**3. Bile acid analysis**

The simultaneous separation and quantification of bile acids and their conjugates are challenging and have always presented technical difficulties due to marked differences in physicochemical properties, the presence of isomeric forms, their structural similarity or their relatively low concentrations in some biological samples [14, 20].

Use of Chromatography in Animal Ecology 39

The collection of samples for analysis is a critical step and constitutes a potential source of variation. For this reason, the procedure must be standardized. To determine the fecal bile acid pattern, it is necessary to collect feces from a known origin of the species under study. These reference feces can be collected in zoos, reserves or any place where one can assure the procedence of the fecal material. Moreover, feces can be collected during the capture and

Feces are put in paper bags with silica gel so as to avoid humidity. They are correctly identified with: number, collector, date and place of collection, GPS coordinates, a microhabitat description, fecal external aspect (fresh, old, very old) and any information that it is considered relevant. Unknown feces from the study area, should match the external

In our study, we analyzed the fecal bile acid patterns of different species from the Magnaorden Xenarthra (Mammalia). Our aim was to identify wild collected feces through their fecal bile acid patterns, and find in chromatography, a simple and rapid ecological tool. For this purpose, samples were collected in zoos, reserves from the wild in different areas of Argentina. The studied species were: pichi (*Zaedyus pichiy*), screaming hairy armadillo or crying armadillo (*Chaetophractus vellerosus*), big hairy armadillo (*Chaetophractus villosus*), southern long-nosed armadillo (*Dasypus hybridus*), giant armadillo (*Priodontes maximus*), southern tamandua (*Tamandua tetradactyla*), giant anteater (*Myrmecophaga tridactyla*), southern three-banded armadillo (*Tolypeutes matacus*) and six-banded armadillo (*Euphractus* 

The method of extraction will depend on the physicochemical properties of the biological material to be analyzed. Solid materials, such as feces, present particular problems due to the need of disrupting the matrix before extraction, either by homogenization, saponification, enzymatic digestion or lyophilization. Bile acids are hydrophobic steroids and hence, capable of extraction from aqueous media by organic solvents. The ease of extraction of bile acids from biological samples depends on four factors: the extent and type of conjugation, the number of hydroxyl groups, the state of ionization of the carboxyl group and the degree of binding to proteins. An increase in the number of hydroxyls or the presence of sulfates, glucuronides and to a lesser extent, glycine and taurine conjugates, will greatly increase the relative polarity (water solubility) of the bile acid. The state of ionization primarily depends upon the pK of the terminal group of the side chain, which varies from around 5.8 for the carboxyl group in the free bile acids to 4.3 for the glycine-conjugates and 1.9 for the sulfonic acid group of the taurine-conjugates. Although it would be expected that a decreased pH would favor extraction of bile acids because of the suppression of ionization, in fact bile acid extraction is, in many instances, favored at alkaline pH, presumably because

physical characteristics of the species under study, shape, size, colour and odour.

**5. Sample pre-treatment and solvent extraction** 

of the reduced protein binding that occurs [12].

**4. Sample collection** 

handling of a wild individual.

*sexcinctus*).

The most used techniques for the analysis of bile acid patterns in different biological samples include TLC coupled to densitometry, colorimetry, fluorimetry [5, 7, 21, 22], GC [16, 23, 24] and HPLC [25-27]. Moreover, non-chromatographic methods have been developed non chromatographic methods such as enzymatic, immunological and electrochemical ones. However, several of those methods are either not sensitive or are non-specific and have been displaced by GC, HPLC and radioimmunoassay. Radioimmunoassay, although it is highly sensitive, is not quite specific and overlaps of different bile acids occur [28]. On the other hand, both GC and HPLC are highly specific and sensitive to the extent of measuring even a few picomoles of bile acids [19].

Enzymatic methods using bile acid hydroxysteroid dehydrogenase are used for routine analysis of physiological fluids due to their simplicity. However, one of the main disadvantages is that differentiation of individual bile acids is not possible because total bile acids, rather than individual bile acids, are quantified [14].

Immunoassays of bile acids in different matrixes have provided relatively simple and sensitive methods for determining the concentration of selected sub-groups of bile acids. However, these assays do not provide full data for each of the individual bile acids.

Bile acids are haptens, nonantigenic low molecular weight, therefore complexes of bile acids covalently bound to large immunogenic molecules, for example bovine serum albumin, are required to obtain specific antibodies. In early experiments, [14C]- or [3H]-labelled bile acids were used as tracers for analysis of serum bile acids, whereas [125I]-labelled tracers were used in later publications. RIA can detect bile acids at the picomol level and are useful for high-throughput analysis. RIA for different bile acids in serum was described early for CA [29] and for CDCA, UDCA and LCA [30].

EIA use a hapten-enzyme conjugate as a tracer instead of radioisotope-labelled antigens. The construction of an effective EIA system requires the preparation of an enzyme-labelled antigen that possesses a high specific activity without decreasing the enzyme activity or immunoreativity. The labelling enzymes that have been employed for these assays include alkaline phosphatase and peroxidase [31]. Therefore, a quality antibody, preferably a monoclonal one, is very important for reliable analysis of bile acids in biological fluids.

The drawback of most RIAs and EIAs remains the limited specificity of the antibody. Usually, antibodies demonstrate high structural specificity for bile acids but often there is a low level of cross-reactivity with bile acids having similar structure to the target bile acid.
