Capillary electrochromatography of sterols and related steryl esters derived from vegetable oils.

Capillary electrochromatographic (CEC) separations of plant sterols and related esters were evaluated under various conditions. Stationary phases included octadecylsilica (C18) and triacontylsilica (C30). Mobile phases comprised acetonitrile, tetrahydrofuran, and tris(hydroxymethyl) aminomethane buffers in aqueous or non-aqueous systems. Apart from notable differences in component resolution, both C18 and C30 phases had dramatic influence on the elution behavior of the title compounds. Generally, C18 had greater selectivity for most components with elution patterns in consistence with the hydrophobicity of side chain structures, while no predictable trend of analyte elution was observed in CEC with C30. In the latter column systems, analyte separations appeared to be improved by conversion to benzoates or ferulates. Twenty-four-epimers of campesterol acetate and 7-campestenol acetate as well as the campesterol-stigmasterol pair were readily resolved by CEC with either phase. However, the cholesterol-stigmasterol pair was barely resolved and had an elution order opposite to that of their acetates or benzoates. Potential applicability of the CEC technique in the analysis of sterols and sterol ferulates in vegetable oil is demonstrated.

Capillary electrochromatographic evaluation of vitamin E-active oil constituents: tocopherols and tocotrienols.

Separations of lipid antioxidants, tocopherols (T) and tocotrienols (T3), on octylsilica (OS), octadecylsilica (ODS), phenylsilica, or silica were studied by capillary electrochromatography (CEC)-UV detection. The homologues and isomers of the vitamin E-active compounds were best separated with an OS column. CEC with an ODS column tended to yield broad peaks with poor resolution. Among the various mobile phases evaluated, [acetonitrile-methanol (64:36)]-[25 mM tris(hydroxymethyl)aminomethane, pH 8] (95:5) eluent systems produced the most satisfactory results. Under these conditions, a baseline separation of an 11-component mixture was obtained with elution order similar to that observed in reversed-phase HPLC: deltaT3 > (gamma+beta)T3 > alphaT3 > epsilonT > (delta+zeta2)T > (gamma+beta)T > alphaT > alphaT-acetate. CEC of the antioxidant acetates led to separations inferior to those of the parent compounds. Effects of CEC experimental variables (e.g., mobile phase solvents and buffers, stationary phases and electric field) on analyte separations were assessed in the context of resolution factors and retention factors.

Chromatographic analysis of plant sterols in foods and vegetable oils.

This paper reviews recently published chromatographic methods for the analysis of plant sterols in various sample matrices with emphasis on vegetable oils. An overview of structural complexities and biological/nutritional aspects including hypocholesterolemic activities of phytosterols is provided in the Section 1. The principal themes of the review highlight the development and application of chromatographic techniques for the isolation, purification, separation and detection of the title compounds. Pertinent gas chromatographic and high-performance liquid chromatographic methods from the literature are tabulated to illustrate common trends and methodological variability. The review also covers specific analyses of natural/synthetic standard mixtures to shed light on potential applicability in plant sample assays. Examples of combined chromatographic techniques linked in tandem for the analysis of complex samples are included. Elution characteristics of sterol components are discussed in the context of analyte substituent effects, structural factors and stationary/mobile phase considerations.

Chromatographic analysis of tocol-derived lipid antioxidants.

This paper provides a comprehensive overview of existing chromatographic methods for the analysis of tocol-derived lipid antioxidants in various sample matrices. After a brief introductory discussion on biological and nutritional aspects of the vitamin E active compounds, the review focuses on various techniques for the isolation, purification, chromatographic separation, and detection of tocopherols and tocotrienols. Compiled published normal-phase (NP) and reversed-phase (RP) high-performance liquid chromatographic (HPLC) methods demonstrate general trends and analytical variability and versatility of HPLC methodology. The relative merits of the two HPLC methods are assessed. NP and RP elution characteristics are delineated to aid in the identification of antioxidant components. Technical novelty of certain analytical procedures for non-food samples warrants their inclusion in this review in light of the potential applicability in food assays.

Separation procedures for phosphatidylserines.

This paper reviews working procedures for the separation of phosphatidylserines (PS) in complex sample matrices. It begins with an introductory overview of important aspects of PS involvement in cellular lipid biochemistry. The main body of the review describes various procedures for the extraction, isolation, purification, and separation of the PS class and its molecular species in tissue samples. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques. Factors influencing normal-phase and reversed-phase separations are delineated. The last section covers selected chemical derivatization procedures useful for enhancing the separation efficiency and detection sensitivity and specificity.

Reversed-phase separations of nitrogenous phospholipids on an octadecanoyl poly(vinyl alcohol) phase.

Molecular species of nitrogenous phospholipids (PLs) phosphatidylcholine (PC), phosphatidylethanolamine (PE), PE-derivatives and sphingomyelin (SP) were separated on an octadecanoyl poly(vinyl alcohol) (ODPVA) column by reversed-phase HPLC with UV and evaporative light scattering detection (ELSD). Mobile phases employed variable proportions of acetonitrile, methanol and water. HPLC-UV of the polar lipids yield components with peak intensities somewhat different from those obtained by HPLC-ELSD despite discernible similarity in the peak profiles observed in the two detection systems. Incorporation of ammonium hydroxide in mobile phases resulted in a decrease in analyte retention. The mobile phase basicity effect on capacity factors of PE species was significantly greater than that of PC counterparts. The new ODPVA-HPLC-ELSD technique was applied to the analysis of PC and PE molecular species in vegetable oils.

High-performance liquid chromatography of phosphatidic acid.

This paper reviews existing high-performance liquid chromatographic (HPLC) methods for the analysis of phosphatidic acid (PA) in various sample matrices. In addition to the introductory background discussion on important aspects of PA in lipid biochemistry, the review provides comprehensive coverage in the areas of derivatization techniques, detection methods, and HPLC separation techniques. Conversions of PA to suitable derivatives enhance the detection sensitivity and improve the chromatographic behavior of the analytes. Detection methods include the use of state-of-the-art detectors and are discussed in terms of sensitivity, specificity, and compatibility with analytical systems. Pertinent normal-phase and reversed-phase HPLC data for PA are compiled from published methods.

High-performance liquid chromatographic separation of molecular species of neutral phospholipids.

Molecular species of neutral phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were resolved by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases of acetonitrile-methanol-water containing tetraalkylammonium phosphates (TAAPs). Competitive interactions of TAAPs and analyte solutes with a reversed-phase HPLC column resulted in reduced retention of PC or PE with concomitant increase in detection sensitivity. The chromatographic data for PC and PE were distinctly different from those for negatively charged phospholipids where ion-pair retention mechanisms prevailed. While PC (or PE) components eluted at longer retention times with a larger size of TAAP, an increase in the TAAP concentration invariably caused a decrease in phospholipid retention times. Optimization of HPLC conditions by using high concentrations (25-100 mM) of tetramethylammonium phosphate in acetonitrile-methanol-water (70:22:8) facilitated elution of components with improved peak symmetry. HPLC separations of neutral phospholipids derived from animal sources were more complex than those from soybeans.

Analysis of antimycin A by reversed-phase liquid chromatography/nuclear magnetic resonance spectrometry.

A mixture of closely related streptomyces fermentation products, antimycin A, is separated, and the components are identified by using reversed-phase high-performance liquid chromatography with directly linked 400-MHz proton nuclear magnetic resonance detection. Analyses of mixtures of three amino acids, alanine, glycine, and valine, are used to determine optimal measurement conditions. Sensitivity increases of as much as a factor of 3 are achieved, at the expense of some loss in chromatographic resolution, by use of an 80-microL NMR cell, instead of a smaller 14-microL cell. Analysis of the antimycin A mixture, using the optimal analytical high-performance liquid chromatography/nuclear magnetic resonance conditions, reveals it to consist of at least 10 closely related components.

High-performance liquid chromatographic separation of subcomponents of antimycin A.

Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, A1a, A1b, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins A1, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpreted based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

Chiral-phase high-performance liquid chromatography of rotenoid racemates.

The high-performance liquid chromatographic (HPLC) behavior of parent rotenoids (type I) and the hydroxy-analogues (type II) on three different chiral stationary phases (CSPs) was studied. Separations of optical isomers were achieved in various degrees depending largely upon the rotenoidal structures and the CSP types employed. Enantiomers of all but elliptone compounds were separable on beta-cyclodextrin-bonded silica (CDS). Without exception, the 12a-hydroxyrotenoid antipodes were resolved on Pirkle's phenylglycine-bonded silica (PGS) despite unsuccessful attempts to resolve the type I rotenoidal racemates. Conversely, optical resolution of the latter rotenoids was accomplished by using a helical polytriphenylmethylacrylate-coated silica (TPS) column and the observed separation factors (alpha values) ranged from 1.14 to 1.90. The results from HPLC of type II rotenoids on TPS (alpha = 1.00-1.63) suggested that variations in E-ring structures had profound influence on the resolution outcome. Conjugated double bonds on the E-ring and the desisopropylation of the five-membered E-ring of type II rotenoids appeared to be important structural features for chiral recognition involving the TPS substrate. In both reversed-phase (CDS) and normal-phase (PGS and TPS) HPLC modes, the less polar enantiomers were the 6a beta,12a beta-rotenoids as observed in most cases, though this relationship was reversed in the cases of deguelin and hydroxyelliptone probably due to conformational effects of rotenoidal ring systems.

Chromatographic investigations of the configurational and geometrical isomerism of allylic N-terpenyl-N-hydroxyethyl-nitrosamines.

A preparative adsorption column chromatographic method is reported for the separation of cis and trans geometrical isomers of two types of N-nitrosamines derived from allylic terpenyl ethanolamines (experimental fish toxicants). Column eluates were monitored by gas chromatography in which a Carbowax 20M stationary phase was used. Further separation of E and Z configurational isomers was achieved by reversed-phase and normal-phase high-performance liquid chromatography. In the reversed-phase high-performance liquid chromatography system (acetonitrile-water), the 6',7'-acetylenic nitrosamines ( NMOA ) were efficiently resolved by using an argentous (AgNO3) mobile phase, whereas the presence of sodium alkanesulfonate in the aqueous acetonitrile mobile phase favored the base-line resolution of the 6',7'-olefinic nitrosamines ( NDOA ). For normal-phase separation on a silica column, addition of tetrahydrofuran to the mobile phase (methylene chloride-2-propanol) resulted in a varying degree of improvement in peak resolution (R) and column selectivity (alpha). Effects of temperature on the chromatographic behavior of the nitrosamine components are described. The high-performance liquid chromatographic separation method has proved to be applicable for the trace analysis of the title nitrosamines in organic tissues by way of thermal energy analysis.

High-performance liquid chromatographic resolution and quantification of a dilactonic antibiotic mixture (antimycin A).

High-performance liquid chromatographic (HPLC) conditions are presented for the separation and quantitative determination of a homologous antibiotic complex (antimycin A). Combined HPLC and chemical ionization mass spectrometry proved to be exceptionally useful for the structural identification of chromatographic components. Using electrochemical, fluorescence, and ultraviolet detectors, the minimum detectable amounts of the antibiotics were found to be in the ranges 0.10-1.12, 0.31-1.69, and 4.10-28.2 ng, respectively. Advantages of the preparation of Dns derivatives for use in fluorescence detection are discussed. Application of the HPLC technique to the analysis of the antibiotic mixture in organic tissues is demonstrated.