**9. Conclusion**

As a non-invasive method, the analysis of feces is a fundamental tool for field work in ecological studies, not only to identify the presence of certain species in a particular area [94], but also for studying threatened species or animals difficult to observe and trap. For the identification, the original fecal shape must be maintained; however, as several factors can corrode it through time, visual identification is not always reliable. Particularly, feces from Xenarthra are sometimes difficult to identify in the wild because they are, commonly, total or partially mixed with the substrate.

52 Chromatography – The Most Versatile Method of Chemical Analysis

chromophore in the bile acid molecule [14, 66, 73, 84].

bile acid types, running times were longer [71].

acids found by HPLC-UV, through ESI-MS/MS.

reflected in the peak sharpness.

**9. Conclusion** 

absorbance values than their corresponding glyco-conjugated ones.

For several years, a great variety of analytical methods for quantitative determination of bile acids in various biological materials have been described [19, 27, 73, 74], including HPLC– UV assays [25, 38, 81, 83]. The different classes of bile acids have different absorption intensities to UV light, showing some limitations in their detection [27, 80]. The main disadvantages of these methods are the limited sensitivity and specificity of UV detection, especially in complex biological matrices, such as tissues and feces, due to the lack of a

In our study, compounds showed greater absorbance at 200 nm, demonstrated by their larger areas. As it was demonstrated before by other authors [27, 69]; free bile acids were harder to detect than conjugated ones. Tauro-conjugated bile acids showed greater

We could demonstrate not only the great resolution power of HPLC even with a UV detector, but also we achieved the resolution of the majority of the identified compounds in a relatively short time. In previous works, although they reported separation of different

The C-18 column was the most appropriated for the resolution of the majority of the compounds. Although when doing HPLC, there are several parameters that should be taken into account to achieve an efficient separation of all bile acids; among the most important ones, are the composition and strength of the mobile phase. The strength is involved in the peaks symmetry control and in the bile acid elution order [40, 93]; in this work, for most retained compounds we increased the mobile phase strength increasing the proportion of acetonitrile, so for example, we could elute cholesterol. Under these conditions, the analysis reproducibility in terms of retention time and areas between different runs, even among long periods of inactivity, was satisfactory, allowing a precise identification of the peaks. Moreover, the high efficiency of this chromatographic system was demonstrated and this is

As we found unidentified compounds which did not coincide with any of the standards used, we are in process of identifying them and also confirming the identity of fecal bile

Finally, we were able to differentiate all Xenarthra species through their fecal bile acid patterns, by HPLC. This study is of great relevance because is the first one in reporting HPLC as an ecological tool for the identification of wild-collected mammal feces. Moreover, it has proved to be a relatively simple method, without large preparation and derivatization steps, achieving resolution and identification of most of the compounds in a short time.

As a non-invasive method, the analysis of feces is a fundamental tool for field work in ecological studies, not only to identify the presence of certain species in a particular area [94], but also for studying threatened species or animals difficult to observe and trap. For the identification, the original fecal shape must be maintained; however, as several factors can The chromatographic determination of fecal bile acids has become a more precise method to identify unknown feces from the wild. The comparison of the whole pattern of fecal bile acids between field-collected scats and scats with known origin allows identifying the species from fecal material, avoiding capture and manipulation of animals.

We were able to establish the fecal bile acid patterns for Xenarthra species, which were different for all of them. Moreover, these patterns were consistent among different individuals of the same species. As it was reported before for other mammal species [3, 5-7, 25, 35, 36], we confirm that chromatographic determination of fecal bile acids is a precise technique to identify unknown wild-collected feces.

Chromatographic techniques are the method of choice for a detailed analysis of bile acid profiles in biological samples. However, there is no a single satisfactory method for the analysis of all bile acids in biological fluids. All techniques present limitations in their specificity, analysis times or simplicity. Some types of samples, such as urine or feces, can contain complex mixtures of bile acids; other samples, such as tissues and cells, can contain small quantities of bile acids, being, then, easier to analyze. Thus, the choice of the analytical method will depend on the particular aim of the study and the type of sample. Certainly, in our case, the use of multiple analytical techniques (TLC, HPLC, HPLC-ESI-MS/MS) allows a precise resolution and confirmation of complex bile acid patterns.

HPLC-UV analysis has been widely used for the determination of bile acids in several biological fluids [68]. The main disadvantage is its limited sensitivity and specificity to UV detection in complex biological samples, such as tissues and feces [66].

In our study, both techniques, TLC and HPLC, presented advantages and disadvantages in the analysis of Xenarthra feces. Although TLC offers advantages such as relative simplicity, short analysis times, ease of operation and simultaneous analysis of a big number of samples, as reported previously [43, 47, 93], it can be affected by external factors such as environmental conditions, humidity and temperature, and by the operability of the researcher. Moreover, it has lower resolution power and reproducibility than HPLC [44].

TLC separation selectivity allowed resolving and visualizing CHOL, free and amidated bile acids in a single run, as it was reported before [95]. TLC could also resolve pairs of bile acids with very similar Rf values, for example CA-GCA and DCA-CDCA. Previous works have reported the performance of both methodologies for bile acid analysis in gallbladder bile or liver. In [95] they reported that TLC produces quick, precise and reproducible results, with shorter analysis times and low costs.

On the other hand, HPLC most important advantages were precision, higher resolution power than TLC, high sensitivity and specificity, as it was observed by other authors before [44, 75]. However, one disadvantage is longer analysis times due to the injection of only one sample at the time.

Diet of animals, and particularly in the case of Xenarthra species, can have an effect on the detectability of some bile acids. In our study, in feces with high contents of vegetal material, pigments appeared as colored bands in the TLC chromatographic plates and unidentified peaks in HPLC. However, they did not interfere with bile acid detection and we were able to correctly identify compounds.

Use of Chromatography in Animal Ecology 55

**Author details** 

Emma B. Casanave

M. Soledad Araujo

Gustavo H. López

**Abbreviation list** 

CDCA: Chenodeoxycholic acid

ELSD: Evaporative Light Scattering Detector

GCDCA: Glycochenodeoxycholic acid

TCDCA: Taurochenodeoxycholic acid

TDCA: Taurodeoxycholic acid TLC: Thin layer Chromatography

HPLC: High Performance Liquid Chromatography HPTLC: High Performance Thin Layer Chromatography

GDCA: Glycodeoxycholic acid

LC: Liquid Chromatography LCA: Lithocholic acid MS: Mass Spectrometry ODS: octadecylsilane RIA: Radioimmunoanalysis RID: Refractive Index Detector

TCA: Taurocholic acid

CME: Cholic-methyl-esther DCA: Deoxycholic acid DHCA: Dehydrocholic acid EIA: Enzyme immunoassays

ESI: Electrospray Ionization FAB: Fast Atom Bombardment FLD: Fluorescence Detector GC: Gas Chromatography GCA: Glycocholic acid

CA: Cholic acid

CHOL: Cholesterol

*Cátedra de Fisiología Animal, Dpto. de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina* 

*Cátedra de Fisiología Animal, Dpto. de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina* 

*Cátedra de Bioanalítica II, Dpto. de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina* 

*Consejo Nacional de Investigaciones Científicas y Técnicas, (CONICET) Argentina* 

With the application of HPLC we corroborate TLC results, not only confirming the presence of the compounds found by TLC but also identifying new compounds which were not resolved by TLC, in Xenarthra feces.

Bile acid detection constituted an essential step in both techniques. The choice of the detection system is governed mainly by bile acid structures. In TLC, spraying the plates with the revealing reagent is the critical step, in which it is important the ability and experience of the operator. Spraying must be uniform to avoid uncolored areas that later interfere with the correct identification of spots. In HPLC, different classes of bile acids have different absorption capabilities to UV light; this is a critical factor especially for free bile acids, showing limitations for their detection. However, in our study we could detect free bile acids.

In relation to the components of the chromatographic system, the choice of the mobile and stationary phase is very important, as they depend on the type of sample and the objectives of the study. In TLC we used silica gel as stationary phase, which is the most used phase for lipid analysis [47]. The solvent system composed of toluene:acetic acid:water (5:5:1.5 v/v) proposed by [5] was the most adequate for our aims, achieving a good resolution of the great majority of the compounds, except for some tauro-conjugated bile acids such as TDCA and TLCA. In HPLC we used a reversed-phase column with a ODS stationary phase, and the mobile phase composed of ammonium carbonate and acetonitrile; under these conditions we were able to separate almost all bile acids, including tauro-conjugated bile acids not resolved by TLC.

Thus, our work demonstrates that both techniques, TLC and HPLC, are complementary, and they should be used together to take advantage of the positive aspects of each one.

The analysis of fecal bile acids is a useful tool not only for ecological and biological studies, but also is of great clinical interest. These determinations can be helpful in the evaluation of intestinal, biliary and hepatic functions, and in the diagnosis and treatment of some related diseases such as colon cancer [46, 96, 97].

Chromatographic techniques have been widely used to identify bile acids in different biological materials, mainly in gallbladder bile of mammal species. However, our study is the first one to report the use of TLC and HPLC to differentiate Xenarthra species through their fecal bile acid patterns.

Finally, our work established the validity of fecal bile acids to differentiate close related species, being useful to assess habitat use and to study food habits of sympatric species. The determination of these species-specific patterns offers robust data for the elaboration of conservation strategies in the long term.
