Bile acids group separation

The formation of acyl-CoAs is catalysed by several acyl-CoA synthetases. The overlapping of their chain length specificities and their tissue distribution are such that any saturated or unsaturated fatty acid in the range 2-22C or more can be activated in animal tissues, though at different rates. These enzymes can also activate branch chain fatty acids, dicarboxylic acids and carboxylic acids with unusual groups. Bile acids require separate, specific enzymes, however. 

Scalia S (1990) Group separation of free and conjugated bile acids by pre-packed anion-ex-change cartridges. J Pharm Biomed Anal 8 235-241... 

Scalia and Games developed a packed column SFC method for the analysis of free bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) [32]. The baseline separation of all five bile acids was achieved on a packed phenyl column with a methanol-modified carbon dioxide in less than 4 min. The elution order showed a normal-phase mechanism because the solutes eluted in the order of increasing polarity following the number of hydroxyl groups on the steroid nucleus. The method was also applied to the assay of UDCA and CDCA in capsule and tablet formulations. The method was found to be linear in the range 1.5-7.5 ng/ml (r > 0.99, n = 6). The average recoveries (n= 10) for UDCA and CDCA were 100.2% with a RSD of 1.7% and 101.5% with a RSD of 2.2%, respectively. The reproducibility of the method was less than 1.5% (n = 10) for both UDCA and CDCA. 

Steroids, bile acids, and similar compounds pose certain problems when they require to be derivatized for separation by GC. The hydroxyl groups in the respective structures differ greatly in their reaction rate, which will depend on their nature (whether they are primary, secondary, or tertiary) and also, to a certain extent, on their steric environment. 

Onishi, S., Itoh, S., and Ishida, Y., Assay of free, glycine- and taurine-conjugated bile acids in serum by high-pressure liquid chromatography by using post-column reaction after group separation, Biochem. J. 204, 135-139 (1982). 

Formation of suitable derivatives from bile acids has also been a subject of numerous studies. Since the earlier procedures have been reviewed [314-316], a continuous interest in the subject of derivatization appears to indicate that there is still a need for improvement. The initial derivatization of the acidic function to form methyl esters seems to be almost universally employed, while the remaining functional groups (hydroxy and keto) can be converted to a number of derivatives. A number of earlier and more recent studies have endorsed silylation, although various types of acylation are also common [219]. Permethylation has also been advocated recently [22S], but no biological separations were demonstrated using this approach. 

The first step should preferably lead to a group separation of bile acids and to elimination of non-bile acid contaminants. In quantitative work it may be useful to add—at this stage or earlier—labeled compounds, which permit an estimation of recovery of bile acids of different polarities. Tauro-cholic and 3-ketocholanoic acids constitute suitable extremes in polarity. If specific bile acids are to be analyzed, one may add a suitable internal standard to the biological material. Thus, Rooversc/ r/. (36) used nordeoxy-cholic acid for gas chromatographic determination of bile acids in feces and plasma. 

Ion-exchange chromatography may be used for purification and group separation. As mentioned, Okishio et al. (24) purified a liver extract by applying it in 0.1 M NaOH to an Amberlyst A-26 column as described above for solid extraction. An extract can also be dissolved in chloroform-methanol, 1 1, saturated with water, and applied with a slow flow rate to an Amberlyst A-26 column in OH" form in the same solvent. A 10-ml column should be used for 250 mg of sample. Neutral lipid contaminants are removed by washing with a suitable organic solvent and bile acids are eluted with 0.2 M ammonium carbonate in 80% ethanol as described above (34). 

Column adsorption chromatography should be applied when one deals with very crude extracts, such as those obtained from feces, and when a group separation between mono-, di-, and trisubstituted hydroxycholanoic acids is desirable. It may also be used for large-scale purification of a specific bile acid, e.g., in synthetic work. Silicic acid should be used for free bile acids since more active adsorbents may give incomplete recoveries. Methyl ester or methyl ester acetates are usually best separated on aluminum oxide. In... 

When a group separation is attempted on silicic acid, a 25-fold excess of adsorbent is used, and four fractions are collected (1) benzene 10 ml/g silicic acid, (2) benzene-acetic acid, 99 1, 30 ml/g silicic acid, (3) benzene-acetic acid, 3 1,40ml/g silicic acid, and (4) chloroform-methanol, 1 1,10 ml/g silicic acid. Fatty acids and sterols appear in the first two fractions where also unsubstituted cholanoic acid will be eluted. The third fraction contains mainly mono- and disubstituted bile acids and the last one the bulk of tri-substituted cholanoic acids (18). Silicic acid is sometimes very efficient for the separation of protected bile acids. Thus Wootton (71) obtained a partial separation of the methyl ester diacetates of deoxycholic and chenodeoxy-cholic acids on silica gel (Davison 923, Davison Chemical Corp., Baltimore). 

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... 

From the discussion above it is apparent that the choice of stationary phase and bile acid derivative must be based upon desired objectives. The usefulness of a preliminary separation of bile acids according to the number of substituent groups prior to gas chromatography seems evident when complex mixtures of bile acids are to be analyzed. 

In the procedure of Grundy ef at. (30) the bile acids are quantitated as a group after analysis of their trimethylsilyl ether methyl esters on SE-30 columns. As can be seen from Table XII only small separation factors are obtained with this phase. Fortunately, these authors found a unit response for all bile acids analyzed in this way and were able to measure bile acids as the entire base line deflection obtained after 5a-cholestane had been eluted. With such a procedure it has to be established that compounds other than bile acid derivatives are not determined. Whether this is the case under different experimental conditions, such as drug treatment or change of diet, should always be fully explored. 

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