**7. Gas chromatography**

GC and GC-MS have been widely used for quantitative and qualitative analysis of bile acids in different biological samples [10, 14, 37, 62-65]. In broad terms, GC works on the same principle as HPLC, however, in GC compounds are in gaseous form and an inert gas, like nitrogen or helium, is used for elution. The various compounds are resolved on the basis of their retention/elution behavior on the stationary phase of the column. There are two basic requirements for GC analysis, an appropriate column and derivatization of the compounds, to cause vaporization under the chromatographic conditions used [19].

Then, those techniques are limited by complex sample preparation, derivatization and most importantly, because requires the hydrolysis of conjugated bile acids into their free forms, prior to their analysis [66]; although GC-MS provides high sensitivity and resolution of isomeric bile acids, it is necessary to isolate and purify bile acids before analysis [14, 19].

44 Chromatography – The Most Versatile Method of Chemical Analysis

placing in a 120°C oven [60].

concentration range 50–75% (v/v).

**7. Gas chromatography** 

separated taurine and glycineconjugated standard bile acids. In [21], authors reported 15

In [59] they reported two solvent systems. The first one (isopropanol:glacial acetic acid, 93:7 v/v) allows separation of free bile acids, glycine conjugates and taurine conjugates from one another, and a second solvent system consisting of hexane:methylethylketone:glacial acetic acid, 56:36:8 v/v (Petcoff's solution), for the separation of the four free bile acids found in human bile. Authors in [48] used a TLC methodology to identify the fecal bile acid patterns in several carnivore species; plates were developed in a paper-lined, equilibrated bath containing Petcoff´s solution. After air drying, plates were visualized by spraying with a solution of acetic acid:sulphuric acid:anisaldehyde 50:1:0.5 (v/v) as revealing agent, and

On the other hand, as cited in [33], they developed plates in Petcoff´s solution and then visualized with a 50:1:0.5 v/v solution of acetic acid:sulfuric acid:p-anisaldehyde to identify bile acids from grizzly and black bear feces. In [35] they used used a developing bath of Petcoff's solution to run the plates and they visualized bile acid bands by spraying the plates with 50% v/v sulfuric acid, which allowed them to observe spot colors in cougar and jaguar feces [3]. Sulfuric acid was also present in our visualizing agent. [61] reported a method to separate standard bile acids consisting of a reversed thin-layer chromatography using a mixture of metanol:water as the solvent system, in the

In [6] they reported a TLC method to determine the fecal bile acid patterns in carnivore feces. It consisted of an alkaline extraction to purify the bile acids from other lipophilic steroidal compounds; plates were eluted with Petcoff's solution and developed with a

From our results, and because TLC of fecal bile acids has proved to offer robust data to establish habitat use and to study food habits of some sympatric mammal species [6, 36], we assume that this technique would also be useful for future ecological studies in Xenarthra and in other species. Considering the scarcity of available information about some ecological and biological aspects of Xenarthra, these results, the first ones on the application of TLC for the identification of their feces, could be very important for future studies about the

GC and GC-MS have been widely used for quantitative and qualitative analysis of bile acids in different biological samples [10, 14, 37, 62-65]. In broad terms, GC works on the same principle as HPLC, however, in GC compounds are in gaseous form and an inert gas, like nitrogen or helium, is used for elution. The various compounds are resolved on the basis of their retention/elution behavior on the stationary phase of the column. There are two basic requirements for GC analysis, an appropriate column and derivatization of the compounds,

mixture of glacial acetic acid, water, sulfuric acid and 3,4-dimethoxybenzaldehyde.

conservation, distribution and eco-physiology of this group.

to cause vaporization under the chromatographic conditions used [19].

solvent systems to separate bile acids, being the most efficient the acidic one.

Bile acids are present in unconjugated and/or conjugated form in biological fluids. They range from 1-2 µg/ml in plasma and urine to significant amounts in the intestinal content and as much as 10 mg/ml in the gallbladder bile. Bile acids are present in association with proteins, sterols and their esters, free or esterified fatty acids, bile pigments and watersoluble small molecules, which must be either removed before chromatography or the chromatographic conditions should be such that these compounds do not interfere in bile acid analysis. Since bile acids are present mainly as glycine or taurine conjugate in plasma and bile, unconjugated in feces and unconjugated as well as conjugated with glycine, taurine, glucuronic acid and sulfuric acid in the urine, the methods for isolation of bile acids from these sources need to be appropriately modified [19].

However, a major difficulty in quantitative analysis of fecal bile acids is their strong binding with the bacterial debris in the stool, and quantitative extraction is difficult. Stool contains bile acids, neutral sterols, cholesterol and its bacterial metabolites, plant sterols and their bacterial metabolites and fatty acids, which need to be removed before GC. Since fatty acids are less strongly retained on the capillary columns, bile acids can usually be quantitated in their presence.

For GC fecal bile acid analysis, several methods have been reported and most of them are quite complex [23, 62, 67].

In [68] they used continuous soxhlet extraction of aliquots of stool using chloroform and methanol. Extracts were derivatized and the methyl esters obtained were subjected to preparative TLC, respective bands were eluted and prepared for GC. On the other hand, in [23] they did a similar procedure but with tedious and longer steps. In alternate methods, bile acids from feces have been extracted with ammoniacal alcohol, methanol-hydrochloric acid, acetic acid-toluene, after removal of neutral sterols [23, 67].

In [19] they digested fecal samples with internal standard, extracted neutral sterols by repeated extractions, extracted bile acids with ethyl acetate, remove mineral acid and the residue was subjected to methyl ester formation; part of the aliquot was used for GC after trimethylsilylation. However, this method was not applicable to samples which contained conjugated bile acids.

Since the basic requirement for GC is that the compounds are in gaseous form at the column temperature, it is necessary to derivatize the polar functional groups. The carboxyl group in bile acids is often converted into the methyl ester. In addition, other bile acid esters have been used for GC, such as ethyl, n-propyl, isobutyl and n-butyl esters. Moreover, other derivatizations are common: of the hydroxyl group or the oxo group.
