Toshihiro Akihisa

Analysis of sterols

1. Nomenclature and Biosynthesis of Sterols and Related Compounds. 2. Extraction of Sterols from Tissues. 3. Initial Separation Methods. 4. HPLC of Sterols. 5. Gas-Liquid Chromatography of Sterols. 6. Infra- Red and Ultra-Violet Spectroscopy of Sterols. 7. Mass Spectrometry of Sterols. 8. 1H NMR Spectroscopy of Sterols. 9. 13C NMR Spectroscopy of Sterols. 10. One-Dimensional and Two-Dimensional NMR Spectroscopy of Sterols. 11. X-Ray Crystallography of Sterols. 12. Sources of Sterols. Tables of Physical Data. References. Index.

Tirucalla-5,24-dien-3β-ol [(13α,14β,17α,20S)-lanosta-5,24-dien-3β-ol] and three other Δ5-unsaturated tirucallanes from the roots of Bryonia dioica Jacq.: the first naturally occurring C-10 methylated tetracyclic triterpene alcohols with a Δ5-monounsaturated skeleton

Four novel triterpene alcohols with a Δ5-unsaturated tirucallane-type skeleton, i.e. tirucall-5-en-3β-ol, tirucalla-5,24-dien-3β-ol, 24-methyltirucalla-5,24(241)-dien-3β-ol and (24S)-24-methyltirucalla-5,25-dien-3β-ol, have been isolated from the roots of Bryonia dioica Jacq. (Cucurbitaceae). The structures have been determined by spectroscopic and chromatographic methods. These compounds are the first examples of naturally occurring C-10 methylated triterpenes with a Δ5-monounsaturated skeleton.

Mass spectrometry of sterols

The potential for the application of mass spectrometry (MS), and especially combined gas chromatography—mass spectrometry (GC—MS), to sterol and steroid analysis was recognized in the 1960s [1–12]. Since that time the applications of mass spectroscopy for sterol analysis have moved in parallel with the considerable advances in instrument design and the development of new techniques. Mass spectrometry, and GC—MS in particular, is an ideally suited method to help with the unravelling of the identities of the components in a complex sterol mixture. Among the factors which have made GC—MS such a good analytical method for sterol analysis are: (a) the high sensitivity of the technique; (b) the excellent resolutions of closely related sterols now achievable on fused-silica bonded stationary-phase capillary gas chromatography columns that are capable of high-temperature operation and therefore also suited to steryl conjugate analysis; (c) improved designs for interfacing the GC with the mass spectrometer ensuring good sample transfer; (d) powerful computer programs for instrument operation and data handling that allow mass chromatography, selected ion monitoring, library searching of spectra, etc.; (e) the introduction of bench-top quadrupole and ion-trap mass spectrometers with automatic sample injection systems which are now permitting GC—MS to become a routine laboratory technique. The development of alternative ionization methods has also benefited the analysis of steryl conjugates which were previously rather intractable to MS examination. The mass spectrometry of sterols and related compounds has been reviewed in books and articles [1, 13–20].

13C NMR spectroscopy of sterols

13C NMR spectroscopy is of considerable value for the determination of the structures of complex molecules such as sterols because of the great sensitivity of 13C chemical shifts to structural changes and the fact that each carbon atom in the molecule can usually be examined individually [1–3]. Tabulations of 13C chemical shift data for over 400 steroids were published in 1977 [4], and 13C NMR spectral data of many more steroids have been published since that time [3, 5–7]. Two reviews [8, 9] have been published which give the 13C chemical shift data for many sterols (3-monohydroxysteroids) and related compounds (triterpene alcohols and 3-oxosteroids and triterpenoids). A compilation of the 13C NMR data of 396 selected naturally occurring triterpenoids has been published [10]. This review includes a brief account of the newer signal assignment techniques and a discussion on the substituent effects on the 13C shieldings of the triterpenoids.

High-performance liquid chromatography of sterols

High-performance (or high-pressure) liquid chromatography (HPLC) developed rapidly after the introduction of reliable high-pressure pumps, efficient analyte detectors and uniform silica-gel microsphere column packings. HPLC can employ either normal-phase (adsorption) chromatography on silica-gel microspheres or reversed-phase chromatography on column packings which have an −O−Si(CH3)2−(CH2)17CH3 group chemically bonded to the silicon atoms of the microspheres. This octadecyldimethylsilyloxylated silica is called an ODS or C18 reversed-phase HPLC packing. After the first successful application of HPLC for the analysis of sterols [1, 2], it has come into extensive use for both analytical and preparative purposes in sterol analysis. It is now a widely used method for sterol separation [3–8] in the same way that GLC has been employed for many years. HPLC is often used only as a final purification step because of the relatively high cost of the stationary phases. Table 4-1 summarizes some of the HPLC systems used in the separation of sterols and some related compounds (3-oxo steroids, triterpene alcohols). Because of the similarity of many of these separations, only representative examples have been given.

4α-Methyl-5α,14β-ergosta-8,24(241)-dien-3β-ol (“triticusterol”): the first naturally occurring 14β(H)-steroid

The structure of triticusterol isolated from the germ oil of wheat (Triticum aestivum L.; Gramineae) was established to be 4α-methyl-5α,14β-ergosta-8,24(241)-dien-3β-ol on the basis of spectroscopic data. This is the first example of a naturally occurring compound with a 14β(H)-steroid skeleton.

Initial separation methods

Various strategies are available for the processing of a lipid extract in order to isolate the sterols and their conjugate derivatives [1–8]. The initial objective is to recover from the lipid a fraction enriched in a sterol, a mixture of structurally related sterols, or a group of sterol conjugates. This material can then be analysed by gas—liquid chromatography (GC), GC—mass spectrometry (GC—MS) or high performance liquid chromatography (HPLC). This may provide all the data required from an experiment or it can serve as a guide to the direction and choice of further purification procedures that need to be employed to provide pure sterol samples for rigorous identification by NMR and mass spectrometry. The separation methods to be chosen will obviously depend upon the objectives of the investigation. If the extraction has been undertaken to obtain a particular known sterol for use in some other investigation then the most efficient and least time-consuming procedure will be desired. In this case an immediate saponification of the total lipid will destroy triacylglycerols, wax esters, phospholipids and other esters which are materials that usually contribute considerably to the bulk of the extract and can interfere with chromatography. Saponification of the total lipid can often lower the mass of an oily liquid or semi-liquid total lipid by as much as 70–80% to yield a solid or semi-solid non-saponifiable lipid fraction which contains the sterols.

Gas—liquid chromatography of sterols

Gas—liquid chromatography (GC) was first applied in 1960 for the analysis of steroids, including sterols, using a thermally stable non-polar liquid phase (SE-30) [1, 2]. Clayton [3] then demonstrated that each substituent, alkyl group or double bond in the steroid molecule had a particular effect on the retention time during GC separation. It was subsequently established that the retention time of a sterol is the product of the retention time of the fundamental sterol nucleus and the individual retention factors contributed by each of the substituent groups on the rings and the side chain. As a consequence GC rapidly became the most frequently used analytical method for the primary identification of sterols, for checking sterol purification, and for the quantification of sterols in biological samples. The use of GC for the analysis of sterols and related steroids has been described in many reviews [e.g. 4–12].

One-dimensional and two-dimensional NMR spectroscopy of sterols

Whereas 13C NMR spectra of sterols and other steroids are normally subjected to a full signal assignment, it has been customary with 1H NMR spectra to assign only the methyl, olefinic, or carbinol protons whose signals are significantly deshielded relative to TMSi. The rest of the spectrum is often complicated by severe peak overlap as the remaining protons are distributed in a narrow spectral range between ~1 and 2.5 p.p.m., the so-called ‘methylene envelope’. Full 1H signal assignment of a steroid, 17β-hydroxyandrosta-l,4-diene, at 400MHz was performed in 1980 [1] by the use of a combination of the then recently developed one- and two-dimensional NMR techniques. Since that time, complete or nearly complete 1H NMR assignments for a number of steroids have been obtained with the aid of these various techniques [2–11]. This chapter describes some of the one-dimensional (1D) and two-dimensional (2D) NMR techniques most frequently used for the structure elucidation and signal assignments of sterols and related compounds.

Extraction of sterols from tissues

The ideal method to utilize for the extraction of lipid from a tissue should be one which will remove all the required lipophilic compounds efficiently without losses or artefact formation due to hydrolysis, autoxidation or other degradation. The most well-established solvent extraction-partitioning methods were those developed originally for animal tissues by Folch et al. [1] and Bligh and Dyer [2]. They are still widely applied to obtain lipids from tissues of animals and have also been adopted by many workers for the extraction of lipids from micro-organisms and plants. These methods are based upon the ability of chloroform—methanol (CHCl3—MeOH) mixtures to form a monophasic system with the water in the tissue which will then disrupt the membrane structures and remove the lipid from the tissues. In addition to these two methods, various other solvent systems and techniques are used for the extraction of sterols from tissues and cells of plant and animal origins. Table 2-1 lists examples of the methods of lipid extraction employed with plant or animal materials, and indicates the methods of material preparation and initial separation of sterol subclasses from the extracted lipid.