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

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 of the reduced protein binding that occurs [12].

Although there are a wide variety of methods available for the extraction of bile acids from biological materials, there is no general method that covers all eventualities. For that reason, the researcher must establish the solvent system which best fits to the aim of each particular study.

Use of Chromatography in Animal Ecology 41

TLC separation mechanism consists of the differential migration of the mixture components through the chromatographic plate, dragged by the moving solvent; the distance travelled by each compound depends on its chemical structure and its affinity for both phases. Several mechanisms are involved in the separation process and predominant forces depend

Although various separation techniques are commonly applied for the determination of bile acids in biological samples, TLC offers practical advantages, mainly its simplicity, economical equipment needed, ease of operation, short analysis time and high efficiency in analysing simultaneously a large number of samples [43, 46]. It enables reliable separation and analysis of a wide variety of compounds from different types of biological samples. Moreover, this technique is versatile because it can be modified using different types of

The choice of the correct stationary and mobile phases is of great importance and it depends upon the type of sample and the objectives of the study. Thus, the greatest problem in using TLC in specific cases, such as bile acid analysis, is the selection of suitable mobile phases

Since there are a great variety of TLC methodologies to separate bile acids, in this section we report the technique used at our laboratory, which allows us to resolve the highest number of compounds in a single chromatographic run. The best results were obtained using HPTLC silicagel 60F254 plates with aluminium base of 20 x 20 cm, with a bed thickness of 0.2 mm. We follow the protocol cited in [5]. Each sample extract and standards for the most common bile acids for mammals are spotted on the plates with the use of a capillary tube: LCA, TCA, GCA, CA, CDCA, DCA, DHCA, GCDCA and CHOL. Bile acid standard stock solutions are prepared in methanol at a concentration of 0.1 %. We tested the use of different sample (75, 90, 105, 120, 150 and 180 µl) and standard (7.5, 15, 22.5 and 30 µl) quantities being spotted on the plates, so as to standardize the optimal concentrations for a better

As development solvent we use a mixture of toluene:aceticacid:water in a proportion of 5:5:1.5 v/v. We first saturate the mobile phase with water vapour and then we extract the rest of the liquid water. This gives more reproducibility to the analysis since variations in

Bile acid spots are visualized by spraying the plates with a revealing solution of anysaldehide:glacial acetic acid:sulphuric acid in a concentration of 0.5:50:1 v/v; plates are

The bile acid pattern of each species is determined by the comparison of Rf values (relation between distance travelled by the compound and distance travelled by the eluent) and colour of the compounds with those of standard solutions. The relative intensity (concentration) of

sample spots helps in the identification of the fecal bile acid pattern of each species.

on the phases and solutes properties [43, 44].

[47].

visualization.

**6.1. Experimental protocol** 

mobile and stationary phases and visualizing methods [43, 45].

ambient humidity and temperature can alter the results.

heated in oven at 150 °C for 15 minutes.

Previously to the extraction step, feces are dried in oven at 30°C for one day to eliminate humidity and are stored in hermetic flasks in a dry and dark place. Feces are crushed with the use of a mortar and pestle and sieved with a fine mesh. They are macroscopically observed and rests of preys (plants, seeds, insects, bones, feathers) are eliminated. Fecal extracts must be filtered and concentrated to an adequate final volume, preferably under nitrogen stream.

For the extraction step, the ratio solvent/sample should be kept high, 20:1 or 10:1 (v/w). In our study, we extracted 1 gram of the fecal powder with a mixture of benzene:methanol (1:1 v/v). With the use of this solvent system, we could extract from feces the highest number of compounds in a single step. In addition, we also tested another extraction solvent system composed of dichloromethane:methanol (1:1 v/v). We replaced benzene with dichloromethane because it has the same extractive properties as benzene but is less toxic and thus easier to handle [3]. For HPLC, dried fecal extracts were resuspended in methanol and filtered with a polytetrafluorethylene filter of 0.45 µm before injection.
