Sterol identification

Sterol Identifications were made by comparing relative retention times of unknowns with those of authentic standards obtained from Applied Science Labs, Inc. and Suppelco, Inc. Identifications were confirmed by gas chromatography/mass spectrometry using a Hewlett-Packard 5890 GC/MS. 

Prior to lipid extraction, plants and spores were lyophilized. Sterols were extracted and analysed by the method described by Costet-Corio and Benveniste (1988), except that the saponifiable matter, containing total fatty acids, was adjusted to a pH of one by adding HCl before being extracted three times with hexane. These extracts were pooled and dried. Fatty acids and sterols were quantified by gas chromatography (GC). Fatty acid and sterols identifications were made by comparing retention times respectively to methyl heptadecanoate (17 0) and cholesterol as internal standards. All sterols isolated were identified by GC / mass spectrometry. 

Although many sterols and bile acids were isolated in the nineteenth century, it was not until the twentieth century that the stmcture of the steroid nucleus was first elucidated (5). X-ray crystallographic data first suggested that the steroid nucleus was a thin, lath-shaped stmcture (6). This perhydro-l,2-cyclopentenophenanthrene ring system was eventually confirmed by the identification of the Diels hydrocarbon [549-88-2] (4) and by the total synthesis of equilenin [517-09-9] (5) (7). 

Chloroform-methanol extracts of Borrelia burgdorferi were used for the identification of lipids and other related components that could help in the diagnosis of Lyme disease [58]. The provitamin D fraction of skin lipids of rats was purified by PTLC and further analyzed by UV, HPLC, GLC, and GC-MS. MS results indicated that this fraction contained a small amount of cholesterol, lathosterol, and two other unknown sterols in addition to 7-dehydrocholesterol [12]. Two fluorescent lipids extracted from bovine brain white matter were isolated by two-step PTLC using silica gel G plates [59]. PTLC has been used for the separation of sterols, free fatty acids, triacylglycerols, and sterol esters in lipids extracted from the pathogenic fungus Fusarium culmorum [60]. 

J.S. Mills, R. White, The identification of paint media from the analysis of their sterol composition, Studies in Conservation, 20, 176 182 (1975). 

Gas chromatography appears to give adequate separation and measurement of the various sterols to be found in the marine environment where it is less than satisfactory is in the identification of the substances being measured. With compounds whose structures can be so similar, only gas chromatography-mass spectrometry can be expected to provide reasonable identifications. 

The potential for the preservation of lipids is relatively high since by definition they are hydrophobic and not susceptible to hydrolysis by water, unlike most amino acids and DNA. A wide range of fatty acids, sterols, acylglycerols, and wax esters have been identified in visible surface debris on pottery fragments or as residues absorbed into the permeable ceramic matrix. Isolation of lipids from these matrices is achieved by solvent extraction of powdered samples and analysis is often by the powerful and sensitive technique of combined gas chromatography-mass spectrometry (GC-MS see Section 8.4). This approach has been successfully used for the identification of ancient lipid residues, contributing to the study of artifact... 

Fig. 2.128. HPLC-MS summed base peak mass chromatograms of total extracts of (a) T. weissflogii culture, (b) control, (c) faecal pellets immediately after grazing (48h) and (d) pellets after ageing in filtered seawater in the dark for 30 days. Peak identification a = phaeophorbide-a b = pyrophaeophorbide-a c = 132-chlorophyllone-a d = 132-epi-chlorophyllone-a (carotenoid) e + f = 132-hydroxyhlorophyll-a, 15 -hydroxylactone, chlorophyll-a g = chlorophyll-a g = chlorophyll-a epimer h = chlorophyll-a-like i = hydroxyphaeophytin-a + unknown i = hydroxyphaeophytin-a epimer j = phaeophytin-a j = phaeophytin-a epimer k = purpurin-18-phytyl ester 1 = pyrophaeophytin-a m = chlorine-a-like t = 132-oxopyrophaeophytin-a u = 132-oxopyrophaeophorbide a-24-methylcholesta-5,24(28)-dien-3/ -yl-ester SCE Sterol n = C272 d.b.a o = C27 2 d.b. +C28 2 d.b. p = C29 2 d.b. q = C27 1 d.b. r = C28 1 d.b. + C29 2 d.b. s = C29 2 d.b. Reprinted with permission from H. M. Talbot et al. [293], (ad.b = number of double bonds. = carotenoid.)...

Endogenous and exogenous androgens can be derivatized with trimethylsilyl (TMS) for hydroxy functions and by 0-methylation for ketones, and analyzed with GC-FID or GC-MS (Shimada et al., 2001). MS is more prevalent due to unequivocal identification and greatly increased sensitivity but FID is still used in laboratories for some steroids. Sterols have typically been analyzed by GC-FID and GC-MS with derivatization to optimize peak shape (Shimada et al., 2001), and bile acids can be derivatized with M-butyl ester-TMS ether and analyzed by GC-FID from plasma samples (Batta et al., 1998). Juricskay and Telegdy (2000) reported an assay capable of analyzing 28 steroids in urine samples using GC-FID. 

CA107 Duplatre, A., C. Tisse, and J. Estienne. CAl 19 Identification of Arabica and Robusta (coffee) species by studying the sterol fraction. Ann Falsif Expert Chim Toxicol 1984 77(828) 259-270. 

As an example of the use of single-crystal photographs for identification, vitamin B4 was shown by Bernal and Crowfoot (1933 b) to be identical with adenine hydrochloride. The extensive survey of the crystallography of substances of the sterol group by Bernal, Crowfoot, and Fankuchen (1940) gives a vast amount of information on these crystals, including unit cell dimensions this paper also contains a discussion of the identification problems in this group. 

HPLC determination (Osada et al., 1999), and HPLC/MS (Redden and Huang, 1991). In general, enzymatic determination is superior to colorimetry to obtain true cholesterol content. When food such as shellfish contains sterols other than cholesterol, the GC determination is the most adequate method. Although GC/MS also accomplishes good separation between and identification of all sterol analogs, the instrument is too expensive to use for routine analyses of cholesterol. 

Full identification of isolated sterols from commercially consumable fats performed by GC/MS, and quantitative estimation of cholesterol content by capillary GC with flame ionization detection. 

There are cases where HPLC separation is performed not in order to quantify the alcohols but as a technique for the purification of the analytes to be subjected to further instrumental analysis. This is the case, for example, with the identification and determination of the structure of an abscisic acid in starfruit extract (Averrhoa carambola L.). The separation and purification of the analytes was carried out also with HPLC using a mobile phase of diethyl ether, whereas the structure was elucidated by H and UC-NMR (6). In a similar way, to separate the sterols and alkanols from the unsaponifiable matter from olive oils on a silica column, a gradient composed of hexane/diethyl ether was chosen in an offline system (7), whereas an online HPLC-HRGC system uses as its mobile phase hexane/isopropanol (8). 

Moon, Y. A., Shah, N. A., Mohapatra, S., Warrington, J. A. and Horton, J.D. (2001). Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins. J. Biol. Chem., 276,45358—45366. 

Canuel et al., 1997). This recurring theme of overlapping sterol markers in different organic matter sources indicates that caution should be advised when using sterols solely to distinguish between land and aquatic sources (Volkman, 1986 Jaffe et al., 2001). Instead, biomarker source identifications should be corroborated across lipid compound classes and using bulk and compound-specific isotope analysis. 

Guyot, A. (1969) Identification of fat mixtures by analysis of fatty acids and sterols. Bull. Rech. Agron. Gembloux, 4, 484—508. 

IUPAC (1987) Identification and determination of sterols by gas-liquid chromatography, in Standard Methods of Analysis of Oils, Fats and Derivatives (eds C. Paquot and A. Hautfenne), Oxford, Blackwell Scientific, pp. 165-169. 

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