Chromatographic analysis of tocol-derived lipid antioxidants

doi:10.1016/S0021-9673(00)00131-X

Journal of Chromatography A

Volume 881, Issues 1–2, 9 June 2000, Pages 197-216

https://doi.org/10.1016/S0021-9673(00)00131-X Get rights and content

Abstract

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.

Introduction

Lipid antioxidants, tocopherols (T) and tocotrienols (T₃) comprise a number of vitamin E active substances derived from a chromanol structure. These compounds are closely related homologues and isomers depending respectively on the number and position of methyl groups on the aromatic ring of the tocol backbone in tocopherols (Fig. 1). The unsaturated analogues of tocopherols are tocotrienols in which the carbon-2 triterpenyl side chain contains three double bonds at carbon-3′, carbon-7′, and carbon-11′ positions. In addition, plastochromanol-8 (the octaterpenyl side chain analogue of γ-tocotrienol), dehydrotocopherols, and tocodienols occur in nature as tocopherol like substances in vegetable oils.

In 1964, Pennock and coworkers first reported the existence in nature of eight tocol-derived compounds, α-, β-, γ-, and δ-tocopherols of 2R,4′R,8′R-configuration along with the corresponding 2R-trans/trans-tocotrienols [1]. Synthetic materials from racemic precursors normally produce mixtures of diastereomers (Table 1). Due to the presence of three asymmetric carbon atoms at carbon-2, carbon 4′, and carbon 8′, there are eight stereoisomers for each tocopherol molecule. Likewise, synthetic tocotrienols consist of an array of enantiomers (2R and 2S forms) and geometrical isomers (cis/cis-, cis/trans-, trans/cis-, and trans/trans-forms). As demonstrated in Table 1, a total of 64 stereoisomers are present in all racemic synthetic tocopherols and tocotrienols.

The vitamin E family of compounds offers multifaceted health benefits and is essential for mitochondrial electron-transport function in physiological systems. Owing to their lipid soluble antioxidative properties, these compounds inhibit lipid peroxidation processes of polyunsaturated fatty acids and other compounds in cell membranes [2]. Biological activities of the lipid antioxidants vary and depend largely on lipid solubility and standardization of bioassays. Nonetheless, it has been established that 2R4′R8′R-α-tocopherol possesses the highest activity among the various antioxidant structures. Other homologues and levorotatory stereoisomers including tocotrienols exhibit relatively weak vitamin E potency. Thus, the general trend of biological activity of some compounds has been reported as follows: αT>βT>αT₃>γT>βT₃>δT [3].

The title lipid antioxidants have been shown [4] to exhibit an increasing order of relative autoxidative stabilities in methyl myristate: αT=αT₃<βT₃<γT₃<δT₃<γT<δT=βT, and in methyl linoleate: αT<αT₃<γT₃<βT<γT<δT. The same study has demonstrated different trends of photolytic stabilities in respective methyl myristate and methyl linoleate: γT₃<αT₃<δT<αT<γT<βT, and αT<αT₃<γT₃<βT<ζT<γT<δT. On the other hand, the relative stabilities of these compounds in frying oils are function of oil varieties: in soybean oil, αT>δT>βT>γT; in corn oil, αT>γT>δT>γT₃; in palm oil, αT>δ T₃>αT₃>γT₃ [5].

The structural complexity and the wide variation in antioxidative activity of the title compounds necessitate reliable analytical techniques for the isolation, separation, differentiation, and quantification of individual components in mixtures derived from various sample matrices. Availability of pure compounds facilitates structure–activity studies and furthers biomedical and pharmaceutical research on their health application. In particular, trace natural occurrence of certain precious members of the compounds in the series render it necessary to recourse to sophisticated analytical procedures for sample enrichment, high-efficiency resolution, and sensitive detection of analytes at very low levels.

Tocopherols and tocotrienols occur in plants in variable abundance. Vegetable oils provide the best sources of these lipid antioxidants. Isolation and enrichment of the compounds from plant tissue matrices entails traditional solvent extraction or supercritical fluid extraction (SFE) of oilseeds followed by various chromatographic procedures. In comparison to solvent extraction, SFE is a sophisticated newer technique (see its discussion in the Other Chromatographic Techniques section) providing a convenient way to fasten up the extraction with reduced loss of tocol-derived compounds during extraction procedure. For characterization and quantification purposes, the crude isolate can subsequently be further purified and separated by a wide variety of chromatographic techniques: column chromatography, thin layer chromatography (TLC), normal-phase high-performance liquid chromatography (HPLC), reversed-phase HPLC, and other chromatographic techniques. With the recent advancement in column technologies, complete separations of mixtures of synthetic lipid antioxidants (which contain more components than the natural compounds) can be achieved.

Based on the large numbers of publications available in the literature, most researchers have preferred normal-phase HPLC techniques as the methods of choice for their separations of lipid antioxidants because of the relatively easy separations of isomeric β-, and γ-tocopherols and tocotrienols. However, recent introduction of several reversed-phase stationary phases excluding octadecylsilica (ODS) phases enables the separation the β-, and γ-isomers of interest. The new reversed-phase column systems can eliminate the use of hazardous solvents. This paper present a critical review on chromatographic methods for the analysis of tocol-derived lipid antioxidants and their application in food analysis with emphasis on vegetable oils.

Section snippets

Isolation and enrichment procedures

Depending on sample matrices, isolation procedures vary among solid and liquid samples of animal or plant origins. Tocol-derived lipid antioxidants in tissues and oilseeds can generally be isolated by solvent extraction and saponification [6], [7], [8]. In a typical procedure, vegetable oilseeds (15 g) were homogenized (60 s) in a coffee been grinder and extracted with absolute ethanol (100 ml) in a soxhlet thimble overnight on a steam bath. Water (100 ml), and light petroleum (50 ml) are added

Thin-layer chromatography and column chromatography

Among the many chromatographic techniques available for the determination of lipid antioxidants, TLC and solid–liquid adsorption column chromatography methods employ the least expensive apparatus and instruments despite the lack of high quantitation precision. They are suitable for sample cleanup, purification [9], [10], qualitative assays, and rough estimates of the antioxidants in assay samples.

Gas chromatography

Prior to the development of HPLC, lipid scientists have relied heavily on GC techniques for the accurate measurement of oil constituents including tocopherols and tocotrienols. Abreast with the progress in chromatographic column technologies, the column systems used in GC analyses of the title compounds have evolved from packed columns to capillary columns. Using the latter coated columns or chemically bonded columns, antioxidant sample assays can be achieved with high degrees of detection

Normal-phase high-performance liquid chromatographic analysis

Since the emergence of the HPLC technology a few decades ago, the majority of investigators in lipid research have used HPLC methods for the analysis of lipid antioxidants. HPLC has outshone GC because less tedious sample cleanup is involved and milder column temperature conditions prevent the loss of labile analytes. By virtue of detection specificity and sample homogeneity, direct HPLC analysis of tocopherols and tocotrienols in vegetable oils can be carried out with little sample

Reversed-phase high-performance liquid chromatographic analysis

Although β-, and γ- isomers of tocopherols and tocotrienols have not been resolved on traditional octadecylsilica (ODS) columns with acetonitrile (or methanol)–water mobile phases (Fig. 4), reversed-phase HPLC techniques have been widely used in the analysis of lipid antioxidants in cases where one component of an isomer pair is absent in samples and the need for the isomer separation is unimportant in specific research projects. Some practical advantages offered by reversed-phase are easy

Detection techniques

There are several commercial HPLC detectors (Table 3, Table 4) available in the market for evaporative light-scattering detection (ELSD), UV absorbance detection, fluorescence (FL) detection, and electrochemical detection (ED) of tocol-derived lipid antioxidants present in HPLC column effluents. Of these, ED is known to provide the highest sensitivity and has proven useful for the trace analysis of the antioxidants by reversed-phase HPLC–ED. In recent years, ELSD methodology has been widely

Other chromatographic techniques

In the past few years, a new breed of chromatographic technology, capillary electrochromatography (CEC), has progressed rapidly and received a great deal of interest from the separation science community. The CEC technique features high-efficiency, high-resolution, and high-speed separations and poses immense analytical potential for the determination of tocol-derived lipid antioxidants which possess no net charge. A preliminary study [74] conducted at the author’s laboratory shows that, except

Nomenclature

ACN

Acetonitrile

BME

tert.-Butyl methyl ether

CEC

Capillary electrochromatography

IPE

Diisopropyl ether

Dipropyl ether

Electrochemical detection

ELSD

Evaporative light scattering detection

Fluorescence

FID

Flame ionization detection

HAc

Acetic acid

HFIP

Hexafluoroisopropanol

Hexane

Isooctane

Isopropanol

Mass spectrometry

ODPVA

Octadecanoyl polyvinyl alcohol

ODS

Octadecylsilica

PFPS

Pentafluorophenylsilica

PDA

Photodiode array

SFC

Supercritical fluid chromatography

TEA

tetraethyl ammonium hydroxide

THF

Tetrahydrofuran

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ReviewChromatographic analysis of tocol-derived lipid antioxidants

Abstract

Introduction

Section snippets

Isolation and enrichment procedures

Thin-layer chromatography and column chromatography

Gas chromatography

Normal-phase high-performance liquid chromatographic analysis

Reversed-phase high-performance liquid chromatographic analysis

Detection techniques

Other chromatographic techniques

Nomenclature

Biochem. Biophys. Res. Commun.

J. Lipid Res.

Anal. Biochem.

J. Chromatogr.

J. Chromatogr. A

J. Chromatogr.

J. Chromatogr.

Anal. Biochem.

J. Chromatogr.

J. Chromatogr. A

Method Enzymol.

Anal. Biochem.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Food Chem.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr.

J. Chromatogr. A

Methods Enzymol.

J. Chromatogr. A

Methods Enzymol.

J. Chromatogr.

J. Chromatogr.

Anal. Biochem.

Lipids

J. Agric. Food Chem.

J. Assoc. Off. Anal. Chem.

J. Am. Oil Chem. Soc.

J. Am. Oil Chem. Soc.

J. Planar Chromatogr.–Mod. TLC

J. Am. Oil Chem. Soc.

J. Am. Oil Chem. Soc.

J. Am. Oil Chem. Soc.

J. Am. Oil Chem. Soc.

Review
Chromatographic analysis of tocol-derived lipid antioxidants