Triterpenes detection

Note The solvents employed should be anhydrous. The esters of phenoxy-alkanecarboxylic acids (detection limits 500 ng) [1] yield brown to violet, terpenes violet-grey [2] and triterpenes yellow to violet [5] colored chromatogram zones. 

B. Buchele, T. Simmet, Analysis of 12 different pentacyclic triterpenic acids from frankincense in human plasma hy high performance liquid chromatography and photodiode array detection, Journal of Chromatography B, 795, 355 362 (2003). 

SPME/GC/MS is an efficient technique to reveal the presence of resinic substances in archaeological samples. Indeed, volatile terpenes are still present in very old archaeological samples (4000 years old), particularly in the case of compact matrixes, and can be trapped by the SPME fibre. In comparison with methylene chloride extraction, SPME is very specific and allows the direct analysis of the volatile terpenes content in complex mixtures including oils, fats or waxes. For this reason, headspace SPME is the first method to use when analysing an archaeological sample it will either allow the identification of the resin or indicate further sample treatment in order to detect characteristic triterpenes. The method is not really nondestructive because it uses a little of the sample but the same sample can be used for several SPME extractions and then for other chemical treatments. 

The use of HMDS as a derivatization reagent in the analysis of triterpenoid resins has been less explored. The TMS derivatives of triterpenoids bearing hydroxyl groups [a-amyrine, p-amyrine and hop-22(29)-en-3p-ol] have been identified in the triterpenic fraction of Burseraceae resins, thus demonstrating that HMDS combined with Py-GC/MS is effective in the derivatization of triterpenoid compounds [59]. However, the range of structures that can be fully derivatized and detected must be extended and, in order to get comprehensive results comparable with those coming from the well assessed off-line GC/MS procedures, general improvements in the on-line trimethylsilylation-pyrolysis method are needed. 

The utility of mass selective detection is greatest when analyzing compounds that do not contain chromophores or when structural information is needed for chemical identification. Triterpene saponins contain very weak chromophores and have long been associated with a variety of biological activities including... 

Three other GC analyses now used in authentication, largely for olive and other oils which should not be refined or solvent extracted, are the determination of waxes, aliphatic alcohols, triterpene alcohols (uvaol and erythrodiol), and stigmastadiene and other sterol-dehydration products (EEC, 1991). These analyses are used at present not to detect adulteration with other oils, but with solvent-extracted or refined oils. However, it is possible that, with solvent-extracted oils, wax, aliphatic alcohol and terpene alcohol compositions, could prove useful in differentiating or detecting different oils. 

Much of the sterol component of shea butter is present as esters of cinnamic acid, which are less readily saponified than esters with glycerol (Peers, 1977) published data for this fat might therefore underestimate the hue value. Surprisingly measurement of cinnamic acid has not been used to test for the presence of shea butter in mixtures. Triterpene alcohols similar to those found in shea also occur in sal fat. Homberg and Bielefeld (1982) showed the presence of triterpene alcohols in illipe and sal fats and in commercial CBEs, and their analysis was proposed as a qualitative measure to detect cocoa butter adulteration. 

Reaction of shea triterpene alcohols with acetic anhydride and sulphuric acid to produce coloured products (Fitelson s reaction) was the basis of a sensitive early test for the presence of shea butter in cocoa butter (Fincke, 1975). Analysis of the triterpene fraction of a commercial cocoa butter by TLC fractionation followed by GC (Fincke, 1976), or argentation TLC followed by GC (Gegiou and Staphylakis, 1985), have been shown to have potential for detecting CBEs in chocolate based on the difference in levels of P-amyrin, butyrospermol and... 

The quantitative determination of sterenes has been used to detect added CBEs or refining in cocoa butter and to establish the presence of CBAs in retail chocolates (Crews et al., 1997). The high quantity of triterpene alcohols in shea butters means that dehydration products of these sterols are formed in detectable levels when shea is bleached. These triterpene sterenes, which have not be characterized, can be detected in the stearin fraction used in CBEs (Crews etal., 1999). 

The fatty/waxy products contained the lipophilic substances, including fatty oils, waxes, resins and colorants. Valuable pharmacological effects were proved for some minor constituents of these products (e.g. triterpenes, diterpenes, sterols and carotenoids). Thin layer chromatography and on-line UV-VIS spectroscopy were used for the quick identification and quantity determination of these compounds using authentic samples as standards. The SFE method proved favorable in terras of both extraction yield and speed of carotenoids. The CO2 extracts of the lavandin, clary sage and thyme have been enriched in triterpenic compounds (a-es P-amyrin, oleanic acid, ursolic acid, etc.) and phytosterols. Both free and esterified triterpenoids were present in the extracts of the different samples. Furthermore camosol and other diterpenes were detected in the SFE extract of Lamiaceae plants. The fatty acid composition was only slightly different for extracts obtained by SFE and conventional hexane extraction. 

The silver nitrate-impregnated silica gel G was used to obtain a separation with a group of tetracyclic triterpene alcohols, which could not be separated on untreated silica gel. However, on silica gel G (Ag impregnated), the Rf values were sufficiently different so as to enable the substances to be identified separately. Chloroform was used as the mobile phase. The tetracychc triterpenes were detected by spraying with one of the three reagents 50% sulfuric acid in water or 10% phosphomohbdic acid in ethanol or chlorosulfonic and acetic acids (1 2) followed by heating in an oven at 150°C. ... 

A new HPLC method permitted the separation of 13 triterpene glycosides isolated from different Astragalus species within 40 min. A water/acetonitrile gradient was used as eluent, RP-18 was used as stationary phase, and evaporative light scattering detection was used. The method facilitated differentiation of different Astragalus species. 

Finally, since many natural product compounds have been investigated with various chromatographic modes and detection techniques, a selection of examples has been summarized in this chapter. This information has been compiled in the form of tables for well-researched classes of secondary metabolites selected from the major subgroups of isoprenoids (mono-, sesqui-, di-, and triterpenes iridoids and secoiridoids carotenoids saponins and ecdysteroids), of phenolics (coumarins, flavonoids, and isoflavonoids), and of alkaloids. 

Fig. 1 This solvent system and the AS reagent are suitable for the separation and the detection of triterpene-saponins, e.g. senegins in Senegae radix (1), as well as steroid (ester) saponins, e.g. Smilax saponins in Sarsaparillae radix (2).

Detection of triterpenes, steroids (.saponins, bitter principles). 

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