Derivatization of Bile Acids

Derivatization of Carboxyl Group

The carboxyl group in bile acids is most often converted into the methyl ester. Treatment with

Figure 2 Gas chromatography of bile acids. Chromatographic conditions are described in Table 2; CP-Sil-5 CB capillary column was employed. (A) n-Butyl ester-trimethylsilyl ether derivatives of standard bile acids. Peak identification, derivatives of: 1, nor-cholic acid; 2, lithocholic acid; 3, deoxycholic acid; 4, cheno-deoxycholic acid; 5, cholic acid; 6, ursodeoxycholic acid. (B) Faecal bile acids in a healthy volunteer. Dried stool (10 mg) containing 20 |ig nor-cholic acid as internal standard was directly subjected to n-butyl ester followed by trimethylsilyl ether formation, dissolved in 200 |L hexane and 1 |L was injected into the GC column. Peak identification: 1-6, same as in (A); a, sitosterol, 3', isodeoxycholicacid; 7, 3-keto, 12a-hydroxy-5^-cholanoicacid; 8, 12-ketolithocholic acid (C) Plasma bile acids in a patient with lipid storage disease, sitosterolemia. Plasma (1 mL) containing 10 |ig nor-cholic acid as internal standard was used. Bile acids were enzymatically deconjugated and bile acids obtained by passing through Sep-pak. Bile acids were derivatized as n-butyl ester-trimethylsilyl ethers, dissolved in 100 |L hexane and 5 |L was injected into the GC column. Peak identification: 1-6, same as in (A); a, sitosterol.

Table 2 GC retention indices of bile acids as their methyl ester-trimethylsilyl ether derivatives3

Retention indexb

Table 2 GC retention indices of bile acids as their methyl ester-trimethylsilyl ether derivatives3

Retention indexb

Bile acid methyl ester-trimethylsilyl ether CP-Sil-5 CB CP-Sil-19 CB

Lithocholic acid

3157

3339

Deoxycholic acid

3221

3373

Chenodeoxycholic acid

3244

3397

Cholic acid

3261

3381

Ursodeoxycholic acid

3279

3439

Hyodeoxycholic acid

3256

3422

Hyocholic acid

3340

3445

^-Muricholic acid

3310

3468

Nor-cholic acid

3140

Nor-deoxycholic acid

3106

Homocholic acid

3346

3a,7a,12a-Trihydroxy-5^-cholestanoic

3468

acid aA Hewlett-Packard model 6890 gas chromatograph equipped with a flame ionization detector and an injector with a split/splitless device for capillary columns was used. A chemically bonded fused silica CP-Sil-5 CB or CP-Sil-19 CB capillary column (25 m x 0.22 mm i.d.) was employed and helium was used as the carrier gas. The column temperature was kept at 100°C for 2 min, then increased at a rate of 35°C min-1 to a final temperature of 278°C.

bRetention indices (Kovats values) were determined by previous injection of an alkane mixture under identical GC conditions.

acid can silylate hydroxyl groups in all positions. Bis(trimethylsilyl)trifluoroacetamide is equally reactive as N,0-bis(trimethylsilyl)acetamide but the reagent and the by-products are more volatile. The older silylating reagent, a mixture of 1,1,1,3,3,3-hexamethyldisilazane, trimethylchlorosilane and pyridine (3 : 1 : 9) is still commonly used for derivat-ization. Retention volumes of several common bile acid methyl ester-silyl ether derivatives on capillary aA Hewlett-Packard model 6890 gas chromatograph equipped with a flame ionization detector and an injector with a split/splitless device for capillary columns was used. A chemically bonded fused silica CP-Sil-5 CB or CP-Sil-19 CB capillary column (25 m x 0.22 mm i.d.) was employed and helium was used as the carrier gas. The column temperature was kept at 100°C for 2 min, then increased at a rate of 35°C min-1 to a final temperature of 278°C.

bRetention indices (Kovats values) were determined by previous injection of an alkane mixture under identical GC conditions.

diazomethane in ether converts bile acids instantly into their methyl esters. However, small amounts of methyl ethers are formed as artefacts, so alternative methods are often employed. Methyl esters are quantitatively formed with methanol-sulfuric acid or anhydrous methanolic hydrochloric acid. Alternatively, 2,2-dimethoxypropane-hydrochloric acid may be used for derivatization. In addition to methyl esters, ethyl, «-propyl, isobutyl and «-butyl esters have all been employed, the advantage being that the retention times of the bile acid derivatives are increased and, sometimes, resolution is improved. In particular, much better separations from neutral sterols can be obtained and, as shown in Figure 2, chemical removal of neutral sterols may be completely unnecessary with the use of butyl esters.

Derivatization of Hydroxyl Groups

The derivative of choice for the hydroxyl group is the trimethylsilyl ether and a variety of silylating reagents are commercially available for quantitative derivatiz-ation of even hindered and tertiary hydroxyl groups. Thus, N,0-bis(trimethylsilyl)acetamide, in combination with trimethylchlorosilane, can convert relatively unhindered hydroxyl groups into their trimethylsilyl ether derivatives and, when mixed with trimethyliodosilane, the reagent is highly potent and

Figure 3 Gas chromatography of 6-hydroxylated bile acids. Chromatographic conditions are described in Table 2. (A) CP-Sil-5 CB capillary column; (B) CP-Sil-19 CB capillary column. Peak identification, derivatives of: 1, 3a,6^,7a-trihydroxy-5^-cholanoic acid; 2, 3a,6^,7a,12a-tetrahydroxy-5^-cholanoicacid; 3, 3a,6a,7a-trihydroxy-5^-cholanoic acid; 4, 3a,6a,7a,12a-tetrahydroxy-5^-cholanoic acid; 5, 3a,6^,7^-trihydroxy-5^-cholanoic acid; 6, 3a,6^,7^,12a-tetrahydroxy-5^-cholanoic acid; 7, 3a,6a,7^-tri-hydroxy-5^-cholanoic acid; 8, 3a,6a,7^,12a-tetrahydroxy-5^-cholanoic acid.

Figure 3 Gas chromatography of 6-hydroxylated bile acids. Chromatographic conditions are described in Table 2. (A) CP-Sil-5 CB capillary column; (B) CP-Sil-19 CB capillary column. Peak identification, derivatives of: 1, 3a,6^,7a-trihydroxy-5^-cholanoic acid; 2, 3a,6^,7a,12a-tetrahydroxy-5^-cholanoicacid; 3, 3a,6a,7a-trihydroxy-5^-cholanoic acid; 4, 3a,6a,7a,12a-tetrahydroxy-5^-cholanoic acid; 5, 3a,6^,7^-trihydroxy-5^-cholanoic acid; 6, 3a,6^,7^,12a-tetrahydroxy-5^-cholanoic acid; 7, 3a,6a,7^-tri-hydroxy-5^-cholanoic acid; 8, 3a,6a,7^,12a-tetrahydroxy-5^-cholanoic acid.

Figure 4 Gas chromatography of acetate-methyl esters and trimethylsilyl ether-methyl esters of bile acids. Chromatographic conditions are described in Table 2; CP-Sil-5 CB capillary column was employed. (A) Trimethylsilyl ether-methyl esters of bile acids. (B) Acetate-methyl esters of bile acids. Peak identification: 1, lithocholicacid; 2, deoxycholicacid; 3, chenodeoxycholicacid; 4, cholicacid; 5, 3a,6a-dihydroxy-5^-cholanoic acid; 6, ursodeoxycholic acid; 7, 3a,6^,7a-trihydroxy-5^-cholanoic acid; 8, 3a,6a,7^-trihydroxy-5^-cholanoic acid; 9, 3a,6^,7^-trihydroxy-5^-cholanoicacid.

Figure 4 Gas chromatography of acetate-methyl esters and trimethylsilyl ether-methyl esters of bile acids. Chromatographic conditions are described in Table 2; CP-Sil-5 CB capillary column was employed. (A) Trimethylsilyl ether-methyl esters of bile acids. (B) Acetate-methyl esters of bile acids. Peak identification: 1, lithocholicacid; 2, deoxycholicacid; 3, chenodeoxycholicacid; 4, cholicacid; 5, 3a,6a-dihydroxy-5^-cholanoic acid; 6, ursodeoxycholic acid; 7, 3a,6^,7a-trihydroxy-5^-cholanoic acid; 8, 3a,6a,7^-trihydroxy-5^-cholanoic acid; 9, 3a,6^,7^-trihydroxy-5^-cholanoicacid.

columns of low and medium polarity are given in Table 2, while the chromatographic resolution of these derivatives of a number of bile acids with additional hydroxyl group at C6 (present in several animal species and in patients with hepatobiliary diseases) is shown in Figure 3. Although trimethylsilyl ethers have gained general applicability in bile acid analysis, other silylating groups sometimes have advantages. Thus, dimethylethylsilyl and dimethylpropylsilyl ether derivatives have longer retention times than the corresponding trimethylsilyl ethers, while t-butyldimethyl-silyl ether derivatives are very stable, and also show significantly longer retention times, so that often better resolutions can be obtained. Hydroxyl groups can also be protected as acyl derivatives and formate, acetate and trifluoroacetate derivatives have all been employed for GC. These derivatives are often more stable than the silyl derivative and sometimes show better resolution, as seen in Figure 4. A one-step derivatization of the hydroxyl and carboxyl groups with heptafluorobutyric anhydride, also resulting in simultaneous hydrolysis of the glycine and taurine conjugates of the bile acids, has been reported.

Derivatization of Oxo Groups

Oxo bile acids are formed by bacterial modification of bile acids during their intestinal transit and are therefore found in the intestinal content. Quantitation of oxo bile acids is beset with problems. Thus, oxo bile acids, in particular, 3-oxo bile acids, are vulnerable to rigorous alkaline hydrolysis conditions and therefore the much milder enzymatic hydrolysis of conjugates is preferred when oxo bile acids are suspected. Oxo bile acid methyl esters can be chromatographed without further derivatization (the hydroxyl groups in the partially oxidized compounds must be derivatized); however, sometimes artefacts are produced and it may be better to protect the oxo groups as the O-methyloxime or the dimethylhydrazone derivatives.

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