High performance reversed-phase chromatography of the triglycerides from human plasma lipoproteins

The triglycerides of human plasma lipoproteins were separated with high performance reversed-phase liquid chromatography. An octadecyl bonded 5-μ silica column was used with a mobile phase of acetonitrile/acetone. Individual triglyceride types and critical pairs may be easily separated and identified.     

Uptake and interconversion of cholesterol and cholesteryl esters byPhytophthora cactorum

When cholesterol, cholesteryl palmitate and cholesteryl acetate were added individually to sterol-free cultures ofPhytophthora cactorum, the free sterol was at first taken up more rapidly. By 24 hr, the uptake of esters and free sterol was similar. The 2 esters apparently are taken up by different mechanisms, since much acetate was found in extracts of the mycelium at early harvests, but very little palmitate. In cultures supplemented with a mixture of cholesterol and cholesteryl palmitate, the palmitate-derived cholesterol was preferentially incorporated into the free sterol fraction of mycelial extracts. Cholesteryl palmitate and acetate were both hydrolyzed, and free cholesterol esterified by filtrates of cultures grown on sterol-free medium. Reverse-phase chromatography on hydroxyalkoxy-propyl-Sephadex resolved the sterol esters of mycelial extracts into 3 zones, the most polar comprising mainly the linolenate ester, the next linoleate, and the least polar mainly oleate. Linoleate was predominant among the first sterol esters synthesized by the mycelium whether the supplement was free sterol, palmitate or acetate. Later, oleate became predominant.     

Determination of cholesteryl esters and of cholesteryl and epicholesteryl silyl ethers by capillary gas chromatography

The capillary gas chromatography of cholesteryl esters after splitless injection into a 25-m, OV-1-coated, fused silica WCOT column and a 7-m Silar 10C-coated glass WCOT column is reported. The nonpolar OV-1 column separated the cholesteryl esters principally on the basis of carbon number, but separation of the saturated esters from the unsaturated esters was also achieved. The polar Silar 10C column separated the esters mainly according to the degree of unsaturation. Thus, the 2 column types complement each other in the analysis of nanogram quantities of cholesteryl esters from small samples, such as those from plasma or single arterial atherosclerosis lesions. This technique therefore obviates some of the difficulties of analyzing such cholesteryl ester samples in the form of methyl esters (incomplete transmethylation, and contamination by solvent impurities and/or plasticizer esters). Both columns were also found to be useful for the separation and quantitaiton of thet-BDMS ethers of cholesterol and epicholesterol in mixtures containing various proportions of these epimers.     

High pressure reverse phase liquid chromatography of fatty acidp-bromophenacyl esters

High pressure reverse phase liquid chromatography has been employed to rapidly separate saturated and unsaturated fatty acids as the correspondingp-bromophenacyl esters. Through the use of a highly efficient C₁₈ reverse phase column packing, it has also been possible to distinguish among geometrical and positional isomers of the unsaturated acids. The use of ultroviolet-sensitive esters has permitted the detection of low (nanogram range) concentrations of fatty acids. The time required for analysis has been further reduced by employing a novel and rapid method for the preparation of the esters.     

反相高效液相色谱法测定化妆品中的3-O-乙基维生素

采用反相高效液相色谱法测定化妆品中的3-O-乙基维生素C,色谱条件:SH IMADZU Sh im-pack VP-C18ODS(150 mm×4.6 mm,5μm)色谱柱;C18ODS保护柱芯;流动相V(甲醇)∶V(0.025 mol/L KH2PO4)=20∶80;流速:1.0 mL/m in;柱温:30℃;紫外检测器检测,检测池温度:40℃,波长:254 nm。结果表明,在此条件下,3-O-乙基维生素C在0~100 mg/L与相应的峰面积具有良好的线性关系(r=0.999 2),线性回归方程为A=31.170 3,ρ回收率在97.75%~100.60%,方法精密度RSD约为2.6%(n=5)。     

反相高效液相色谱法测定化妆品中几种防腐剂

采用反相高效液相色谱法测定化妆品中重氮烷基脲、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯3种防腐剂,用Waters XTerra Ms C18柱(4.6 mm i.d ×250 mm,5μm),乙腈-水作为流动相,梯度洗脱,检测波长254 nm.回收率为97.30%～102.00%,相对标准偏差为3.6%～8.2%.     

反相高效液相色谱法测定化妆品中防腐剂

采用反相高效液相色谱法测定化妆品中山梨酸、苯甲酸、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯和对羟基苯甲酸丁酯等6种防腐剂,用ZorbaxODS(5μm,4 6mmi d ×150mm)柱,V(磷酸二氢钾)∶V(乙腈)为90∶10作流动相,加入磷酸调pH值为2 0,检测波长为254nm。样品用乙醇-水-冰乙酸(体积比为70∶29 5∶0 5)提取,回收率为90 8%～105 7%,相对标准偏差为3 1%～8 5%。     

High performance liquid chromatography of fatty methyl esters: Analytical separations

Fatty methyl esters are separated on the basis of unsaturation and chain length on an analytical scale by high performance liquid chromatography with a C18/Corasil column and aqueous acetonitrile solvent. Analysis by this method includes polymerized and oxidized esters which may not be detected by gas chromatography.     

High Performance reversed phase chromatography of Natural triglyceride mixtures: Critical pair separation

The separation of the critical triglyceride pairs C48:0, C50:l, C54:3ccc and C54:3ttt as well as C54:2, C52:l and C50:l has been accomplished without the aid of any interacting ion such as silver. A theoretical carbon number (TCN) for the unsaturated triglycer-ides can be calculated from the carbon number (CN) and capacity factor (k’) relationship of the saturated triglycerides, and used to predict the separation of critical pairs. A mathematical equation was derived for the identification of not only the triglycerides by their carbon number and number of double bonds but also the possible acyl groups present in these triglycerides. The pattern of triglyceride elution sequence within each triglyceride category with the same equivalent carbon number (ECN) starts with the triglyceride with the highest number of double bonds and terminates with those with the lowest number of double bonds, with the lower ECN triglycer-ides eluting ahead of those with higher ECN. A possible mechanism for the separation of these triglycerides on highly efficient columns packed with 5n silica bonded with the octadecyl stationary phase is postulated.     

Cholesterol metabolism in human monocyte-derived macrophages: Stimulation of cholesteryl ester formation and cholesterol excretion by serum lipoproteins

The role of lipoproteins and serum in the formation and accumulation of cholesteryl esters in human monocyte-derived macrophages (HMD macrophages) was investigated; studies were also carried out with IC21 cells (a cell line derived from mouse peritoneal macrophages). Following preincubation of HMD macrophages with lipoprotein-depleted serum (LPDS), both native and acetylated low density lipoprotein (LDL and AcLDL, respectively) stimulated the formation of cholesteryl esters with a resultant increase in cellular cholesteryl ester content. Cholesteryl ester formation and accumulation was also stimulated in macrophages exposed continuously to 25-hydroxycholesterol. However, the stimulation of cholesterol esterification by either lipoproteins or 25-hydroxycholesterol was not inhibited by progesterone in HMD macrophages, but was in the IC21 cells. Cholesterol efflux and the hydrolysis of cellular cholesterol ester, promoted by serum components, were studied in HMD macrophages preloaded with cholesteryl ester by incubation with 25-hydroxy cholesterol. Replacement of the medium with one devoid of 25-hydroxycholesterol resulted within 24 hr in at least a 30% decrease in the cholesteryl ester content of the HMD macrophages; replacement with a medium high in cholesterol acceptor content (LPDS or high density lipoprotein) and incubation for three days led to the most marked decreases in cellular cholesterol content. Thus, hydrolysis of the cholesteryl esters by HMD macrophages was not dependent on the presence of cholesterol acceptors in the medium, but cellular cholesterol content was.     

Triglyceride analysis by combined argentation/nonaqueous reversed phase high performance liquid chromatography

Full analysis of triglycerides of natural fats and oils has been investigated by the combination of argentation high performance liquid chromatography (HPLC) with nonaqueous reversed phase (NARP) HPLC. An infrared detector was used in argentation HPLC, because it indicated molar responsibility to all triglycerides. After peak trapping with argentation HPLC, each triglyceride fraction was analyzed with NARP chromatography using the glyceride-selective post-column reactor detector. The results of the analyses of triglycerides of palm oil and cocoa butter by the proposed method agreed well with those reported earlier.     

The influence of phthalate esters on human plasma lecithin/cholesterol acyltransferase

The effect of various phthalate esters on the lecithin/cholesterol acyltransferase activity in man was studied in vitro. The enzymatic activity was strongly reduced with all phthalates except for the dimethyl phthalate. The inhibition rate depends on the phthalate concentration and also on the carbon number of the alkyl groups of phthalates.     

Transesterification of cholesteryl esters

The transesterification of cholesteryl stearate, oleate, linoleate, linolenate and arachidonate to the fatty acid methyl ester and free cholesterol under mild conditions is described. In adaptation of a published procedure the transesterification was carried out for two hr at room temperature in 1N NaOH in methanol:benzene (60:40, v/v). After addition of saturated sodium chloride solution the reaction mixture was extracted with ethyl acetate. The product mixture was checked for cholesterol oxides by HPLC fractionation followed by measurement of the oxides by direct on-column capillary GC. Although transesterification of cholesteryl stearate and oleate was complete (>99%) in 30 min, a uniform reaction period of two hr, required by the polyunsaturated esters, was used for all cholesteryl esters. 7-Ketocholesterol, a principal oxidation product, was unaffected by the reaction conditions. Presented in part at the AOCS meeting in New Orleans, LA, in May 1987.     

反相高效液相色谱法测定肉制品中残留的盐酸克伦特罗

建立了快速简便测定肉类产品中盐酸克伦特罗含量的反相高效液相色谱法。简化了样品前处理方法,优化了色谱分析条件。结果表明,以Inertil/WondaSil C18(5μm,4.6 mm×150 mm)为分离柱,流动相V(0.1%磷酸)∶V(甲醇)=6∶4,流速1.0 mL/min,进样量10μL,柱温40℃,检测波长244 nm时,可以获得良好的色谱峰。绞碎的样品经饱和硼砂和硫酸锌处理除去脂肪和蛋白质,用水定容。样品加标回收率在95%¹03%之间,保留时间的相对标准偏差为0.14%,峰面积的相对标准偏差为6.73%。此方法前处理简单,分析时间短,测定结果准确可靠,适于肉类产品中盐酸克伦特罗的测定。     

The identity of the cholesteryl esters in human milk

Milk samples were collected from 11 mothers who were at least 4 weeks postpartum. The amounts of fat and the fatty acid compositions of cholesteryl esters (CE) and triacylglycerols (TG) in the milk were determined. The mean concentration of total milk lipid was 3.01 gm/100 ml of milk±.42 SD. The major fatty acids esterified with CE and TG were 16∶0,cis 18∶1 and 18∶2. The patterns were similar except for a greater proportion ofcis 18∶1 in the CE. The majortrans fatty acid detected was the 18∶1 isomer which accounted for 4.48% of the TG fatty acids and 2.96% of the CE fatty acids. Scientific Contribution No. 821, Storrs Agricultural Experiment Station, University of Connecticu, Storrs, CT. 06268     

Glycosphingolipids of human plasma lipoproteins

The content and structure of glycosphingolipids (GSL) in human plasma lipoproteins were studies. The quantitative distribution of the neutral GSL(Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer) and the principal ganglioside (AcNeu-Gal-Glc-Cer) within the different lipoprotein classes was similar to that of whole plasma. The total amounts (μmol glucose/100 ml plasma) of GSL in the plasma lipoproteins of three normal subjects were VLDL (very low density lipoproteins) (trace to 0.46), LDL (low density lipoproteins) (1.08–1.48), HDL₂ (high density lipoproteins₂) (0.62–0.85), and HDL₃ (high density lipoproteins₃) (trace to 0.28). In subjects with Lp(a) lipoproteins, HDL₂ rather than HDL₃ contained most of the GSL in HDL. When the data were corrected for differences in the plasma concentrations of the lipoproteins, the total amounts of GSL(nmol glucose/mg lipoprotein cholesterol) were VLDL(trace to 21.20), LDL(11.70–15.36), HDL₂(8.50–9.10), and HDL₃(3.12). No GSL were detected in lipoprotein deficient plasma. Mass spectrometry of the trimethylsilyl derivatives of the GSL in LDL showed major fragment ions characteristic of their individual structural components. The elevated plasma levels of the GSL(2–18 fold), in a homozygote for familial hypercholesterolemia, resided in LDL which contained an absolute increase (per mg lipoprotein cholesterol) of GSL. Most, if not all, of the plasma GSL are associated with plasma lipoproteins and may have an important role in their biological functions.     

Measurement of intraparticle diffusion in reversed phase liquid chromatography

The intraparticle diffusion coefficient was measured using a method based on the fitting of a set of experimental chromatographic profiles to the lumped pore diffusion model. For this purpose, both the analytical solution of the model in the Laplace domain and a numerical method were used. There was an excellent agreement between the results given by the two methods. These results are compared to those obtained by moment analysis of the same set of chromatographic profiles and by the determination of the intraparticle diffusion coefficient from the second central moment of these bands. Nearly identical results were obtained with these two independent methods. The values of the intraparticle diffusion coefficient, D_e, for rubrene in pure methanol was found to be by the modeling method and by the moment analysis method. These values increase with increasing water concentration, to 1.10×10⁻⁶ and , respectively, in a methanol/water solution and to 1.63×10⁻⁶ and , respectively, in a solution.These results confirm the validity and the consistency of the lumped pore model and the moment analysis theory. They show that both approaches describe correctly the mass transfer kinetics in the particles of packing material during the chromatographic process. Systematic determinations of the intraparticle diffusion coefficient can now be undertaken and the influence of various experimental parameters on this important property of packing materials can be investigated.     

Argentation high performance liquid chromatography of methyl esters

The technique of argentation chromatography with silver ion on a macroreticular exchange resin has been applied to high performance liquid chromatography (HPLC) to separate fatty methyl esters and their isomers. Elution of methyl linolenate from the column and more rapid separation of dienes are made possible by programming column temperature from 25 to 70 C. Samples from ca. 0.025 μ to 8 μl can be analyzed on a 2-mm id x 61-cm column. Two 7-mm id x 61-cm columns in series have been used to separate 100-μl samples into fractions for further analysis by capillary gas chromatography. Various forms of argentation chromatography have been widely used for analysis and for separation of compounds from oils and fats including hydrogenated soybean oil. This paper describes the application of argentation procedures to modern HPLC to obtain faster, more efficient separations. It also describes the application of temperature programming to give more rapid separations and to extend our previous method to include methyl linolenate and its isomers. Presented at 1980 ISF-AOCS Congeress, New York.     

Synthesis of cholesterol esters in the plasma and liver of sheep

A study was made with sheep on the formation in vitro of long chain fatty acid esters of cholesterol by the lecithin-cholesterol-acyltransferase system present in the plasma and the acyl CoA-cholesterol-acyltransferase system present in the liver. The rate of cholesterol esterification in the plasma was 0.024 μmoles/ml/hr. The relative pattern of fatty acids esterified during incubation of the plasma remained constant over the 8 hr period of incubation and was similar to the fatty acids in the plasma cholesteryl esters before incubation began and to the fatty acids in the 2-position of the plasma lecithin. The predominant cholesteryl esters synthesized contained monoenoic and dienoic fatty acids. Unlike the bovine, there was no apparent discrimination in favor of the 18∶2 containing species of plasma lecithin as donors of fatty acids. This difference could be accounted for by the similarity in the 18∶2 content of the phospholipids present in the high density (density >1.062 and < 1.21) and the low density (density > 1.006 and <1.063) lipoprotein fractions of the sheep plasma. The possibility of some discrimination against 20∶4 during cholesterol ester synthesis in the plasma of the sheep cannot be excluded. In the liver, the predominant cholesteryl esters synthesized contained saturated and monoenoic fatty acids; cholesteryl linoleate was synthesized to a very much less extent. There was considerable similarity between the composition of the unesterified fatty acid fraction of the liver before incubation began and the fatty acid composition of the cholesteryl esters synthesized during incubation. Addition of sonicated suspensions of free fatty acids altered markedly the fatty acid pattern of the cholesteryl esters synthesized by the liver slices. From the evidence presented it is concluded that the cholesteryl esters in sheep plasma are syntheized mainly by the plasma lecithin-cholesterol-acyltransferase system. The results are discussed in relation to cholesterol esterification systems demonstrated in the plasma and liver of monogastric animals.     

Thermodynamics and mass transfer kinetics of phenol in reversed phase liquid chromatography

The thermodynamics and the mass transfer kinetics of the chromatographic system made of phenol, in a water-acetonitrile mobile phase, on a C18 RPLC column, were studied in the temperature range from 21 to and the interstitial velocity range of 0.021 to 1.27 cm/s. The equilibrium isotherm was accurately approximated by a multilayer model assuming lateral interactions between adsorbed molecules. The parameters of the kinetics of the phenol mass transfer in this column were measured by the method of moments. These data were analyzed using the available models and correlations. It was proven that the parameters of the mass transfer kinetics measured under linear conditions could be successfully used for the prediction of the concentration profiles obtained under overloaded conditions.