**6.1. Experimental protocol**

40 Chromatography – The Most Versatile Method of Chemical Analysis

study.

nitrogen stream.

acid patterns [8].

**6. Thin-layer chromatography** 

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

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

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

TLC of bile acids has been used to differentiate feces of several mammal species, mainly carnivores such as the lesser grisson (*Galictis cuja*), guiña cat (*Leopardus guigna*), red fox (*Lycalopex culpaeus*), grey fox (*Lycalopex griseus*), pampas's fox (*Lycalopex gymnocercus*), puma (*Puma concolor*)*,* jaguar(*Panthera onca*), snow leopard(*Panthera pardus ciscaucasica*), pandas and different species of bears [6, 7, 32-37]. It had also been applied to a wide variety of other species: manatees [38], sperm whales [32], storks and herons [39]. Our study was the first one which reported the use of TLC to differentiate Xenarthra species, through their fecal bile

TLC is a type of planar chromatography in which the stationary phase is a solid adsorbent of fine particles, and the mobile phase is liquid. Aluminum or glass plates are used and they are covered with a fine and uniform layer of the stationary phase which is generally, silica. In some cases, it is chemically modified so as to provide suitable resolution conditions depending on the analyte. The correct choice of the mobile or stationary phase is essential to obtain an efficient separation of compounds to be analyzed [40-42]. Mobile phase generally consists of a solvent system composed of 2, 3 or 4 components which vary in their polarity and selectivity and they can include water, organic solvents and buffers [43, 44]. Silica gel is the most common adsorbent used in TLC as stationary phase, especially for the identification and separation of steroids such as bile acids in several biological samples, due

and filtered with a polytetrafluorethylene filter of 0.45 µm before injection.

to its adsorptive properties, great active surface and high pore size [45].

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 visualization.

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 ambient humidity and temperature can alter the results.

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 heated in oven at 150 °C for 15 minutes.

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.

## **6.2. Results and discussion**

In our study, we could differentiate all the Xenarthra species through their fecal bile acid patterns. We detected 15 compounds in feces; seven bile acids, seven unidentified compounds and cholesterol.

Use of Chromatography in Animal Ecology 43

*L. griseus* in Chile; they argued that there was too much variability in the spot pattern even among feces from the same individual. In spite of that, in [3] they could discriminate feces from both species. Moreover, in [6] authors demonstrated that bile acid patterns were

Several factors should be considered when analysing TLC results. One of them is the concentration of the sample; in the case of our study, samples from captive animals showed more intense bands while samples from wild animals had little concentration of some bile acids, making the correct identification sometimes difficult. However, we could find the optimal concentration, i.e., the concentration that allowed the detection of the compounds in

A second factor that should be considered, especially when working with wild animals, is the effect of the type of diet, which has been reported in other investigations. It is possible that the presence of chemical substances in the feces, as products of the diet, has an effect on the detectability of bile acids, masking the spots [34] as it was observed previously, for

Xenarthra species are omnivores and carnivores-omnivores [51], including invertebrates, small vertebrates, carrion, plant roots, tubers and seeds in their diets [52-55]. During plate running, other colour bands were observed; they corresponded to plant pigments which were yellow or orange for captive animals and green for wild ones. Differences in extract colours among species may reflect variations in the diet composition, mainly due to vegetal pigments which are naturally colored contrary to bile acid which are uncolored. Although we found some plant material in the feces, they had only small amounts and there was no evidence that prey

Age of wild-collected scats and thus, weathering, plays an important role in the analysis of feces by TLC [7, 36]. Some authors [7, 35, 48, 56] suggested that feces weathering can lower the concentration of bile acids in scats, leading to their erroneous identification. In our study, it was demonstrated that bile acids were clearly identified even in two-year old

Further, small sample size can worsen TLC performance [36, 56, 57]; however, this was not the case of our study since we used sufficient quantity of dry pulverized fecal material to

The experience of the analyst in applying TLC is also important. For example, the spraying with the visualizing agent is not always uniform and some areas of the chromatographic

Several authors reported different TLC methods for the analysis and separation of bile acids. In [58] they reported a solvent system which allows group separation; free bile acids are separated with the first solvent system acetic acid:carbón tetrachloride:di-isopropyl ether:iso-amyl acetate:n-propanol:benzene (5:20:30:40:10:10 v/v), then with the second solvent system propionic acid:iso-amyl acetate:water:n-propano (15:20:5:10 v/v) they

plate may be uncoloured, resulting in an incorrect interpretation of the bands.

the chromatographic plates, for wild and captive animals, being higher for wild ones.

specific for some threatened carnivore species in Chile.

example in coyotes [50].

feces.

items interfere with bile acid identification.

allow a good detection of the compounds [5].

Moreover, we showed the resolution of TLC because some pairs of bile acids with very similar Rf values, CDCA-DCA and CA-GCA, were discriminated. Although the Rf value is the main parameter to identify a compound, several other characteristics of the bands had to be carefully analysed in the chromatographic plates to determine bile acid patterns.

Some standard Rf values partially overlapped; however, we established a Rf range for each compound and, together with their colour, the spots were correctly identified. The concentration of the compounds was proportional to the relative intensity of each band in the chromatographic plate, as determined visually. Specific colours obtained through the oxidation by anisaldehyde for each bile acid allowed the identification of the spots in the plates. DHCA differed from the other standards because it showed two and, in some cases, three bands with a distinctively orange colour. Standards which showed more intense colours were LCA (dark green), TCA (blue-grey) and CHOL (dark pink); the rest of the bile acids had less intense colours (Fig. 3).

**Figure 3.** A chromatographic plate revealed with anisaldehyde:glacial acetic acid:sulphuric acid (0.5:50:1 v/v), shows bands of different colours. Left: 12 sample lines. Right: 8 standard bile acids.

Therefore, we stress the importance of spotting standard solutions together with the samples in each chromatographic plate to correctly identify compounds.

Even if TLC of fecal bile acids offers practical advantages such as simplicity and ease of operation [43, 46], like any laboratory technique it needs much of practical knowledge and skills [7] and it requires a careful and detailed analysis from the researcher.

In our study, TLC was useful to identify feces from Xenarthra species as it allowed the extraction, visualization and identification of fecal bile acids from individuals of all the studied species.

For several years, TLC has been used to identify wild-collected feces, mainly for species with low contents of vegetal material in their diets, such as carnivores [5, 35, 48, 49]. Nevertheless, in [34] they were not able to differentiate feces from *Lycalopex culpaeus* and *L. griseus* in Chile; they argued that there was too much variability in the spot pattern even among feces from the same individual. In spite of that, in [3] they could discriminate feces from both species. Moreover, in [6] authors demonstrated that bile acid patterns were specific for some threatened carnivore species in Chile.

42 Chromatography – The Most Versatile Method of Chemical Analysis

In our study, we could differentiate all the Xenarthra species through their fecal bile acid patterns. We detected 15 compounds in feces; seven bile acids, seven unidentified

Moreover, we showed the resolution of TLC because some pairs of bile acids with very similar Rf values, CDCA-DCA and CA-GCA, were discriminated. Although the Rf value is the main parameter to identify a compound, several other characteristics of the bands had to

Some standard Rf values partially overlapped; however, we established a Rf range for each compound and, together with their colour, the spots were correctly identified. The concentration of the compounds was proportional to the relative intensity of each band in the chromatographic plate, as determined visually. Specific colours obtained through the oxidation by anisaldehyde for each bile acid allowed the identification of the spots in the plates. DHCA differed from the other standards because it showed two and, in some cases, three bands with a distinctively orange colour. Standards which showed more intense colours were LCA (dark green), TCA (blue-grey) and CHOL (dark pink); the rest of the bile

be carefully analysed in the chromatographic plates to determine bile acid patterns.

**Figure 3.** A chromatographic plate revealed with anisaldehyde:glacial acetic acid:sulphuric acid (0.5:50:1 v/v), shows bands of different colours. Left: 12 sample lines. Right: 8 standard bile acids.

samples in each chromatographic plate to correctly identify compounds.

skills [7] and it requires a careful and detailed analysis from the researcher.

Therefore, we stress the importance of spotting standard solutions together with the

Even if TLC of fecal bile acids offers practical advantages such as simplicity and ease of operation [43, 46], like any laboratory technique it needs much of practical knowledge and

In our study, TLC was useful to identify feces from Xenarthra species as it allowed the extraction, visualization and identification of fecal bile acids from individuals of all the

For several years, TLC has been used to identify wild-collected feces, mainly for species with low contents of vegetal material in their diets, such as carnivores [5, 35, 48, 49]. Nevertheless, in [34] they were not able to differentiate feces from *Lycalopex culpaeus* and

**6.2. Results and discussion** 

compounds and cholesterol.

acids had less intense colours (Fig. 3).

studied species.

Several factors should be considered when analysing TLC results. One of them is the concentration of the sample; in the case of our study, samples from captive animals showed more intense bands while samples from wild animals had little concentration of some bile acids, making the correct identification sometimes difficult. However, we could find the optimal concentration, i.e., the concentration that allowed the detection of the compounds in the chromatographic plates, for wild and captive animals, being higher for wild ones.

A second factor that should be considered, especially when working with wild animals, is the effect of the type of diet, which has been reported in other investigations. It is possible that the presence of chemical substances in the feces, as products of the diet, has an effect on the detectability of bile acids, masking the spots [34] as it was observed previously, for example in coyotes [50].

Xenarthra species are omnivores and carnivores-omnivores [51], including invertebrates, small vertebrates, carrion, plant roots, tubers and seeds in their diets [52-55]. During plate running, other colour bands were observed; they corresponded to plant pigments which were yellow or orange for captive animals and green for wild ones. Differences in extract colours among species may reflect variations in the diet composition, mainly due to vegetal pigments which are naturally colored contrary to bile acid which are uncolored. Although we found some plant material in the feces, they had only small amounts and there was no evidence that prey items interfere with bile acid identification.

Age of wild-collected scats and thus, weathering, plays an important role in the analysis of feces by TLC [7, 36]. Some authors [7, 35, 48, 56] suggested that feces weathering can lower the concentration of bile acids in scats, leading to their erroneous identification. In our study, it was demonstrated that bile acids were clearly identified even in two-year old feces.

Further, small sample size can worsen TLC performance [36, 56, 57]; however, this was not the case of our study since we used sufficient quantity of dry pulverized fecal material to allow a good detection of the compounds [5].

The experience of the analyst in applying TLC is also important. For example, the spraying with the visualizing agent is not always uniform and some areas of the chromatographic plate may be uncoloured, resulting in an incorrect interpretation of the bands.

Several authors reported different TLC methods for the analysis and separation of bile acids. In [58] they reported a solvent system which allows group separation; free bile acids are separated with the first solvent system acetic acid:carbón tetrachloride:di-isopropyl ether:iso-amyl acetate:n-propanol:benzene (5:20:30:40:10:10 v/v), then with the second solvent system propionic acid:iso-amyl acetate:water:n-propano (15:20:5:10 v/v) they

separated taurine and glycineconjugated standard bile acids. In [21], authors reported 15 solvent systems to separate bile acids, being the most efficient the acidic one.

Use of Chromatography in Animal Ecology 45

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

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

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

For GC fecal bile acid analysis, several methods have been reported and most of them are

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

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

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

from these sources need to be appropriately modified [19].

acid, acetic acid-toluene, after removal of neutral sterols [23, 67].

derivatizations are common: of the hydroxyl group or the oxo group.

analysis [14, 19].

their presence.

quite complex [23, 62, 67].

conjugated bile acids.

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 placing in a 120°C oven [60].

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 concentration range 50–75% (v/v).

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

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 conservation, distribution and eco-physiology of this group.
