Chromatography methyl cholanoates

Fig. 2. Gas-liquid chromatography of methyl cholanoates on 3% OV-17, 3% QF-1, and 3 % OV-1. Methyl 5 -cholanoates are plotted on the left side and methyl 5a-cholanoates are plotted on the right side of each diagonal line representing the phase on the column. Time of elution in minutes is read on the abscissa. The phases were mounted on Gas Chrom Q (100-120 mesh) (60).

In a recent study Hofmann et al. (116) studied the bile acid composition of bile from germ-free rabbits. Deproteinized bile was first analyzed by thin-layer chromatography for a tentative identification of the conjugated bile acids. After alkaline hydrolysis and methylation, bile acid methyl esters were analyzed by thin-layer and gas chromatography. Trifluoroacetate esters and trimethylsilyl ethers of methyl cholanoates were run on QF-1 or Hi-Eff-8B columns, respectively. Gas chromatography-mass spectrometry was used for the final identifications. 

After purification of human serum bile acids on an Amberlyst A-26 anion e.xchanger, Sjovall and co-workers (31, 120, 121) hydrolyzed the concentrated bile acids in alkali and extracted the free acids with ethyl acetate. After methylation, the methyl cholanoates were purified on an aluminum oxide column. The bile acid fraction then obtained was subjected to gas-liquid chromatography on CNSi columns after conversion to trifluoroacetate... 

Ali et al. (128 and 129) suspended freeze-dried human feces in alkaline-50 % ethanol and extracted with petroleum ether-diethyl ether, 1 1, to remove neutral lipids. The bile acids in the aqueous phase were next subjected to conditions for hydrolysis of conjugates. After acidification, the free bile acids were extracted with diethyl ether, methylated, and purified by preparative thin-layer chromatography. Mono-, di-, and trisubstituted methyl cholanoates are eluted together from the thin-layer plate and then quantitated on QF-1 columns before and after conversion into trifluoroacetates. 

In substituted methyl cholanoates (Table I) loss of water or its equivalents (acetic acid, trifluoroacetic acid, trimethylsilanol, etc.) is pronounced. In gas chromatography-mass spectrometry this process is partly thermal, partly due to electron impact when a direct probe is used, thermal elimination can be avoided. Since the mechanisms of the two types of elimination are different (26) the spectra will differ to some extent. However, in work with biological materials, the complexity of the mixtures and the small amounts of bile acids available usually make it necessary to use the former method and to accept the thermal component in the fragmentation process. When methyl esters of di- and trihydroxy bile acids and their acetates or trifluoroacetates are analyzed by gas chromatography-mass spectrometry, a molecular ion is usually not seen. This is partly due to the high temperatures used. Trimethylsilyl ethers usually give a molecular ion peak but it may be quite small. 

Because of the trans fusion of rings A and B the allo-acids are more strongly adsorbed and consequently exhibit a slower mobility than their corresponding 5 -epimers. This is particularly well illustrated in thin- aycr chromatography on silica gel G (Table III) in a comparison of the mobilities of the methyl esters in varying proportions of acetone in benzene. With the exception of the 3a-ols, the 7,12-diones, and the 3-keto-12a-ols, the methyl 5/3-cholanoates are more mobile (larger / /) than their 5a-isomers. Because... 

With a purified enzyme preparation from Clostridium perfringens, Roovers et al. (36) cleave conjugated bile acids directly in plasma. Proteins are then precipitated with Ba(OH)2-saturated ethanol (123) and the supernatant is taken to near dryness. The residue is dissolved in a toluene-isopropanol-methanol-30 % aqueous NaOH (10 20 20 6, v/v) mixture, water is added, and neutral lipids removed by light petroleum extraction. Bile acids are then obtained by acidification and diethyl ether extraction. The bile acids are then methylated and analyzed as above except that the bile acid methyl esters are acetylated before chromatography on 1 % XE-60 columns at 250°C. With this column the following retention times relative to that of the diacetoxy derivative of methyl deoxycholate were found for the following acetate methyl ester derivatives lithocholic, 0.60 23-nor-deoxycholic acid (internal standard), 0.77 chenodeoxycholic acid, 1.24 and cholic acid, 1.88. The deoxycholic acid derivative was eluted after 9.0 min and methyl 5 3-cholanoate after 0.32 min. 

Examples of mass spectra of derivatives of 3,6-, 3,7- and 3,12-dihydroxy-cholanoates are shown in Figs. 4 and 5. At the temperatures used in gas chromatography-mass spectrometry, molecular ions are seen only with the free hydroxy compounds and the trimethylsilyl ether derivatives (intensity usually less than one percent of base peak). Molecular ions with a relative intensity of 5-10% are obtained from the methyl esters or acids when a direct probe is used (14, 15). Loss of 2 and 4 mass units from the molecular ions of compounds with free hydroxyl groups is most probably thermal and is not seen at low temperatures. 

Spectra of O-methyloximes of methyl 3,6-diketo-5a- and 3,6-diketo-5/3-cholanoates have been reported by Allen et al. (30). The O-methyloximes, prepared as described by Fales and Luukkainen (49), were found to be particularly useful derivatives since the 5a- and 5 -isomers could be separated by gas chromatography, which was not the case with the free ketones. Considerable differences in relative intensities of the peaks were noted between the spectra of the two isomers. The 5y3 compound gave a base peak at mje 138 (probably ring A) whereas the molecular ion was base peak in the spectrum of the 5a-epimer. These results show the value of O-methyloximes in mass spectrometry of ketonic bile acids. It should be recognized that partial derivative formation may occur, and that formation of syn- and o //-isomers may result in a complex mixture of products from a pure compound. 

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