Bile acid methyl esters

Bile acid conjugates la 376 Bile acid methyl esters lb 446,448 Binding agent, influence on staining la 123 S-Bioallethrine la 359... 

With steroids bearing hydroxyl groups the linear behaviour between Tx and molar concentration no longer holds. Cholesterol and a series of bile acid methyl esters have been studied. (36) The curves are concave... 

Gas-liquid chromatography Capillary GLC analysis of bile sterols and bile acid methyl esters (as their trimethylsilyl derivatives) was performed on Hewlett-Packard model No. 4890 (equipped with a flame ionization detector) and a split column injector using a CP sil 5 (CB) WCOT capillary colunm (25 m x 0.22 mm with 0.13-mm Him thickness). Helium was used as a carrier gas at a flow rate of 20.2 mL/min (135 kPa). 

Esters, Carboxylic Acids, and Ethers.—The rates of hydrolysis of 3(3- and 6(3-acetoxy-4 ,5 -epoxides and 1 a-acetoxy-2(3,3 (3-epoxides were observed to be accelerated relative to those acetates not containing a neighbouring epoxide group.24 Bile acid methyl esters were readily prepared from the carboxylic acids by reaction with methanol in the presence of toluene-p-sulphonic acid.25 Bile acids were readily converted into the amino-amides (14) by successive reaction with Bu N-... 

Szczepanik, P. A., Hachey, D. L., and Klein, P. D., Evaluation of Poly S-179 as a stationary phase for the gas-liquid chromatography-mass spectrometry of bile acid methyl ester acetates. /. Lipid Res. 19, 280-283 (1978). 

H12. Holmes, W. L., and Stack, E., Gas chromatography of squalene, sterols and bile acid methyl esters. Biochim. Biophys. Acta 66, 163-165 (1962). 

G.c.-m.s. with selected ion monitoring provides a very sensitive determination of oestrogens on a scale of picograms. Standards with high specific deuterium labelling (e.g. [ Hgjoestradiol) were prepared for this purpose. Various dimethyl-alkylsilyl ethers (alkyl = Et, Pr", or Pr ) have proved superior to trimethylsilyl ethers for the g.c. separation of bile-acid methyl esters. G.c.-m.s. has been applied to the separation and identification of unsaturated bile acids found in natural extracts,and, with computerized recognition, to a series of sterols and bile-acid esters.G.c. separation of various steroids and bile acids has been affected on the nematic liquid crystal N,N -bis(p-phenylbenzylidene)-a,a -bi-p-toluidine as stationary phase. 

In another study where DDO was employed for tert-C-H hy-droxylation of several di- and triacetates of (5 8)-bile acid methyl esters, it was reported that derivatives bearing a 7-acetoxy group give 17a- or 14a-hydroxylated products in addition to the 5/8-hydroxylated ones. ... 

Fales and Pisano (1964) have discussed the gas chromatography of amines, alkaloids, and amino acids. Pollock and Kawauchi (1968) have resolved derivatives of serine, hydroxyproline, tyrosine, and cysteine, as well as racemic aspartic acid and tryptophan. VandenHeuvel and Horning (1964) have listed derivatives of steroids that can be separated. VandenHeuvel et al. (1960) first described the separation of bile acid methyl esters and Sjovall (1964) has extended the methods to bile acids. Gas liquid chromatography (GLC) is useful in the analysis of pesticides, herbicides, and pharmaceuticals (Burchfield and Storrs, 1962). Analysis of alkaloids, steroids, and mixtures of anesthetics and expired air are other examples of the application of this very useful technique. Beroza (1970) has discussed the use of gas chromatography for the determination of the chemical structure of organic compounds at the microgram level. 

Another 1,3-dicarbonyl equivalent is keto-enol. Treatment of the ketone with ethyl formate in the presence of NaH afforded the keto-enol as the 2-hydroxy-methylene ketone. Condensation of the keto-enol with hydrazines provided the corresponding pyrazole fused bile acid methyl ester... 

It has been shown that the trifluoroacetates of 3,6,7-trihydroxy bile acids are subject to thermal decomposition in gas chromatographs (30). Oxidation of the bile acids to their keto derivatives and subsequent gas chromatography should also be avoided (31). In our laboratory, we have been unable to gas chromatograph any oxidized 3,6,7 bile acid methyl esters they are either destroyed or will not elute in a reasonable amount of time. 

Fig. 1. GLC of fecal bile acids from germfree, gno-tobiotic, and conventional rats. The first peak in each chromatogram is the internal standard 5a-cholestane. Instrument, Hewlett-Packard 402 gas chromatograph. Column, 2-m glass Utube packed with 1% SE-30 on 80-120 mesh Gas Chrom Q. Isothermal 220°C. Nitrogen carrier gas 75 ml/min. Flame ionization detector. Bile acids were analyzed as the trimethylsilyl ethers of the bile acid methyl esters. From Kellogg et al. (44). Reprinted with permission of the Journal of Lipid Research.

An alternative reagent, trimethylsilyldiazomethane in hexane, is commercially available and appears useful for preparation of bile acid methyl esters [62], Like carboxylic acid groups, hydroxyl groups also require protection prior to GC-MS analysis. A common and mild method to prepare trimethylsilyl (TMS) ethers is to react the sample with a mixture of dry pyridine, hexamethyldisilazane (HMDS), and trimethylchlorosilane (TMCS), 3 2 1 or 9 3 1 (by volume) with, or without heating [32], However, if 0X0 groups are present in the bile acid, this reaction can yield enol-TMS ethers and multiple products. This artifact can be avoided by converting the 0X0 group into an oxime, usually a methyloxime (MO). To do this, the sample is dissolved in 50 pL pyridine with 5 mg methoxyammonium chloride and heated for 30 min at 60°C [32]. 

Silicic acid chromatograpy of fecal bile acid fractions Aluminum oxide chromatography of mono- and disub-stituted bile acid methyl esters from feces ... 

Acetone (% in benzene) Bile acids eluted Ethyl acetate (% in benzene) Bile acids eluted Ethyl acetate (% in benzene) Bile acids eluted Heptane or ethyl acetate (% in benzene) Bile acid methyl ester eluted... 

Sometimes the methyl esters are better separated than the free bile acids. This is especially true when neutral solvents are used. In acidic solvents the difference is small, particularly with trisubstituted bile acids. Thus, the same types of acidic solvents can be used for free and methylated bile acids. When bile acid methyl esters are acetylated or converted into trimethylsilyl ethers their mobility is considerably increased. The separation of configurational isomers is then often impaired whereas the separation of positional isomers may be improved (4). This is often the case when neutral derivatives are analyzed in neutral solvent systems (12). The influence of the nature and position of nuclear substituents on the mobility of a bile acid can be estimated... 

Bile acids Bile acid methyl esters ... 

Trifluroroacetates (100) are prepared by dissolving the bile acid methyl ester (o> the partial silyl ether) in trifluoroacetic anhydride, 15 min, 35 °C (101). The reagent is removed under a stream of nitrogen. Milder conditions yield partial derivatives. Enol esters of 3-keto-J- bile acids may be formed as side products. Trifluoroacetates should be analyzed within 1-2 days since signs of decomposition may appear on storgae for more than 48 hr at room temperature (7, 102). 

Dimethylhydrazones (103) are prepared by dissolving the bile acid methyl ester in 0.1 ml of 1,1-dimethylhydrazine in screw-capped vials. The vials are flushed with nitrogen, closed, and left at room temperature overnight (77). The reagent is evaporated under a stream of nitrogen, the residue is dissolved in acetone and the sample is analyzed after 1 hr. Under these conditions only unconjugated 3-keto groups react (77, 104). 

Sodium borohydride reduction is carried out with 1 mg reagent in 0.5 ml isopropanol. The rates of reduction differ with the position of the keto group (108). Treatment with lithium aluminum hydride in diethyl ether results in formation of C-24-ols from bile acid methyl esters. This may be valuable in chromatographic identification work, especially if combined with mass spectrometry. 

TABLE XII. Relative Retention Times of Different Bile Acid Methyl Ester Derivatives on Methyl (SE-30 and OV-1) and Methyl-Phenyl (SE-52, PhSi-20, and OV-17 5, 20, and 50% Phenyl Groups, Respectively) Substituted Silicones... 

TABLE XIII. Relative Retention Times" of Different Bile Acid Methyl Ester Derivatives on QF-1... 

Improved separations of bile acid methyl esters can be observed with increasing amounts of phenyl substituents in the stationary phase (5% in SE-52, 20% in PhSi-20, and 50% in OV-17). Positional and configurational isomers are better resolved than on SE-30 or OV-1 columns. For instance the valuable separation of the trimethylsilyl ethers of 3,6,7-substituted methyl cholanoates from the corresponding cholic acid derivative on OV-17 should be noted. The pronounced effect of a 7/3-hydroxy substituent on retention times on columns of SE-52 and PhSi-20 is noteworthy. The large separation factors between the diacetate derivatives of chenodeoxy- and ursodeo.xycholic acids may be most useful in work with bile acids of biological origin. 

Bile acid methyl esters with unprotected hydroxy groups are best separated on QF-1 columns (Table XIII). It is advisable to use 3 % columns since it is often difficult to prepare 0.5-1 % QF-1 columns that do not give tailing of polar compounds. This difficulty has also been experienced by Okishio and Nair (24) who therefore developed a column having a mixture of QF-1, SE-30, and NGS as stationary phase. On QF-1 columns epimeric alchohols... 

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