Molecular species analysis of phosphatidylcholine by reversed-phase ion-pair high-performance liquid chromatography

The retention behavior of molecular species of phosphatidylcholine (PC) is studied by reversed-phase (RP) ion-pair high-performance liquid chromatography (HPLC). Mobile phases contain tetraalkyl ammonium phosphates (TAAPs) in methano-acetonitrile-water. The stationary phase is alkyl-bonded silica. Competitive interactions of TAAPs, analyte solutes, and an RP-HPLC column result in reduced retention of PC molecular species. PC molecular species are eluted at longer retention times with a larger size of TAAP in the mobile phase, and an increase in the TAAP concentration invariably causes a decrease in PC molecular species retention times. There is a linear correlation between the logarithmic retention factors (k) of PC molecular species and the total number of carbon atoms of TAAP, and the logarithm of k values of PC molecular species can be approximated as a linear function of the logarithm of the counter-ion concentration. There is found to be no distinct dependence between k values of PC molecular species and the mobile phase pH.     

Reversed-Phase Ion-Pair High-Performance Liquid Chromatography of Phosphatidylinositols

Abstract

Reversed-phase ion-pal, high-performance liquid chromatographic (HPLC) separations of molecular species of phosphatidylinositols (PI) were studied. Mobile phases of acetonitrile-methanol-water containing various tetraalkylammonium phosphates (TAAP) were used for optimization. Stationary phases of macroporous polystyrene divinylbenzene (MPD), octylsilica, and octadecylsilica exhibited arying degrees of retentivity toward PI solutes. Without exception capacity factors (k') of PI on either alkylsilica or MPD increased with the alkyl chain length and the concentration of the quaternary ammonium counter ions evaluated. the results can be interpred in terms of an ion-pair retention mechanism. Logarithmic k' values were linearly related to the total number of carbons in TAAP. Perbenzoylation of PI yielded derivatives readily resolvable without the use of any mobile phase additive. Incorporation of TAAP to a mobile phase facilitated component separations of early-eluting perbenzoates. In HPLC with     

Thin Layer Chromatography of Phospholipid Composition in Mouse and Rabbit Spermatozoa

Abstract

The time course of the loss of different components of the phospholipids of rabbit and mouse epididymal spermatozoa during spontaneous lipid peroxidation was determined, using thin layer chromatography with a specific situ hydrolysis method to differentiate the acyl and alkenyl (plasmalogen) moieties. The components followed were phosphatidylethanolamine (PE), phosphatidylcholine (PC), the PE and PC phasmalogens, sphingomyelin (SP), cardiolipin (CL), and phosphatidyl-glycerol (PG). In both mouse and rabbit sperm, the PE component was found to be more than 90% diplasmalogen: 1,2-di(0-1′-alkenyl) glycerophosphoethanolamine. This component was lost rapidly during peroxidation. All PE has disappeared from rabbit sperm after 4 h aerobic incubation, at which point the other phospholipids had been little affected. In both mouse and rabbit sperm, the PC component was found to be 50% monoplasmalogen. The decrease in PC plasmalogen of rabbit sperm amounted to 74% after 20 h, compared to 42% loss of total PC. Similar observations were made with mouse sperm, except that rates of loss of all components were approximately twice those in rabbit. Distribution of the phospholipid components between sperm heads and tails was also determined: PE diplasmalogen was almost entirely found in the tail fraction, in both mouse and rabbit sperm. This mode of analysis allows the differentiation of sensitivities towards spontaneous peroxidation of the different types of phospholipid present in sperm membranes.     

红细胞膜上各种磷脂中脂肪酸的测定

应用自制硅胶板的薄层色谱法分离了红细胞膜上四种主要磷脂。将分离后的每种磷脂斑点硅胶涂层刮下，不经萃取，直接在无水甲醇－苯－乙酰氯溶液中进行转移甲基化，然后用毛细管相色谱分离测定其脂肪酸组成和含量。上述方法已成功地用于先天愚型病人和正常人红细胞膜上各种磷脂中脂肪酸轮廓分析，获得有意义的结果。     

用高效液相色谱法测定热应激时大鼠肺及肝组织中细胞膜磷脂的变化

 应用高效液相色谱法（ＨＰＬＣ）测定了正常大鼠肺及肝组织中细胞膜磷脂的含量及热应激时膜磷脂的含量变化。流动相为甲醇∶乙腈∶８５％磷酸（３∶１００∶１，Ｖ／Ｖ／Ｖ），色谱柱为μ－Ｐｏｒａｓｉｌ柱（３．９ｍｍｉ．ｄ．×３００ｍｍ）。通过测定膜磷脂的变化，可以为阐明机体的发病机理提供可靠的数据。     

Carbon fiber bundle-Au-Hg dual-electrode detection for capillary electrophoresis

Normal-phase high-performance liquid chromatography was used on a preparative scale to seperate phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC) phospholipids from soybean. Separation was achieved using mixtures of three solvents, hexane, methanol and isopropanol. The optimized mobile phase compositions were experimentally determined while operating in a linear gradient mode using 15, 5-20, 25-40, and 40-63 microm preparative particles as well as 4 microm analytical particles. A gradient mobile phase was established on a commmercially available analytical Nova-Pak column such that hexane linearly decreased from 85 to 0 as isopropanol and methanol linearly increased in two gradient steps from 10 to 30 and 5 to 70 respectively. The total run time was 25 min at a flow-rate of 1.5 ml/min. A slight change in mobile phase composition was required to increase the resolution of phospholipids. The 15 microm particle size gave the best separation of the preparative particle sizes examined based on their resolutions between PE and PI and PI and PC. Finally, the retention factors of PE and PC were correlated in terms of mobile phase composition.     

High-performance liquid chromatography of biotin and analogues

Biotin, analogues, and chemical intermediates were separated by high-performance liquid chromatography (HPLC) using reversed-phase and anion-exchange chromatographic conditions. Reversed-phase separations provided a wide range of retention times and resolution of nearly all the biotin compounds from mixtures of the analogues. Anion-exchange separations gave generally shorter retention times as compared to reversed-phase separations and greater resolution between biotin l- and d-sulfoxide. However, fewer analogues were resolved from mixtures of the compounds with anion-exchange HPLC.     

兔骨骼肌肌质网磷脂组分及脂肪酸的分析

分析了兔骨骼肌肌质网磷脂的组成及摩尔分数,并用气相色谱法对两种主要磷脂的脂肪酸组成及摩尔分数进行了测定。     

高效液相色谱法检测食品中多种香料共存时的罂粟壳

 采用高效液相色谱法检测食品中多种香料共存时的罂粟壳。样品经碱化后采用氯仿 乙醇 (体积比为 9∶1)提取 ,在苯基柱上以乙腈 甲醇 水为流动相进行分离 ,外标法定量。罂粟碱 ,吗啡 ,可待因在 1 2mg/L～ 10 0mg/L范围内线性关系良好 ,平均回收率为 79.3%～ 86 .1%。该方法的专属性和准确度较高。     

兔骨骼肌肌质网磷脂组分及脂肪酸的分析

 分析了兔骨骼肌肌质网磷脂的组成及摩尔分数,并用气相色谱法对两种主要磷脂的脂肪酸组成及摩尔分数进行了测定。     

Characterization of dynamically prepared phospholipid-modified reversed-phase columns.

We have modified a reversed-phase (RP8) column by passing through it an aqueous solution of phosphatidylcholine-based liposomes. The phospholipids from the liposomes adsorb onto the octyl chain of the stationary phase, thus altering the nature of the stationary phase and of the chromatographic interactions. The properties of the phospholipid-modified column were investigated using solutes belonging to several chemical classes. We found that the retention factors of negatively and positively charged solutes decreased as the amount of phospholipid in the column was increased. For the solutes studied here the extent of the decrease was smaller for the positive solutes. With neutral solutes, the retention factors of some (benzenediols) increased markedly while with others (ketones) the retention factors decreased. The selectivities between the various solutes on the phospholipid-modified column were different than on the original reversed-phase column. The retention behavior of the solutes can be explained in terms of (1) effective shielding of the hydrophobic part of the stationary phase by the polar head groups of the phospholipids and (2) hydrogen bond formation between the solutes and the carbonyl oxygens as well as the non-ester phosphate oxygens in the polar head groups of the phospholipids.     

Profiling and quantification of Drosophila melanogaster lipids using liquid chromatography/mass spectrometry

We present here the findings of global profiling of Drosophila lipids using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on an LTQ-Orbitrap instrument. In addition, we present a multiple reaction monitoring (LC-MRM) method for the absolute quantification of the major phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids of Drosophila. Using both normal- and reversed-phase LC followed by accurate mass analysis and MS/MS on an LTQ-Orbitrap instrument, we evaluated the lipid composition of the fruit fly Drosophila melanogaster. A total of 74 lipid species were identified consisting of glycerphospholipids belonging to the PE, PC, phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) classes including several plasmanyl PE species, as well as triacylglycerides, cardiolipins, ceramides, and PE ceramides. Individual PE and PC phospholipids were then quantified using an LC-MRM approach. Reversed-phase chromatography followed by monitoring on a QTrap 4000 instrument of 21 MRM transitions combined with calibration curves constructed using internal standards enabled the absolute quantification of 28 PE and PC lipid species with limits of quantification of 3 and 5 pg/μL, respectively. Internal standards accounted for the differences in ionization efficiencies of PE and PC phospholipids, facilitating more accurate lipid abundance measurements. The method presented here builds on previous Drosophila work by making the quantification of absolute lipid abundance possible and will be of interest to scientists who study variation and changes in the degree of unsaturation, fatty acid carbon length, and head-group concentration among individuals of different genotypes in response to environmental, genetic, or physiological perturbation in small insects. It will also be particularly useful to biologists interested in adaptation and acclimation of cellular membranes in response to thermal heterogeneity.     

Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.     

Effect of phospholipase A2 on purified gastric vesicles.

The phospholipid and fatty acid composition and role of phospholipids in enzyme and transport function of gastric (H+ + K+)-ATPase vesicles was studied using phospholipase A2 (bee venom). The composition (%) was phosphatidyl-choline (PC) 33%; sphingomyelin (sph) 25%; phosphatidylethanolamine (PE) 22%; phosphatidylserine (PS) 11%; and phosphatidylinositol (PI) 8%. The fatty acid composition showed a high degree of unsaturation. In both fresh and lyophilized preparations, even with prolonged incubation, only 50% of phospholipids were hydrolyzed, but the amount of PE and PS disappearing was increased following lyophilization. There was a marked decrease in K+-ATPase activity (75%) but essentially no loss of the associated K+ p-nitrophenyl phosphatase was found. ATPase activity could be largely restored by various phospholipids (PE greater than PC greater than PS). There was also an increase in Mg2+-ATPase activity, partially reversed in fresh preparations by the addition of phospholipids (PE greater than PS greater than PC). Proton transport activity of the preparation was rapidly inhibited, initially due to a large increase in the HCl permeability of the preparation. Associated with these enzymatic and functional changes, the ATP-induced conformational changes, as indicated by circular dichroism spectra were inhibited.     

高效液相色谱法和蒸发光散射检测器分析大豆磷脂分子种

高效液相色谱法和蒸发光散射检测器分析大豆磷脂分子种     

Separation of pesticides on an octadecyl-coated alumina column

The retention times of 33 commercial pesticides were determined on an octadecyl alumina HPLC column using water-methanol mixtures. The retention of the pesticides decreased with an increasing concentration of methanol in the eluent proving the regular retention behaviour of the solutes. The retention characteristics of pesticides did not correlate to their hydrophobicity parameters indicating that the absorption centers of alumina not covered by the hydrophobic octadecyl ligand considerably influence the retention. The opposite linear correlation between the retention of pesticides on alumina and octadecyl alumina supports proves that octadecyl alumina supports behave as real reversed-phase supports, however, their retention characteristics differ considerably from those of traditional octadecylsilica supports. Received: 2 August 1996 / Revised: 12 December 1996 / Accepted: 19 December 1996     

The use of dielectric spectroscopy for the characterisation of polymer-induced flocculation of core-shell particles

This work is aimed at investigating the influence of a plant stanol (β-sitostanol) on Langmuir monolayers from various phospholipids and comparing the effect of phytostanol versus its unsaturated analog--phytosterol (β-sitosterol). The studied phospholipids differed in the structure of polar head (phosphatidylcholine--PC, phosphatidylethanolamine--PE, phosphatidylserine--PS) as well as in the number of monounsaturated chains in PC molecule. It was found that the introduction of stanol into PC monolayers is thermodynamically favorable, contrary to its effect on PE and PS films. The magnitude of condensing and ordering effect of stanol depends both on the number of monounsaturated chains in PC molecule and on the composition of stanol-PC mixture. The analysis of BAM images evidenced phase separation of immiscible components in stanol/DPPS systems. The results of Langmuir monolayer studies for stanol/phospholipids mixtures compared with those for corresponding sterol/phospholipids systems proved quite a similar effect of both compounds on the investigated phospholipid monolayers, despite differences in the structure of tetracyclic ring skeleton.     

Separation of phosphatidylcholine and phosphatidylethanolamine by using high-performance displacement chromatography

A binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was successfully separated by high-performance displacement chromatography (HPDC) on an 150 mm x 4.6 mm analytical silica column (3-5 microm packing), using dichloromethane-methanol (9:1, v/v) as carrier and ethanolamine as displacer. The effects of displacer concentration, flow-rate, loading amount and the composition of the sample on separation efficiency were studied. Eighty-four milligrams sample (PE:PC 1:1.16) was separated perfectly by using 83 mM ethanolamine (in carrier) as displacer at the flow-rate of 0.1 ml/min. The yields of the pure PE and PC (100% purity) were 94.8% and 87.9%, respectively and the cycle time for a single separation was about 195 min. It was valuable that the optimum loading amount (the allowed maximum of sample loading) was investigated only by using the sample to be simulated the composition of the separated actual one, because the separation efficiency was significantly affected by the composition of the sample. For the same loading amount of 175 mg, the yields of the pure PE and PC were improved greatly from 31.4 and 16.9 to 56.0 and 77.6%, respectively, when the proportion of PE to PC was adjusted from 1:1.16 to 1:4. Furthermore, the separation of PE and PC in an actual sample (soybean phospholipids) was achieved using the proposed HPDC method.     

High performance liquid chromatography-tandem mass spectrometry of phospholipid molecular species in eggs from hens fed diets enriched in seal blubber oil

The total lipid fraction of eggs from hens fed diets enriched in seal blubber oil (1.25-5.0% SBO) was directly analysed with normal-phase high performance liquid chromatography coupled on-line with electrospray ionization ion-trap tandem mass spectrometry (HPLC-ESI-MS-MS) for the identification of the molecular species of phospholipids (PLs). The species of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were all detected as the [M-H](-) ions. The phosphatidylcholine (PC), sphingomyelin (Sph) and lysophosphatidylcholine (LPC) classes, were detected as formate adducts [M+HCOO](-). Tandem MS of PE and PI showed the loss of the carboxylate anions, and, for PI, also the loss of water and inositol. Product ion spectrum of PC, LPC and Sph contained only the [M-CH(3)](-) ion fragment. Feeding different levels of SBO for 5 weeks resulted in a significant increase of PE, PC and PI molecular species carrying eicosapentaenoic acid (C(20:5 omega3), EPA), docosapentaenoic acid (C(22:5 omega3), DPA) and docosahexaenoic acid (C(22:6 omega3), DHA), but not Sph nor LPC. The highest increase of the omega3/omega6 ratio occurred for PE and PC. On the contrary, PI was less affected by the increase of SBO in the diet.     

Simultaneous determination of cations, zwitterions and neutral compounds using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography

A novel mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography (HPLC) method is described to simultaneously determine four related impurities of cations, zwitterions and neutral compounds in developmental Drug A. The commercial column is Primesep 200 containing hydrophobic alkyl chains with embedded acidic groups in H(+) form on a silica support. The mobile phase variables of acid additives, contents of acetonitrile and concentrations of potassium chloride have been thoroughly investigated to optimize the separation. The retention factors as a function of the concentrations of potassium chloride and the percentages of acetonitrile in the mobile phases are investigated to get an insight into the retention and separation mechanisms of each related impurity and Drug A. Furthermore, the elution orders of the related impurities and Drug A in an ion-pair chromatography (IPC) are compared to those in the mixed-mode HPLC to further understand the chromatographic retention behaviors of each related impurity and Drug A. The study found that the positively charged Degradant 1, Degradant 2 and Drug A were retained by both ion-exchange and reversed-phase partitioning mechanisms. RI2, a small ionic compound, was primarily retained by ion-exchange. RI4, a neutral compound, was retained through reversed-phase partitioning without ion-exchange. Moreover, the method performance characteristics of selectivity, sensitivity and accuracy have been demonstrated to be suitable to determine the related impurities in the capsules of Drug A.