Determination of phospholipid molecular species in pork meat by high performance liquid chromatography-tandem mass spectrometry and evaporative light scattering detection

Normal phase high performance liquid chromatography has been optimized for both evaporative light scattering detection and tandem mass spectrometry in order to characterize the natural phospholipids (PL) (classes and molecular species) of raw and cooked pork meat. The PL fraction included phosphatidylcholine (PC) (42.9%±4.5 for raw vs 42.6%±8.0 for cooked meat), plasmalogen-phosphatidylethanolamine (pPE) and phosphatidylethanolamine (PE) (26.7%±3.1 vs 28.5%±2.3), cardiolipin (CL) (8.3%±2.9 vs 6.3%±0.7), sphingomyelin (Sph) (7.5%±0.9 vs 8.3%±2.1), phosphatidylinositol (PI) (6.8%±0.7 vs 6.5%±2.1) phosphatidylserine (PS) (4.9%±0.5 vs 4.6%±1.4) and lysophosphatidylcholine (LPC) (2.9%±1.3 vs 3.3%±2.6). Arachidonic acid (absent in Sph) was mainly present in pPE and PI and formed molecular species with a saturated fatty acid, such as stearic (as in PI, PS, PE and PC) or palmitic acid (as in PE and PC), or the respective vinyl ethers in pPE (p18:0 and p16:0); however, in PC, arachidonic acid also formed combinations with oleic and linoleic acid. Palmitic acid formed the most abundant molecular species in PC, but not in CL, PE, PI and PS. Unexpectedly, the cooked pork meat showed an increased content of the molecular species of PI and LPC with more unsaturated fatty acids (18:0/20:4 and 18:2, respectively) with respect to raw meat.     

Characterisation of the glycerophospholipid fraction in flaxseed oil using liquid chromatography–mass spectrometry

A high-performance liquid chromatographic method coupled with mass spectrometry was used to characterise the natural phospholipid (PL) classes and molecular species in flaxseed oils. The PL fraction included phosphatidylethanolamine (PE) (27–40%), phosphatidylinositol (PI) (29–32%), phosphatidylcholine (PC) (7–18%), lysophosphatidylcholine (LPC) (8–21%), phosphatidylglycerol (PG) (1–4%) and phosphatidic acid (PA) (1–9%). The distribution of fatty acids was found to differ between phospholipids. Stearic acid was mainly present in the form of PC and LPC. Palmitic acid was present in the most abundant molecular species in PI, PG and PA whereas linoleic acid formed the most abundant molecular species in PE.     

Lipid and fatty acid composition of rabbit meat: Part 2.-Phospholipids

The phospholipid contents (and their fatty acid composition) of the meat of two rabbit breeds (New Zealand white and the commercial hybrid HYLA) fed with two commercial diets of different protein and fibre contents, have been determined. The phospholipid contents in the meat of both rabbit breeds ranged from 9% to 19% total lipid. In this fraction seven different phospholipid classes were detected but phosphatidylcholine (PC) and phosphatididylethanolamine (PE) had average percentages of 50 and 20 of the total phospholipids. The major fatty acids of both phosphatidylcholine and phosphatidylethanolamine were C-16:0, C-18:0, C-18:1 and C-18:2, these together representing more than 70% of the total fatty acids. In general, the PC had an higher saturated fatty acid content than PE, which had a lower percentage of C-16:0 but higher C-20:4. Although differences in the fatty acid composition of the phospholipid fractions of the rabbit meat were observed, the only clear influences of age, sex, breed, and/or feeding were found with the C-16:0, C-18:0 and C-18:2 fatty acids.     

Phospholipid changes in the course of beef meat storing at 4 °C

The mean molecular weight of beef meat phospholipids (738) and the recalculating factor (23.8), making possible to determine the total phospholipid content from lipid phosphorus, were estimated experimentally. The total amount of phospholipids decreases from 569 to 486 mg per 100 g of meat during 14 days of meat storing at 4 °C. The content of phosphatidylcholine, sphingomyelin, phosphatidylserine and phosphatidylinositol decreases, too. In contrast to this the content of lyso-phosphatides and temporarily also phosphatidylethanolamine is increasing. This temporary increase of phosphatidyiethanolamine (PE) concentration is due to enzymatic demethylation of phosphatidylcholine (PC). The course of this conversion has been followed by PE PC ratio changes in the course of meat storing.     

Studies on phospholipids in non-glutinous and glutinous rice bran

The chemical nature of phospholipids of rice bran prepared from two strains, one being Shin-ei, the non-glutinous rice strain, and the other Kamui, the glutinous rice strain, was studied. The main phospholipids were phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in both strains. The minor phospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine were present much more in Shin-ei than in Kamui. The major component fatty acids were palmitic, oleic and linoleic acids, and unsaturated fatty acids were mostly located in the 2-C position of the phospholipid molecules in the two strains. The composition and positional distribution of fatty acids of PC were somewhat different between Shin-ei and Kamui, but those of PE and PI were quite similar in the two strains. The representative molecular species of PC were 1-palmitoyl-2-linoleoyl and 1-oleoyl-2-oleoyl in decreasing order for Shin-ei, and 1-oleoyl-2-oleoyl and 1-palmitoyl-2-oleoyl for Kamui. In both Shin-ei and Kamui, the major molecular species of PE and PI were 1-palmitoyl-2-linoleoyl and 1-palmitoyl-2-oleoyl, and the compositions of the molecular species were similar to each other.     

Changes of intramuscular phospholipids and free fatty acids during the processing of Nanjing dry-cured duck

In this study, changes of intramuscular phospholipids and free fatty acids were tracked during the processing of Nanjing dry-cured duck. Phospholipids were identified and quantified by high performance liquid chromatography combined with UV and evaporative light scattering detectors. The types and quantities of free fatty acids and fatty acids derived from phospholipids were analyzed by capillary gas chromatography. The results showed that raw duck meat had high quantities of phosphatidylethanolamine, and phosphatidylcholine (37.95% and 54.07% of total phospholipids, respectively), which contained high percentages of polysaturated fatty acids. The percentages of total phospholipids, phosphatidylethanolamine, and phosphatidylcholine decreased during processing, with a concomitant increase in quantities of free fatty acids. The lipolysis of phospholipids, especially phosphatidylethanolamine is the main contributor to the increase of free fatty acids.     

Seasonal variation in the chemical composition of horse-mackerel (Trachurus trachurus)

Chemical analysis was performed on samples of horse mackerel (Trachurus trachurus L.) caught monthly off the Portuguese coast throughout a period of one year (May 1997-April 1998). In the period between August and January the amount of total lipids present was recorded showing a minimum in February. Protein content remained fairly constant during the whole one-year period. Seasonal variation of lipid classes was followed and fatty acid profiles of total, polar and non-polar lipids as well as that of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were determined. The non-polar lipids were the ever dominant group, mainly composed of triacylglycerols. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the main phospholipids mainly containing 20:5̣ and 22:6̣, but PE was richer in 22:6̣. Thus this species is all year long an adequate element of the traditional Portuguese diet, and contributes to the ̣ intake.     

六种鱼鱼头磷脂的组成及脂质脂肪酸分析

为研究海水鱼和淡水鱼鱼头磷脂及脂质脂肪酸组成的差异,以海水鱼(马鲛鱼、巴浪鱼、金鲳鱼)和淡水鱼(罗非鱼、鲫鱼、鲢鱼)鱼头为研究对象,利用Floch法提取6种鱼鱼头总脂;硅胶柱层析法将总脂分离收集中性脂、糖脂和磷脂;薄层层析(thin layer chromatography,TLC)分析比较6种鱼鱼头磷脂种类的分布、组成;气相色谱(Gas chromatography,GC)分析6种鱼鱼头脂质的脂肪酸组成。结果表明,6种鱼鱼头中提取率最高的脂质类型为中性脂,占总脂的48.98%~77.84%,其次为磷脂,占总脂的5.93%~22.86%,糖脂提取率最低,占总脂的3.67%~15.91%。鱼头磷脂中共检出4种磷脂组分,分别为磷脂酰乙醇胺(Phosphatidylethanolamine,PE)、磷脂酰胆碱(Phosphatidylcholine,PC)、鞘磷脂(Sphingomyelin,SM)、溶血磷脂酰胆碱(Lysophosphatidylcholine,LPC),其中PC明显高于其他磷脂组分。脂质中均含有较高含量的多不饱和脂肪酸(Poly-unsaturated fatty acid,PUFA),其中以二十二碳六烯酸(docosahexaenoic acid,DHA)为主,且均含有二十碳五烯酸(eicosapntemacnioc acid,EPA),海水鱼和淡水鱼鱼头磷脂中DHA和EPA占总脂肪酸的比例最高,分别为18.39%~21.43%和5.61%~10.38%,且海水鱼鱼头中以DHA和EPA为主的PUFA含量高于淡水鱼鱼头。因此,海水鱼鱼头是提取n-3多不饱和脂肪酸的潜在资源,尤其是制备n-3多不饱和脂肪酸型磷脂的良好来源。     

油菜、菊花和荷花蜂花粉中磷脂的色谱分析

以油菜、菊花、荷花蜂花粉为原料,采用薄层层析法(thin layer chromatography,TLC)法分离纯化蜂花粉中的磷脂,并用高效液相色谱法(high-performance liquid chromatography,HPLC)法测定磷脂酰肌醇(PI)、磷脂酰丝氨酸(PS)、脑磷脂(PE)、卵磷脂(PC)及溶血卵磷脂(LPC)的含量。结果表明：3种蜂花粉中总磷脂含量为1.19～3.98g/100g,3种花粉存在极显著差异(P＜0.01);PC是蜂花粉磷脂的主要成分,占总磷脂的34.30%～59.69%;在油菜蜂花粉中检测到PI、PS、PE、PC、LPC,菊花蜂花粉未测出PI,荷花蜂花粉未测出PI、LPC。结论：油菜蜂花粉中磷脂种类最丰富、总含量最高,是花粉磷脂的较好来源。     

草鱼头磷脂制备工艺优化及抗氧化性能分析

为了得到较高纯度的草鱼头磷脂并分析其抗氧化性能,采用乙醇溶液浸提冷冻干燥草鱼头粉末制备磷脂,正交试验确定其最优工艺后,利用薄层层析(TLC)分析比较草鱼头磷脂种类的组成及含量,利用气相色谱-质谱联用技术分析磷脂的脂肪酸组成,并进行体外抗氧化能力测定。结果表明:磷脂制备最优工艺方案为:乙醇浓度80%,料液比1:6 g/mL,提取温度65℃,提取时间3 h,在此条件下,得到草鱼头磷脂的纯度为84.20%±0.65%,提取率为3.03%±0.06%。草鱼头磷脂主要含有磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)、鞘磷脂(SM)、溶血磷脂酰胆碱(LPC)四种磷脂,其中PC的含量最高(60.00%±0.26%),其次为PE(20.00%±0.17%)。草鱼头磷脂脂肪酸组成以多不饱和脂肪酸(PUFA)为主(52.09%±0.59%),其中,C20:5n-3(EPA)和C22:6n-3(DHA)含量较高。体外抗氧化测定试验结果表明,草鱼头磷脂的羟自由基(·OH)清除能力和还原力能力都显著(P<0.05)优于对照的商品大豆磷脂。     

Isolation and characterization of lecithin from squid (Todarodes pacificus) viscera deoiled by supercritical carbon dioxide extraction

Marine lecithin was isolated and characterized from squid (Todarodes pacificus) viscera residues deoiled by supercritical carbon dioxide (SC-CO(2)) extraction. SC-CO(2) extraction was carried out to extract the oil from squid viscera at different temperatures (35 to 45 °C) and pressures (15 to 25 MPa). The extraction yield was higher at highest temperature and pressure. The major phospholipids of squid viscera lecithin were quantified by high-performance liquid chromatography (HPLC). Phosphatidylcholine (PC; 80.5% ± 0.7%) and phosphatidylethanolamine (PE; 13.2% ± 0.2%) were the main phospholipids. Thin layer chromatography (TLC) was performed to purify the individual phospholipids. The fatty acid compositions of lecithin, PC and PE were analyzed by gas chromatography (GC). A significant amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were present in both phospholipids of PC and PE. Emulsions of lecithin in water were prepared through the use of a homogenizer. The oxidative stability of squid viscera lecithin was high in spite of its high concentration of long-chain polyunsaturated fatty acids. PRACTICAL APPLICATION: Squid viscera are discarded as a waste by fish processing industry. Since lecithin from squid viscera contains higher amounts of polyunsaturated fatty acids, it may have promising effect to use in food, pharmaceutical, and cosmetic industries.     

低温风干型酱鸭加工过程中脂质氧化特性

为研究酱鸭加工过程中脂质氧化特性,在传统酱鸭加工工艺的基础上,引入低温风干工艺制备酱鸭,并分析其加工过程中pH值、磷脂、游离脂肪酸（free fatty acid,FFA）、过氧化值（peroxide values,POV）、硫代巴比妥酸（thiobarbituric acid reactive substances,TBARS）值和挥发性盐基氮（total volatile base nitrogen,TVB-N）值的变化。结果表明：pH值从原料肉的5.81升高至杀菌肉的6.09;酱鸭加工过程中肌内磷脂的分子种类结果表明,脑磷脂（phosphatidylethanolamine,PE）有5 种分子含量较高,分别是PE（C16:0/C20:4）、PE（C16:0/C18:1）、PE（C16:0/C22:5）、PE（C16:0/C22:4）和PE（C16:0/C22:6）;POV和TBARS值均先升高后下降;TVB-N值在腌制、酱制和杀菌阶段显著降低,而风干使其升高（P＜0.05）。相关性结果表明,总FFA与单不饱和脂肪酸（R=0.92,P＜0.01）,多不饱和脂肪酸与pH值（R=0.90,P＜0.01）,多不饱和脂肪酸与TBARS值（R=0.80,P＜0.01）,TBARS值与POV（R=0.89,P＜0.01）,POV与TVB-N值（R=0.81,P＜0.01）均呈高度正相关,卵磷脂与PE呈显著负相关（R=－1.00,P＜0.01）。本研究为低温风干工艺下酱鸭的脂质分解氧化机理研究提供理论依据。     

3种经济类海胆的脂质组成差异分析

海胆为我国名贵海珍品,营养丰富、经济价值高,但不同种类的海胆品质存在差异,本文对国内三种常见经济类海胆的脂质组成进行分析。以性腺基本指数、总脂含量、胆固醇含量、磷脂含量、甘油酯含量、脂肪酸组成、磷脂组成等为评价指标,结合统计分析技术比较不同种类海胆脂质组成的差异。结果显示,光棘球紫海胆、黄海胆和虾夷马粪海胆总脂占干基的(21.83±0.12)%~(27.21±0.10)%;其中磷脂和甘油酯是最主要的脂类组分,分别占(39.62±0.49)%~(49.39±0.73)%和(30.08±0.01)%~(38.65±0.03)%;磷脂中的主要组分为磷脂酰胆碱(Phosphatidylcholine,PC)和磷脂酰乙醇胺(Phosphatidylethanolamine,PE)和磷脂酰肌醇(Phosphatidylinositol ,PI);总脂中脂肪酸主要为C16:0、C18:0、C20:1及C20:5(Eicosapentaenoic acid,EPA)。对脂类相关数据进行标准化处理,并结合聚类分析和主成分分析,结果显示三种海胆性腺脂类物质中脂质组成、磷脂组成及脂肪酸组成差异明显。研究表明,光棘球紫海胆相对于其他两种海胆具有较高的营养价值和脂质优势,可作为C20:4n3、C20:4n6、C20:5n3、C22:6n3及磷脂等功能性脂质分子的重要膳食来源。     

Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae. Fatty acid pattern of phospholipid classes and selective acyl turnover at sn-1 and sn-2 positions

Acyl chains linked to phospholipids of the yeast, Saccharomyces cerevisiae, are mainly C16:1 and C18:1 accompanied by minor amounts of C14:0, C16:0 and C18:0. In view of this rather simple fatty acid composition, the question arose whether in yeast, as in higher eukaryotes, fatty acyl groups were characteristically distributed among the sn-1 and sn-2 positions of distinct phospholipid classes. Analysis of fatty acids linked to the sn-1 and sn-2 positions of the major phospholipids showed that indeed saturated fatty acyl groups predominated in the sn-1 positions. While the percentage of saturated fatty acids was low (10%) in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from cells grown on rich medium, it was higher in phosphatidylserine (PtdSer) (25%) and highest in phosphatidylinositol (PtdIns) (41%). Oleate was mainly linked to position sn-2, while palmitoleate predominated in position sn-1. Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g. PtdSer and PtdEtn, PtdEtn and PtdCho, and PtdIns and PtdSer) suggest that pathways must exist for the generation of distinct phospholipid molecular species within the different phospholipid classes. The highly selective incorporation of exogenous [¹⁴C]palmitic acid (90%) and [³H]oleic acid (99%) into the sn-2 position of PtdCho, and the preferential incorporation of these fatty acids into the sn-2 position of PtdEtn (70 and 90%, respectively, for palmitic and oleic acid) are compatible with the postulate that phospholipase A₂-mediated deacylation followed by reacylation of the lysophospholipids is involved in the generation of phospholipid species in yeast.     

Liquid chromatographic–mass spectrometric analysis of glycerophospholipids in corn oil

The phospholipid composition of corn oil was determined by LC/ESI-MS. The content of phospholipids in the oils varied from 5.2% to 8.7%. The major components in the phospholipids fraction were phosphatidylcholine (57.5–68.1%), phosphatidylinositol (14.5–19.8%), and phosphatidylethanolamine (10.3–13.9%). Phosphatidic acid and phosphatidylglycerol accounted for less than 10% of total phospholipid content. Various molecular species within each class were detected. The phosphatidylcholine class was mainly composed of phosphatidylcholine-C18:2/C18:1 (40–45%) and phosphatidylcholine-18:1/C18:1 (40–51%). Phosphatidylinositol-C16:0/C18:2 (50–69%) was the most abundant phosphatidylinositol. The two phosphatidylethanolamines were phosphatidylethanolamine-C16:0/C18:2 (34–42%) and phosphatidylethanolamine-C18:2/C18:2 (31–36%). Phosphatidylglycerol species were mainly phosphatidylglycerol-C16:0/C18:2 (42–48%) and phosphatidylglycerol-C16:0/C18:1 (36.7–40.3%). Identification of PC molecular species suggests the possibility that corn oil can have therapeutics effects.     

A method for separation and quantification of phospholipid classes in human milk

A simple, isocratic method for separating the major phospholipid classes of human milk by HPLC is described. Resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine, and spingomyelin from human milk phospholipids was achieved in 30 min on a silica column. Total phospholipids were injected in 50 microliter of chloroform:diethyl ether (1:2, vol/vol) and eluted with a solvent mixture of acetonitrile:methanol:sulfuric acid (100:3:.05, vol/vol/vol) at a flow rate of 2.5 ml/min. Fractions were collected and each phospholipid class quantified by analysis of inorganic phosphorus after sulfuric acid digestion. A repeatability study with 19 samples had a coefficient of variation of 5.3%. The analytical recoveries of phospholipid standards averaged 98%. Recoveries varied with phospholipid class; variation was greatest with spingomyelin.     

Fatty acid and aldehyde composition of individual phospholipid classes of rabbit skeletal muscles is related to the metabolic type of the fibre

The fatty acid composition of individual phospholipid classes as related to metabolic type of fibre in the rabbit was studied. The fatty acid composition of the individual phospholipid classes of five muscles were compared: two glycolytic ones (Longissimus lumborum and Psoas major), two oxidative ones (Soleus and Semimembranosus propriosus,) and an intermediate one (Gastrocnemius laterale). It was shown that except for phosphatidyl inositol (PI), the fatty acid compositions of the main phospholipid classes were strongly related to the metabolic type of the fibres; phosphatidyl ethanolamine (PE) of oxidative muscles contains less 18:2 n-6 and more 18:0 and long chain PUFA of the n-6 and n-3 series than that of glycolytic ones; phosphatidyl choline (PC) of oxidative muscles contains more 18:0 and less 16:0 and 18:2 n-6 than that of glycolytic ones; cardiolipin of the oxidative muscles contains less 18:2 n-6 than those of the glycolytic ones. These differences in fatty acid composition of PE, PC and cardiolipin explain a large part of the differences in fatty acid compositions of the total phospholipids of glycolytic and oxidative muscles.     

Lipid changes during germination of fenugreek seeds (Trigonella foenum-graecum)

Fenugreek seeds were germinated in the dark for 96 h. Total lipid extracts were prepared and found to decrease on germination. Ultraviolet, visible and infra-red spectra were estimated for the lipids of ungerminated and germinated fenugreek seeds. Free fatty acids (FFA), total chlorophyll and carotenoid pigments increased greatly after germination. On the other hand, triglycerides, phospholipids and unsaponifiable matter decreased. Determination of individual phospholipids showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constitute about 67% of the total phospholipids of ungerminated seeds. After germination PC and PE decreased whilst phosphatidic acid (PA) and phosphoglyceric acid (PG)—degradation products of phospholipids by phospholipases—increased. The fatty acid composition showed that the total unsaturated fatty acids decreased whilst the total saturated fatty acids increased on germination. The fatty acids 18:2 and 18:3 were the most abundant acids in the lipids of the ungerminated seeds and fell after germination from 41·2% to 31·8% and 23·2% to 14.4%, respectively. The minor constituent fatty acids 20:0, 22:0 and 20:1 increased by 3·3-, 3·0- and 7·8-fold, respectively after germination.     

Improvement of a solid phase extraction method for separation of animal muscle phospholipid classes

The solid phase extraction (SPE) method used by Banni et al. (2001) [Banni, S., Carta, G., Angioni, E., Muru, E., Scanu, P., Melis, M. P., et al. (2001). Distribution of conjugated linoleic acid and metabolites in different lipid fraction in the rat liver. Journal of Lipid Research, 42, 1056–1061] for fractionation of liver phospholipids (PLs) into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) was modified to improve its separation efficiency. A mixture of PL standards, containing PC, PE, PS and PI, was separated into individual classes by using aminopropyl minicolumns, following the method of Banni et al. (2001). The obtained eluted fractions were further analysed by thin layer chromatography (TLC) and PL classes were identified. TLC revealed the co-elution of PC and PE, that of PE with PS and no elution of PI. The SPE method was subsequently modified in order to obtain a correct PL fractionation into PC, PE, PS and PI. The principal modifications consisted of increased solvent volumes for PC, PE and PS elution, and a different solvent mixture to allow PI elution. The effectiveness of the modified SPE method was checked by TLC, using both standards and muscle samples, showing a correct elution of PC, PE, PS and PI.     

卵磷脂类保健食品中PE、PC的高效液相色谱测定

为准确快速地对卵磷脂保健食品中有效成分进行定量分析 ,我们建立了卵磷脂保健食品中磷脂主要组分磷脂酰胆碱 (PC)、磷脂酰乙醇胺 (PE)的高效液相色谱测定方法。方法的日间精密度为 5 4 %～ 9 0 %;PE、PC的加标回收率分别为 83 0 %～ 10 5 6 %和 81 3%～ 86 0 %,均可满足卫生分析的要求。对 5种卵磷脂保健食品中的PC、PE的含量进行了测定 ,PC、PE的含量分别为15 2 %～ 2 4 2 %和 3 1%～ 6 1%。方法的样品前处理方法简单 ,且检测灵敏度高 ,分析速度快 ,适于在国内推广使用