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.     

超高效液相色谱-静电场轨道阱高分辨质谱鉴定核桃仁的脂质构成

利用超高效液相色谱-静电场轨道阱高分辨质谱系统鉴定了核桃的脂质组.在正负离子模式下获得脂质的一级质谱和二级质谱信息后,共鉴定出4大类525种脂质分子,包括250种甘油酯、221种磷脂、18种糖脂、36种鞘脂类,含量分别占总脂质的87％、12.97％、0.02％和0.01％.甘油二酯(diacylglycerol,DG)...     

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.     

Lipid Content and Fatty Acid Composition in Bamboo Shoots

Lipids from bamboo shoots (Phyllostachys pubescens), peeled and divided from top to base, were extracted and fractionated into three classes, and each class separated into constituent components by thin-layer chromatography (TLC). Fatty acid composition and amount of separated lipids were determined. Total lipids (TL) ranged from 800 (top) to 380 mg (base) per 100g fresh weight and the ratio of nonpolar lipids (NPL):glycolipids (GL):phospholipids (PL) was about 17:27:56. The main fatty acids of the three lipid classes were palmitic, linoleic and linolenic acids, but composition was remarkably different among these fractions. The fatty acid composition of triglycerides (TG) was similar to the original NPL. Palmitic acid was almost all located in 1-, 3-position, linoleic acid mainly located in 2-position of TG, while linolenic acid was distributed in each position. Digalactosyl diglyceride (DGDG) and monogalactosyl diglyceride (MGDG) were the main components of GL; the average of the former had about 37% linoleic and 29% linolenic acids, while the latter had about 25% linoleic and 62% linolenic acids. Bamboo shoots contained 9 PL fractions, the major being phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE). PC contained about 48% linoleic, 31% palmitic and 11% linolenic acids, and PE also had the similar tendency as PC.     

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。     

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.     

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.     

Phospholipids determination in vegetable oil by thin-layer chromatography and imaging densitometry

A rapid reliable method was developed to measure vegetable oil phospholipid content by thin-layer chromatography–imaging densitometry. Phospholipid samples were obtained from crude soybean oil by water degumming and then analyzed and quantified by thin-layer chromatography and imaging densitometry. Phosphatidyl choline (PC), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) standard curves were generated. The total phospholipid content of the oil was 1.12–1.26%, of which 27.1 ± 1.86% was PC, 22.7 ± 0.45% was PE, while PI accounted for 17.2 ± 0.85% of the total. A high correlation coefficient of 0.985 was found for reproducibility of pixel area (OD*mm) at a constant concentration of standard phospholipid. Regression analysis of the pixel area vs the phospholipid (PC, PE, and PI) weights (μg) generated R² greater than 0.983. Thin-layer chromatography–imaging densitometry proved a useful tool for rapid determination and quantification of the major phospholipids in soybean oil.     

Lipid metabolism in Fusarium oxysporum

Certain aspects of lipid metabolism in Fusarium oxysporum were studied using acetate-1-C¹⁴ and H₃P³²O₄ for incorporation of radioactivity into the fungal mycelium. During initial stages of growth of the organism (up to 1 h of incubation) the phospholipid fraction exhibited relatively higher specific activity values indicating its rapid rate of synthesis. Higher turnover rates were observed for phosphatidic acid (PA) and polyglycerophosphatides (PGP). It is postulated that PGP represents a labile pool where PA synthesised in excess of the metabolic needs of the cell is stored to be subsequently used during periods of intense phospholipid biosynthesis. In pulse and chase experiments, higher specific activities were observed for PA, PGP and cardiolipin (CL) fractions after 1-day chase on unlabelled media. Rapid turnover rates were observed for PA, PGP and cardiolipin (CL) fractions. It is suggested that these are involved in the growth of new cells and (or) in the cell repair processes. In cells grown both under normal and carbon-starvation conditions, the specific activity pattern of phosphatidyl choline (PC), and phosphatidyl ethanolamine and phosphatidyl glycerol (PE + PG measured collectively) was very similar indicating similarity in the metabolism of these two classes of phosphatides. Addition of sodium arsenite, alloxan, p. benzoquinone and citric acid in the incubation media was found to affect the synthesis of lipid classes in different ways. For example, among the polar lipids, PA, PC, PE + PG, CL and SGP + FFA fractions were inhibited by p.benzoquinone, while the incorporation of label from C¹⁴ was found to be stimulated into the phosphatidyl inositol fraction.     

Characterization of phospholipid composition of black cumin (Nigella sativa L.) seed oil

Black cumin (Nigella sativa L.) seed oil was extracted with two different solvents, n-hexane (H) and a mixture of chloroform/methanol (CM) (2:1, by volume). Amount of total lipid (TL) was higher in the CM miscelle (39.2% of seed fresh weight) than in the H extract (37.9%). Chemical characteristics as well as fatty acid profile of the TL extracts were compared and the analysis revealed that the major fatty acid was linoleic acid C18:2n-6 (ca. 57% of total fatty acid methyl esters (FAME)) followed by oleic acid C18:1n-9. Palmitic acid C16:0 was the major saturated fatty acid and detected in appreciable level. Chromatography on a silica column with solvent of increasing polarity yielded 96.1-97.2% neutral lipids (NL) and ca. 3% of polar lipids. Gas liquid chromatography with flame ionization detector (GLC/FID) showed that the major fatty acid present in all lipid classes was C18:2n-6 followed by C18:1n-9 and C16:0 acids, respectively. Phospholipid (PL) classes were separated via normal-phase HPLC. Separation was achieved on a silica column by gradient elution from isooctane/2-propanol (6:8, by volume) to isooctane/2-propanol/water (6:8:0.6, by volume) lasting 35 min with UV detection at 205 nm. The major individual PL classes were found to be phosphatidylcholine (PC; ca. 46-48% of total PL) followed by phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), respectively. Phosphatidylglycerol (PG), lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC) were isolated in smaller quantities. The level of saturated fatty acids, namely palmitic C16:0 and stearic C18:0 acids, was considerably higher in PL classes than in the corresponding triacylglycerols. Characterization of PL profile from Nigella sativa L. seed oil as well as the development of new source of PL was the primary aim of this study.     

Effect of vacuum-packaged frozen storage, after cooking, on the phospholipids of Hereford and Bison

Changes in the neutral lipid (NL), phospholipid (PL) and their fatty acid (FA) profiles in lean samples of cooked and cooked, vacuum-packaged, frozen (28 days) Hereford and Bison L. dorsi muscle were analyzed. Samples from Herefords contained more saturated fatty acids (SFA) in the neutral lipid fraction than Bison. Storage decreased the C_18:2, C_18:3, C_20:1 and C_20:4 content of the NL. Bison samples contained more total PL and phosphatidylethanolamine (PE) than Hereford. Storage decreased the content of the least polar of the PL which were eluted in the solvent front (fraction 1), phosphatidylinositol (PI), phosphatidylserine (PS), PE and sphingomyelin (SPH). In the combined PL fraction, there were no differences in the FA profile of Bison and Hereford. Storage resulted in a decrease in the content of C_18:2, C_20:4, unsaturated fatty acid (UFA) and polyunsaturated fatty acid (PUFA) in the combined PL fraction. In the PE fraction, Bison samples contained more C_20:4 and PUFA than Hereford. Storage had no effect on the FA profile of PE. The LPC fraction of Bison samples contained more saturated SFA, UFA and PUFA than Hereford, and with storage these FA decreased. Cooked frozen storage also decreased SFA, UFA and PUFA in PC/LPE.     

Influence of Phospholipid Content and Fatty Acid Composition of Individual Phospholipids in Muscle from Bison, Hereford and Brahman Steers on Flavor

Neutral lipid (NL) and phospholipid (PL) fractions and their corresponding fatty acid profiles from the L. dorsi of Bison, Hereford, and Brahman steers of similar age finished on identical diets were determined. Compositional differences were related to sensory characteristics of the samples. Phosphatidylcholine + lysophosphatidylethanolamine (PC + LPE) existed in the highest concentration in all samples evaluated followed by lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), sphingomyelin (SPH), fraction one and phosphatidylserine (PS). Bison contained more total PL, particularly PC + LPE and LPC, than the other two bovine species. Polyunsaturated fatty acid (PUFA) composition of these PL was greater for Bison. Off-flavor and aftertaste increased with increasing levels of PC + LPE and LPC due to their greater composition of PUFA.     

Seasonal changes in phospholipids of mussel (Mytilus edulis Linne)

The phospholipids of mussels (Mytilus edulis Linne) from the coast of Qingdao were extracted, fractionated and analysed over a 12 month period. The contents of total lipids, neutral lipids, polar lipids and phospholipids were measured. The composition of phospholipids was determined by high‐performance liquid chromatography, and their fatty acid composition was analysed by gas chromatography. The phospholipid content ranged from 3.6 to 6.4 g kg^?1 soft tissue. PE (phosphatidyl ethanolamine) and PC (phosphatidyl choline) were the major constituents. C16:0, C20:5 and C22:6 were the major fatty acids. C20:5 (5.25–23.10%) and C22:6 (6.05–20.42%) varied regularly with the seasonal factors. Their total amounts were high from January to June, which would be an optimal time for the utilisation of the phospholipids of mussels. © 2002 Society of Chemical Industry     

配位吸附柱层析法对卵磷脂分离纯化的研究

采用配位吸附柱层析法分离纯化市售粉末大豆磷脂(磷脂酰胆碱含量40%),得到高纯度卵磷脂产品(磷脂酰胆碱含量96.2%)。重点建立了Cu2+型配体交换树脂柱,并优化了分离纯化条件。结果表明,该方法能简便有效地分离出高纯度卵磷脂。     

养殖大黄鱼各部位磷脂组分及其脂肪酸组成分析

为明确大黄鱼各部位磷脂组分及其脂肪酸组成,本实验选取大黄鱼头部、背肌、腹肌、内脏、尾部以及鱼卵为研究对象,分别测定各部位的磷脂含量、组分及其脂肪酸组成。结果表明:鱼卵中磷脂含量最高,为5.50 g/100 g。头部、背肌和腹肌磷脂组分一致,为溶血磷脂酰胆碱(LPC)和磷脂酰胆碱(PC);头部磷脂中LPC和PC的含量分别为39.23%、45.12%,背部分别为62.74%、36.12%,腹部分别为66.69%、33.31%。内脏磷脂组分为溶血磷脂酰胆碱(LPC)、磷脂酰肌醇(PI)和鞘氨醇磷脂(SM),含量分别为59.37%、12.77%、29.83%。鱼尾磷脂为LPC、PC和磷脂酰乙醇胺(PE),含量分别为21.41%、59.37%、19.22%。鱼卵磷脂为LPC、PI、PE和PC,含量分别为12.30%、1.09%、9.12%、76.36%。脂肪酸组成分析表明大黄鱼各部位富含多不饱和脂肪酸,其中鱼卵磷脂中多不饱和脂肪酸占比43.1%,明显优于大黄鱼其他各部位。研究结果说明大黄鱼是一种富含磷脂功能因子的海洋鱼类。      

C NMR as a tool for authentication of different gadoid fish species with emphasis on phospholipid profiles

The aim of this study was to evaluate if phospholipid profiles obtained by ¹³C nuclear magnetic resonance (NMR) spectroscopy is characteristic enough to separate species of lean gadoid fish. ¹³C NMR data were obtained from muscle lipids of five categories of lean gadoid fish, namely, north-east arctic cod and Norwegian coastal cod (Gadus morhua), haddock (Melanogrammus aeglifinus), saithe (Pollachius virens), and pollack (P. pollachius). A total of 27 fish caught at the same location on the Norwegian coast in the traditional fishing season (March/April) in 2006 were analysed. The sn-2 position specificity of 22:6n-3 (docosahexaenoic acid, DHA) in phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) for the different species/stocks were investigated, and the full ¹³C NMR spectra applied in multivariate analysis. Stereospecific distribution calculations showed significant differences among species in the distribution of 22:6n-3 in PC and PE, and the pollack group displayed the lowest values for 22:6n-3 in sn-2 position, both in PC and PE. This first screening showed that by using the ¹³C NMR fingerprint of muscle lipids, linear discriminant analysis gave a correct classification rate of 78% according to the five categories of lean gadoid fish, while successful classification (100%) was achieved with Bayesian belief networks (BBN) predictions.     

Determination of intramuscular phospholipid classes and molecular species in Gaoyou duck

In this study, intramuscular phospholipid classes and molecular species in Gaoyou duck meat were determined. Classes of phospholipids were identified and quantified by normal phase HPLC combined with UV and evaporative light scattering detectors (ELSD). The main phospholipid classes (phosphatidylcholine and phosphatidylethanolamine) were prepared on a semi-preparative silica gel column by HPLC. Reverse phase HPLC was coupled in parallel with both an ELSD and a mass spectrometry in order to characterise molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The results showed that Gaoyou duck meat had high quantities of PC and PE (64.66% and 28.10% of total phospholipids, respectively). Arachidonic acid was mainly present in PE and formed molecular species containing a saturated fatty acid, such as stearic or myristic acid; however, oleic acid together with palmitic or stearic acid formed the main molecular species in PC. The content of the molecular species with polyunsaturated fatty acids in PE accounted for 98.33%, while that in PC only 46.20%.     

Muscle individual phospholipid classes throughout the processing of dry-cured ham: Influence of pre-cure freezing

This paper aims to study the profile of phospholipid (PL) classes of Iberian ham throughout its processing and the changes it underwent due to the influence of the pre-cure freezing treatment. The general profile of each PL class did not vary during the ripening stage. Phosphatidylcholine (PC) showed the highest proportion, followed by phosphatidyletanolamine (PE) and phosphatidylserine (PS) and phosphatidylinositol (PI) being the minor PL. The four PL classes were highly hydrolysed during the salting stage and their degradation continued during the rest of the processing. Pre-cure freezing of Iberian ham influenced the levels of the four PL classes at the initial stage, all of them being higher in refrigerated (R) than in pre-cure frozen (F) hams. Moreover, the pattern of hydrolysis was not the same in these two groups.     

不同煮制时间对水煮鸡蛋质构及蛋黄脂质成分的影响

将新鲜鸡蛋分别在沸水中煮制10、15、20、25 min,通过测定蛋清水分含量、蛋黄水分含量、水分T₂驰豫分布、质构以及蛋黄脂质组成,探究不同煮制时间对鸡蛋质构及脂质营养成分的影响。结果表明,蛋清蛋白在煮制15 min后完全凝胶,而蛋黄则在20 min后完全凝胶。蛋黄中具有生物活性功能的磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)、磷脂酰己醇(PI)、磷脂酸(PA)、磷脂酰甘油(PG)和神经酰胺(Cer)均在煮制15 min的样品中含量最高,只有鞘磷脂(SM)的含量在煮制时间10 min的样品中含量最高,其次是煮制15 min的样品。单不饱和脂肪酸(∑MUFA)含量煮制前15 min下降显著(p<0.05),之后延长煮制时间变化不显著(p>0.05);而多不饱和脂肪酸(∑PUFA)则在煮制前15 min含量变化不显著,之后随煮制时间延长含量显著下降(p<0.05)。由此可得,煮制时间为15 min时,鸡蛋的质构及蛋黄脂质营养最好,这将为工业化生产早餐水煮鸡蛋或者卤蛋生产提供一定的理论指导。