.04 0.02 0.02a

0.00 40.94 0.00 0.00 37.26 0.00 6.74 0.00 0.00 6.52 2.52 2.56 1.95 0.41 0.95 0.12 0.05

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.00 0.13 0.00 0.00 0.18 0.00 0.03 0.00 0.00 0.15 0.03 0.07 0.08 0.01 0.02 0.00 0.01b

Data are mean ± SD of triplicate determinations. The contents of TAG species were determined according to eq 1.

a

sunflower oil were primarily trilinolein (LLL), dilinoleoyl oleoyl glycerol (OLL), and linoleoyl palmitoyl glycerol (PLL), accounting for 63.3% and 85.0%, respectively. Both soybean oil and sunflower oil contained minor amounts of dioleoyl linoleoyl (OLO), palmitoyl linoleoyl oleoyl glycerol (PLO), dipalmitoyl linoleoyl glycerol (PLP), OOO, dioleoyl palmitoyl glycerol (POO), dipalmitoyl oleoyl glycerol (POP), dioleoyl 3856

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Journal of Agricultural and Food Chemistry stearoyl glycerol (SOO), and stearoyl oleoyl palmitoyl glycerol (SOP). Similarly, there were several TAGs containing linolenic acid in the soybean oil profile in comparison with the sunflower oil profile, such as dilinoleoyl linolenoyl glycerol (LLnL), oleoyl linolenoyl linoleoyl glycerol (OLnL), and palmitoyl linolenoyl linoleoyl glycerol (PLnL). The main TAGs in soybean oil and sunflower oil were consistent with those found in previous research.31 PCs are biologically active lipids and are the principal constituents of biological membranes. To date, publications regarding the characterization of PC molecular species in the food industry are still limited. Chen et al. screened the PC compositions of phospholipase-A2-catalyzed enzymatic reaction products by LC ESI-tandem MS.18 Hence, RP-HPLC-ELSD could be used to monitor the fatty acid composition of structured PCs during enzymatic interesterification. Recently, sunflower lecithin has been regarded as a promising replacement for soybean lecithin due to concerns about genetically modified products. As shown in Table 2, the major PC species of soybean lecithin and sunflower lecithin are quite similar. Moreover, sunflower lecithin was rich in the bioactive LLPC and may have the potential to replace soybean lecithin to produce LLPC from a non genetically modified source. Additionally, on the basis of the significant differences in minor PC species such as LLnPC, OLnPC, and PLnPC, determining the content of these PC molecular species was sufficient to differentiate soybean and sunflower lecithins. These various PC species could be viewed as prospective indicators to discriminate plant lecithins. In conclusion, a method for PC molecular species profiling by RP-HPLC-ELSD was introduced. The PC molecular species were identified by positive-ion MALDI-TOF-MS. The relationship between the TCNs and retention times of standard PC species was developed alongside the RP-HPLC-ELSD analysis. The quantitation of PC molecular species was achieved on the basis of area normalization. This method can be used to characterize PC molecular species from crude lecithins, which would provide further information regarding the properties and functions of lecithins.

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ABBREVIATIONS USED

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REFERENCES

PC, phosphatidylcholine; PPC, polyenylphosphatidylcholine; RP, reverse phase; HPLC, high performance liquid chromatography; PE, phosphatidylethanolamine; ELSD, evaporative light scattering detector; UV, ultraviolet; GC, gas chromatography; FID, flame ionization detector; TAG, triacylglycerol; TCN, theoretical carbon number; 2,5-DHB, 2,5-dihydroxybenzoic acid; TLC, thin-layer chromatography; Nd:YAG, neodymiumdoped yttrium aluminum garnet; Rf, retention value; AOCS, American Oil Chemists’ Society; LPC, lysophosphatidylcholine; TCNPC, TCN of PC; TCNTAG, TCN of TAG; ECN, equivalent carbon number; ECNTAG, ECN of TAG; TCNPC, TCN of PC; LLnL, dilinoleoyl linolenoyl glycerol; LLL, trilinolein; OLnL, oleoyl linolenoyl linoleoyl glycerol; PLnL, palmitoyl linolenoyl linoleoyl glycerol; OLL, dilinoleoyl oleoyl glycerol; OLnO, dioleoyl linolenoyl glycerol; PLL, dilinoleoyl palmitoyl glycerol; PLnO palmitoyl linolenoyl oleoyl glycerol; PLnP, dipalmitoyl linolenoyl glycerol; OLO, dioleoyl linoleoyl glycerol; PLO, palmitoyl linoleoyl oleoyl glycerol; PLP, dipalmitoyl linoleoyl glycerol; OOO, triolein; POO, dioleoyl palmitoyl glycerol; POP, dipalmitoyl oleoyl glycerol; SOO, dioleoyl stearoyl glycerol; SOP, stearoyl oleoyl palmitoyl glycerol; LLPC, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine; PPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; OOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; OPPC, 1-oleoyl-2palmitoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine; LLnPC, 1-linoleoyl-2-linolenoyl-sn-glycero-glycero-3-phosphocholine; OLnPC, 1-oleoyl2-linolenoyl-sn-glycero-3-phosphocholine; PLnPC, 1-palmitoyl2-linolenoyl-sn-glycero-3-phosphocholine; OLPC, 1-oleoyl-2linoleoyl-sn-glycero-3-phosphocholine; PLPC, 1-palmitoyl-2linoleoyl-sn-glycero-3-phosphocholine; SLPC, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine; SOPC, 1-stearoyl-2-oleoylsn-glycero-3-phosphocholine

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ASSOCIATED CONTENT

S Supporting Information *

Correlation between TCN and retention time (Figure S1), calibration curves (Figure S2), and chromatograms of isolated PCs (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*N.-W. Su: tel, +886-2-33664819; fax, +886-2-23632714; email, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The MS spectra were obtained at the Joint Center for High Valued Instruments, National Sun Yat-Sen University, supported by the Ministry of Science and Technology, Taipei, Taiwan. The authors thank Prof. Jentaie Shiea and Dr. Hung Su for technical assistance with MS analysis. 3857

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