S.L. Abidi

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.

Reversed-phase high-performance liquid chromatographic separations of tocopherols

Abstract The retention behavior and separation characteristics of five natural products α-, β−, γ-, δ-, and ξ 2 -tocopherols and their acetyl derivatives were studied by reversed-phase high-performance liquid chromatography (HPLC)-fluorescence detection. Investigated stationary phases included an octadecyl polyvinyl alcohol (ODPVA) column, several octadecylsilica (ODS) columns, and a pentafluorophenylsilica column. Mobile phases comprised methanol (or acetonitrile) and water at variable proportions. Separation factors for β- and γ-tocopherol isomers and capacity factors of the five tocopherols were determined under various conditions. The β- and γ-tocopherols were separable on ODPVA but not on ODS. However, the βγ pair was resolved with the latter column only as their ester derivatives. HPLC with mobile phases containing alkanols with carbon atoms greater than 2 favored the separation of the βγ pair on ODS and yielded results diametrically different but complementary with those obtained with the ODPVA phase. The combined effects of mobile phases, stationary phases and antioxidant structures on component separations were delineated.

Elution behavior of unsaponifiable lipids with various capillary electrochromatographic stationary phases

Capillary electrochromatographic (CEC) separations of unsaponifiable lipids, tocopherols (T), tocotrienols (T3), and plant sterols were studied under various conditions. Investigated stationary phases include pentafluorophenylsilica (PFPS), triacontylsilica (TCS), and octadecylsilica (ODS) phases. A baseline separation of four sterols (ergosterol, lanosterol, sitosterol and stigmasterol) on ODS was achieved and their elution order was found to be dictated by side-chain structures. CEC of the tocol-derived compounds on PFPS in aqueous methanol yielded the most satisfactory results with complete resolution of all components eluting in the order deltaT3>beta3>gammaT3>epsilonP>alphaT3>deltaT>zeta2T>betaT>gammaT>alphaT, while a reversal in elution of the epsilonT-alphaT3 pair was observed in aqueous acetonitrile. CEC with a TCS phase in non-aqueous methanol led to a different elution pattern deltaT3>gammaT3>betaT3>alphaT3epsilonT>deltaT>(zeta2+gamma)T>betaT>alphaT, despite favorable resolution of the (gamma-zeta2)T pair along with the observation of inseparable(beta-gamma)T and (beta-gamma)T3 pairs in non-aqueous dimethylformamide. Non-aqueous acetonitrile mobile phases provided unique selectivity for the (gamma-zeta2)T pair and isomer separations on TCS. Variations in separation and retention factors of relevant antioxidant species with CEC variables were evaluated. Examples of CEC quantification of unsaponifiable fractions of rice bran oils and soybean oils are presented.

High-performance liquid chromatography of phosphatidic acids and related polar lipids

The retention behavior of phosphatidic acids (PA) and phosphatidic acid methyl esters (PM) was studied by reversed-phase ion-pair high-performance liquid chromatography (HPLC). The HPLC systems consisted of mobile phases of acetonitrile-methanol-water containing tetraalkylammonium phosphates (TAAP) and stationary phases of alkyl-bonded silica and polystyrene-divinylbenzene resins. The lipid solutes were more strongly retained when using mobile phases containing larger TAAP at higher concentrations. The results are indicative of an ion-pair retention mechanism. Molecular species of PM were readily resolved despite the complete inseparability of PA under all conditions used. Except for tetrabutylammonium phosphate, there was a linear correlation between the logarithmic capacity factors (k′) of PM (or PA) and the total number of carbon atoms of TAAP. The significant concentration dependence of separation factors for certain PM components was related to the size effect of TAAP. A normal-phase HPLC method for the separation of PA from other polar lipids is described.

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.

Separations of tocopherols and methylated tocols on cyclodextrin-bonded silica

Abstract α-, β-, γ-, and δ-tocopherols (methyl-substituted tocols) and 5,7-dimethyltocol were separated by normal-phase HPLC on β- or γ-cyclodextrin-bonded silica (CDS) with fluorescence detection. The HPLC behavior of the tocol components was studied under various mobile phase conditions. Hexane or cyclohexane was used in combination with oxygen-containing solvents (alcohol, ether, and esters) in binary and ternary mobile phases. Capacity factors ( k ′) and separation factors (α) for adjacent tocol components were determined. Incorporation of non-polar ethers in the hydrocarbon (hexane or cyclohexane) mobile phases favored the separation of β- and γ-tocopherols with improved α values, which enabled trace analysis of the β-isomer present in soybean oil. Analyte solutes tended to be more strongly adsorbed in mobile phases containing branched-chain alcohols and ethers than in those containing the corresponding straight-chain solvents. Generally, the k ′ values obtained with hexane mobile phases or with the β-CDS phase were greater than those observed in HPLC with cyclohexane mobile phases or with the γ-CDS phase.

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.

Reversed-phase retention characteristics of tocotrienol antioxidants

Abstract Mixtures of α-, β-, γ- and δ-tocotrienols were separated by reversed-phase (RP) high-performance liquid chromatography (HPLC). Four sets of subcomponents (cis/cis-, cis/trans-, trans/cis- and trans/trans-geometrical isomers) were further resolved under various HPLC conditions. Retention characteristics of the β- and γ-isomers on octadecylsilica (ODS) were indistinguishable. However, complete separations of all sixteen tocotrienol components were achieved by RP-HPLC with a nonsilica-based octadecanoyl polyvinyl alcohol (ODPVA) column. In this system, β-tocotrienol peaks were found to interpose alternatively with those of γ-tocotrienols, which was attributable to apparent differences in intrinsic molecular polarity and hydrophobicity. HPLC with a pentafluorophenylsilica (PFPS) column led to partial separations of tocotrienols, of which two subclass species remained unresolved. Peak profiles obtained with PFPS columns of different packings were similar but not identical. Nonetheless, the component elution patterns on PFPS were markedly different from those on ODPVA. Furthermore, the use of a triacontylsilica column enabled separations of fifteen components and yielded an elution order parallel to that of an ODPVA column. In general, the elution sequence of tocotrienols appeared to depend largely on the type of organic modifier used, despite the subtle influence of stationary phases and the isomeric characteristics of alkenyl chains. While poorly resolved on silica, geometrical isomers were readily separated on the RP phases studied with acetonitrile–water as the preferred eluent. Factors affecting analyte retention and component resolution were delineated.

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.