**8. High performance liquid chromatography**

The first report of the application of HPLC for bile acid separation was in 1976 [68 in 12]; and although its usefulness has been demonstrated for bile acid analysis in a wide variety of biological samples, up to the present it has never been used for the identification of species through their fecal bile acid pattern.

Use of Chromatography in Animal Ecology 47

Reversed-phase HPLC is the most used type of liquid chromatography for compound separation; it has been reported that almost 75% of all HPLC separations are done in the reverse mode. It is one of the most reliable and powerful methods for a direct and no destructive analysis of those compounds which are less volatile than others, polar and particularly, steroids and their conjugates [27]. Particularly, the resolution of compounds with similar polarities to bile acids results efficient if reversed-phase HPLC is used [78, 79].

The main characteristic of reversed-phase HPLC is that the mobile phase is more polar than the stationary phase, and therefore the components of the sample elute from the column in order of their hydrophobicity, number, position and configuration of hydroxyl groups in the

The retention behavior of a certain compound in reversed-phase HPLC depends on its solubility in and the partition between mobile and stationary phases. The polarity of a molecule controls the solubility in the mobile phase while the hydrophobic surface plays a key role in the interaction with the stationary phase. Bile acids have a hydrophobic region in the β face of the steroid nucleus, an area which is constant among common bile acids. Therefore, when increasing the number of OH groups in the α face of the molecule, increases the polarity and improve the solubility in an aqueous mobile phase. On the other hand, the presence of a β OH or carbonyl group, reduces the hydrophobic area of β face, resulting in a diminished of retention time [79]. The most polar bile acids are trihydroxilated tauroconjugated ones and the least polar are free monohydroxylated bile acids [74, 79].

The choice of the mobile phase will depend not only on the characteristics of the analyte, but also on the detection mode. Almost all methods use an elution gradient with a binary or tertiary solvent system, which are combined depending on their polarities; so, a satisfactory

In different biological samples, bile acids are present as a complex mixture with a wide variety of polarities due to the presence of conjugated and free forms. For that reason is that the choice of the detection system will be largely governed by the structures of the analytes

There are several detectors that can be coupled to an HPLC system. However, at the present it is not available a simple, sensitive and direct method for the simultaneous analysis and

UV detectors have been widely used for the determination of bile acids in biological samples [25, 38, 81-83], mainly due to their relative low costs [44] and also because it allows the direct analysis of conjugated bile acids without prior derivatization [27, 71] and the reduction of the analysis time. However, HPLC-UV does not provide the required sensitivity and selectivity to detect the trace amounts of some bile acids or bile acid-sulfates in biological matrices [14, 20, 66, 73, 84], for example free bile acids [27, 80]. The simultaneous analysis of

quantification of individual bile acids in different tissues and fluids [20].

steroid nucleus [44, 46, 80].

**8.1. HPLC detectors** 

[14, 27].

resolution of different lipid classes is achieved [44].

During the last years, HPLC has been one of the most used techniques for the analysis and identification of different lipid classes [44] and, particularly, for the separation and quantification of bile acids and their derivatives in different biological materials, due to its possibility to be coupled to a great variety of detectors [14, 27, 69-74]. HPLC presents several advantages in relation to other techniques such as high resolution, high sensitivity and specificity [44, 75].

A chromatographic system consists of four main components: a device for sample introduction, a mobile phase, a stationary phase (column) and a detector. The injector is simply required to allow introduction of the analytes into a flowing liquid stream without introducing any discrimination effects, being manually or automatically operated. The two components which are associated with the separation that occurs in a chromatographic system are the mobile and stationary phases. In HPLC the mobile phase is a liquid delivered under high pressure to ensure a constant flow rate, and reproducible retention times. The stationary phase is packed into a cylindrical column with a particulate support to which is bonded the stationary phase and it is capable of withstanding the high pressures which are necessary.

Detectors and columns vary depending on the objectives of the study and the type of sample. A small quantity of liquid sample is injected into the mobile phase which is flowing through the column, so the individual components of the mixture are separated [76, 77].

To take advantage of an HPLC column, it is necessary to use integrated chromatographic systems; all the components are specifically designed and coupled to get a maximum efficiency.

A chromatographic separation occurs if the components of a mixture interact to different extents with the mobile and/or stationary phases (Fig. 3), and therefore take different times to move from the position of sample introduction to the position at which they are detected. So, there are two extremes, as follows: a) all analytes have total affinity for the mobile phase and do not interact with the stationary phase; all analytes move at the same rate as the mobile phase, they reach the detector very quickly and are not separated, and b) all analytes have total affinity for the stationary phase and do not interact with the mobile phase; all analytes are retained on the column and do not reach the detector. The role of the analyst is therefore, based on a knowledge of the sample, to manipulate the properties of the stationary and/or mobile phases to move between these extremes and reach the desired separation [77].

Reversed-phase HPLC is the most used type of liquid chromatography for compound separation; it has been reported that almost 75% of all HPLC separations are done in the reverse mode. It is one of the most reliable and powerful methods for a direct and no destructive analysis of those compounds which are less volatile than others, polar and particularly, steroids and their conjugates [27]. Particularly, the resolution of compounds with similar polarities to bile acids results efficient if reversed-phase HPLC is used [78, 79].

The main characteristic of reversed-phase HPLC is that the mobile phase is more polar than the stationary phase, and therefore the components of the sample elute from the column in order of their hydrophobicity, number, position and configuration of hydroxyl groups in the steroid nucleus [44, 46, 80].

The retention behavior of a certain compound in reversed-phase HPLC depends on its solubility in and the partition between mobile and stationary phases. The polarity of a molecule controls the solubility in the mobile phase while the hydrophobic surface plays a key role in the interaction with the stationary phase. Bile acids have a hydrophobic region in the β face of the steroid nucleus, an area which is constant among common bile acids. Therefore, when increasing the number of OH groups in the α face of the molecule, increases the polarity and improve the solubility in an aqueous mobile phase. On the other hand, the presence of a β OH or carbonyl group, reduces the hydrophobic area of β face, resulting in a diminished of retention time [79]. The most polar bile acids are trihydroxilated tauroconjugated ones and the least polar are free monohydroxylated bile acids [74, 79].

The choice of the mobile phase will depend not only on the characteristics of the analyte, but also on the detection mode. Almost all methods use an elution gradient with a binary or tertiary solvent system, which are combined depending on their polarities; so, a satisfactory resolution of different lipid classes is achieved [44].

## **8.1. HPLC detectors**

46 Chromatography – The Most Versatile Method of Chemical Analysis

through their fecal bile acid pattern.

specificity [44, 75].

necessary.

efficiency.

separation [77].

**8. High performance liquid chromatography** 

The first report of the application of HPLC for bile acid separation was in 1976 [68 in 12]; and although its usefulness has been demonstrated for bile acid analysis in a wide variety of biological samples, up to the present it has never been used for the identification of species

During the last years, HPLC has been one of the most used techniques for the analysis and identification of different lipid classes [44] and, particularly, for the separation and quantification of bile acids and their derivatives in different biological materials, due to its possibility to be coupled to a great variety of detectors [14, 27, 69-74]. HPLC presents several advantages in relation to other techniques such as high resolution, high sensitivity and

A chromatographic system consists of four main components: a device for sample introduction, a mobile phase, a stationary phase (column) and a detector. The injector is simply required to allow introduction of the analytes into a flowing liquid stream without introducing any discrimination effects, being manually or automatically operated. The two components which are associated with the separation that occurs in a chromatographic system are the mobile and stationary phases. In HPLC the mobile phase is a liquid delivered under high pressure to ensure a constant flow rate, and reproducible retention times. The stationary phase is packed into a cylindrical column with a particulate support to which is bonded the stationary phase and it is capable of withstanding the high pressures which are

Detectors and columns vary depending on the objectives of the study and the type of sample. A small quantity of liquid sample is injected into the mobile phase which is flowing through the column, so the individual components of the mixture are separated [76, 77].

To take advantage of an HPLC column, it is necessary to use integrated chromatographic systems; all the components are specifically designed and coupled to get a maximum

A chromatographic separation occurs if the components of a mixture interact to different extents with the mobile and/or stationary phases (Fig. 3), and therefore take different times to move from the position of sample introduction to the position at which they are detected. So, there are two extremes, as follows: a) all analytes have total affinity for the mobile phase and do not interact with the stationary phase; all analytes move at the same rate as the mobile phase, they reach the detector very quickly and are not separated, and b) all analytes have total affinity for the stationary phase and do not interact with the mobile phase; all analytes are retained on the column and do not reach the detector. The role of the analyst is therefore, based on a knowledge of the sample, to manipulate the properties of the stationary and/or mobile phases to move between these extremes and reach the desired In different biological samples, bile acids are present as a complex mixture with a wide variety of polarities due to the presence of conjugated and free forms. For that reason is that the choice of the detection system will be largely governed by the structures of the analytes [14, 27].

There are several detectors that can be coupled to an HPLC system. However, at the present it is not available a simple, sensitive and direct method for the simultaneous analysis and quantification of individual bile acids in different tissues and fluids [20].

UV detectors have been widely used for the determination of bile acids in biological samples [25, 38, 81-83], mainly due to their relative low costs [44] and also because it allows the direct analysis of conjugated bile acids without prior derivatization [27, 71] and the reduction of the analysis time. However, HPLC-UV does not provide the required sensitivity and selectivity to detect the trace amounts of some bile acids or bile acid-sulfates in biological matrices [14, 20, 66, 73, 84], for example free bile acids [27, 80]. The simultaneous analysis of

individual bile acids from a mixture presents several technical difficulties due to their similar and complex chemical structures, to their low UV absorbance, their low volatility, the presence of isomeric forms and the low concentration in certain type of samples [20, 27], particularly feces [46, 73, 84].

Use of Chromatography in Animal Ecology 49

**8.2. Liquid chromatography-mass spectrometry** 

acids in feces resulting from colonic bacterial metabolism [88].

87].

mass spectrometer.

complex matrix as feces [46].

**8.3. Experimental protocol** 

Xenarthra species.

Methods using FAB-MS and ESI-MS are applied in bile acid analysis. FAB-MS, however, has inferior quantitative capabilities and yields less intact ions (more in-source fragmentation) compared to ESI. Therefore, ESI-LC–MS remains a powerful technique for direct quantitative analysis of bile acids in biological matrices. Several methods have been developed and used to quantify bile acids in biological matrices using ESI-LC–MS [73, 86,

The persistent need for rapid and sensitive methods has motivated efforts to exploit the high sensitivity, specificity and the minimal sample preparation requirements of HPLC–MS/MS for bile acid analysis in biological fluids, including the complex profile of secondary bile

In conventional ESI the sample eluting from HPLC is pumped through a thin capillary (internal diameter approximately 0.1 mm) which is raised to a high potential (4 kV). Small charged droplets are sprayed from the ESI capillary into a stream of inert gas, generally nitrogen, at atmospheric pressure and travel down towards an orifice in the massspectrometer high-vacuum system. As the droplets traverse this path they become desolvated and reduced in size to such an extent that surface-coulombic forces overcome surface-tension forces and the droplets break up into smaller droplets. This process continues until they reach a point in which either an ion desorbs from a droplet or solvent is completely removed. This mechanism results in a beam of ions, which are directed to the

In a quadrupole mass analyzer MS/MS instrument, a precursor ion is mass-selected by first mass analyzer and focused into a collision cell preceding a second mass analyzer. The mass analyzers are arranged in series. Inert gas is generally introduced into the collision region and collisions occur between the precursor ion and inert gas molecules. In these collisions part of the precursor ions translational energy can be converted into internal energy, and as a result of single or multiple collisions an unstable excited precursor ions decompose to

MS/MS of steroids and bile acids has been an area of considerable study [90, 91]. When analyzed by negative-ion FAB or ESI-MS, bile acids, steroid sulphates and steroid glucuronides give abundant [M-H]−ions and few fragment ions. To gain structural information, MS/MS spectra are recorded. Any modification of the steroid skeleton or side

LC-MS/MS is a simple, sensitive and rapid technique for the analysis of bile acids in

In this section, we will describe the HPLC methods used to identify fecal bile acids in

product ions. Product ions are mass-analyzed by the second mass analyzer [89].

chain would result in a change in the pattern of fragment ions [91].

In several studies, it is necessary to pretreat samples which involve extraction, purification and derivatization so as to increase sensitivity and specificity [40].

Moreover, HPLC has been coupled to other types of detectors, for example RID, FLD and Electrochemical Detector.

The use of RID is limited by poor sensitivity and unstableness of base-line in gradient elution conditions, which is necessary for the separation of a complicated mixture with relatively short analysis time. Furthermore, the use of FLD or electrochemical detection does not detect non-chromophoric bile acids without a suitable pre-column derivatization [27]. Methods for sample derivatization are complex, with fluorescing chemicals [71, 85] or other complex derivatizations [27], which can introduce contaminants and produce secondary incomplete reactions, involving more complexity and longer analysis times [14, 27, 44, 73]. That is why derivatization methods are not the choice for a comprehensive analysis of fecal bile acid patterns [84].

The majority of lipids show a maximum absorbance in the range of wavelengths from 190 to 210 nm [44]; particularly, bile acids show different capabilities of absorption to UV light depending on their structure. UV detection can be used at 200–210 nm with moderate sensitivity for glycine- and taurine-amidated bile acids, which can be analyzed directly without previous derivatization steps, using conventional UV detectors [27, 71]. Unconjugated bile acids and their sulfated and glycosylated conjugates have a markedly low absorbance in this range [27, 80].

For that reason, in certain cases, depending on the aim of the study, is necessary to pretreat samples. This involves steps of extraction, purification and derivatization to increase sensitivity and specificity [40]. Although a great variety of pre-column derivatizations have been used to increase detection of bile acids to UV light, the complete conversion of compounds it is not assured and in general, required long steps that finally led to the loss of sample [14, 27].

In recent years, another detector used is ELSD. Theoretically, this detector responds to all solutes that are not volatile and the response is proportional to the mass of the solute present. The eluent is atomized in a stream of nitrogen and the finely divided spray passes down a heated chamber during which time the solvent is evaporated. The removal of the solvent produces a stream of particles which pass through a collimated beam of light. The scattered light at an angle in relation to the incident light, is focused onto a photomultiplier tube and the output is processed in an appropriate manner electronically. For a linear response, the droplet size must be carefully controlled. The device is fairly compact and relatively simple to operate. Detector sensitivity is in the range of 10-20 ng of solute. However, the magnitude of the response varies widely between different substances.

## **8.2. Liquid chromatography-mass spectrometry**

48 Chromatography – The Most Versatile Method of Chemical Analysis

and derivatization so as to increase sensitivity and specificity [40].

particularly feces [46, 73, 84].

Electrochemical Detector.

bile acid patterns [84].

sample [14, 27].

low absorbance in this range [27, 80].

individual bile acids from a mixture presents several technical difficulties due to their similar and complex chemical structures, to their low UV absorbance, their low volatility, the presence of isomeric forms and the low concentration in certain type of samples [20, 27],

In several studies, it is necessary to pretreat samples which involve extraction, purification

Moreover, HPLC has been coupled to other types of detectors, for example RID, FLD and

The use of RID is limited by poor sensitivity and unstableness of base-line in gradient elution conditions, which is necessary for the separation of a complicated mixture with relatively short analysis time. Furthermore, the use of FLD or electrochemical detection does not detect non-chromophoric bile acids without a suitable pre-column derivatization [27]. Methods for sample derivatization are complex, with fluorescing chemicals [71, 85] or other complex derivatizations [27], which can introduce contaminants and produce secondary incomplete reactions, involving more complexity and longer analysis times [14, 27, 44, 73]. That is why derivatization methods are not the choice for a comprehensive analysis of fecal

The majority of lipids show a maximum absorbance in the range of wavelengths from 190 to 210 nm [44]; particularly, bile acids show different capabilities of absorption to UV light depending on their structure. UV detection can be used at 200–210 nm with moderate sensitivity for glycine- and taurine-amidated bile acids, which can be analyzed directly without previous derivatization steps, using conventional UV detectors [27, 71]. Unconjugated bile acids and their sulfated and glycosylated conjugates have a markedly

For that reason, in certain cases, depending on the aim of the study, is necessary to pretreat samples. This involves steps of extraction, purification and derivatization to increase sensitivity and specificity [40]. Although a great variety of pre-column derivatizations have been used to increase detection of bile acids to UV light, the complete conversion of compounds it is not assured and in general, required long steps that finally led to the loss of

In recent years, another detector used is ELSD. Theoretically, this detector responds to all solutes that are not volatile and the response is proportional to the mass of the solute present. The eluent is atomized in a stream of nitrogen and the finely divided spray passes down a heated chamber during which time the solvent is evaporated. The removal of the solvent produces a stream of particles which pass through a collimated beam of light. The scattered light at an angle in relation to the incident light, is focused onto a photomultiplier tube and the output is processed in an appropriate manner electronically. For a linear response, the droplet size must be carefully controlled. The device is fairly compact and relatively simple to operate. Detector sensitivity is in the range of 10-20 ng of solute.

However, the magnitude of the response varies widely between different substances.

Methods using FAB-MS and ESI-MS are applied in bile acid analysis. FAB-MS, however, has inferior quantitative capabilities and yields less intact ions (more in-source fragmentation) compared to ESI. Therefore, ESI-LC–MS remains a powerful technique for direct quantitative analysis of bile acids in biological matrices. Several methods have been developed and used to quantify bile acids in biological matrices using ESI-LC–MS [73, 86, 87].

The persistent need for rapid and sensitive methods has motivated efforts to exploit the high sensitivity, specificity and the minimal sample preparation requirements of HPLC–MS/MS for bile acid analysis in biological fluids, including the complex profile of secondary bile acids in feces resulting from colonic bacterial metabolism [88].

In conventional ESI the sample eluting from HPLC is pumped through a thin capillary (internal diameter approximately 0.1 mm) which is raised to a high potential (4 kV). Small charged droplets are sprayed from the ESI capillary into a stream of inert gas, generally nitrogen, at atmospheric pressure and travel down towards an orifice in the massspectrometer high-vacuum system. As the droplets traverse this path they become desolvated and reduced in size to such an extent that surface-coulombic forces overcome surface-tension forces and the droplets break up into smaller droplets. This process continues until they reach a point in which either an ion desorbs from a droplet or solvent is completely removed. This mechanism results in a beam of ions, which are directed to the mass spectrometer.

In a quadrupole mass analyzer MS/MS instrument, a precursor ion is mass-selected by first mass analyzer and focused into a collision cell preceding a second mass analyzer. The mass analyzers are arranged in series. Inert gas is generally introduced into the collision region and collisions occur between the precursor ion and inert gas molecules. In these collisions part of the precursor ions translational energy can be converted into internal energy, and as a result of single or multiple collisions an unstable excited precursor ions decompose to product ions. Product ions are mass-analyzed by the second mass analyzer [89].

MS/MS of steroids and bile acids has been an area of considerable study [90, 91]. When analyzed by negative-ion FAB or ESI-MS, bile acids, steroid sulphates and steroid glucuronides give abundant [M-H]−ions and few fragment ions. To gain structural information, MS/MS spectra are recorded. Any modification of the steroid skeleton or side chain would result in a change in the pattern of fragment ions [91].

LC-MS/MS is a simple, sensitive and rapid technique for the analysis of bile acids in complex matrix as feces [46].

## **8.3. Experimental protocol**

In this section, we will describe the HPLC methods used to identify fecal bile acids in Xenarthra species.

We used a HPLC Thermo Finnigan made up of a gradient quaternary pump, on-line degassifier, a thermostatic module for the column and an UV-Vis detector with double wavelength, set at 200 and 210 nm. Analyses were performed on a reversed-phase C-18 column and a similar pre-column. We adapted the HPLC experimental protocol from [25] to our objectives.

Use of Chromatography in Animal Ecology 51

however, their quantities vary between them. This can be due to intra and/or interindividual

Variations in the composition of bile acids were reported for other biological samples. In [71] authors found differences in the hepatic composition of bile acids among different individuals of rats. Moreover, in [92] they recently reported diurnal variations, between morning and afternoon, in liver and plasma bile acids in laboratory mice. Bile acid circadian rhythm has been recognized for several years by various authors in different biological materials such as plasma, liver, gallbladder and intestinal contents. Our results coincide with those studies in which they did not report variations in the bile acid pattern of the

Individual retention times for all 15 standard bile acids and CHOL, were obtained. A typical chromatogram of the mixture of standards shown in Fig. 4. Free bile acids, taurineconjugated and glycine-conjugated bile acids were resolved in less than 32 minutes, achieving also the resolution of DCA and GDCA acids which could not be separated in

**Figure 4.** A typical HPLC chromatogram of the mixture of all the standards used. CA, GCA, TCA, CME, DHCA, DCA, CDCA, GDCA, GCDCA, UDCA, TDCA, TCDCA, LCA, TLCA, CHOL.

species but only in the quantity of some of them [92].

variations.

previous works [25, 80].

Standard bile acids were CA, DCA, DHCA, CDCA, LCA, UDCA, GCA, sodium glicocholate, GDCA, GCDCA, TCA, TDCA, TLCA and CME, and CHOL. All were prepared in methanol at a concentration of 0.1%; and, when it was necessary, at 0.2% and 0.4%, and filtered through a 0.45 µm syringe driven HPLC filter.

As mobile phase we used a solution of 0.3% ammonium carbonate in water/acetonitrile. The analyses were performed under a linear gradient constituted as follows: 73:27 v/v for 10 minutes, 68:32 v/v for 10 minutes, 50:50 v/v for 10 minutes and from 50 to 100% acetonitrile until 60 minutes. The flow rate was 0.8 ml/min. Column temperature was set at 25-27°C, and the sample volume injected was 20 µl. For the most retained compounds, solvent gradient was modified, gradually increasing from the minute 30 to the minute 60 from 50% to 100% acetonitrile.

We run a mixture of all standards before each series of samples, so as to compensate any possible variation in retention times due to column efficiency loss or environmental conditions. Sample peaks were tentatively assigned comparing their retention times with those of standard solutions, previously injected in the same conditions.

For purposes of quantification, calibration curves for all standards in methanol were constructed. Regression equations were obtained through linear regression analysis and applied to the peak area of each bile acid as a function of concentration.

In each chromatogram we measured the retention time (Rt) expressed in minutes which is defined as the time that each compound spends in eluting from the column, the peak area (A) expressed in absorbance units (mAU), which determines the quantity of each compound present in the sample and the peak width measured at the base line (W).

## **8.4. Results and discussion**

HPLC has been applied in a great variety of studies with different aims; however it has never been used as an ecological tool for differentiating species through fecal bile acids. In our work, we could identify the fecal bile acid pattern for all Xenarthra studied species, allowing the differentiation of wild-collected feces. No differences were observed between males and females, or between captive and wild individuals of the same species. All the species presented DHCA, TCA, TLCA and LCA, besides from CHOL, and three unidentified peaks.

When we calculated the percentage composition of each bile acid in samples from different individuals of the same species, we could observe that the compounds were the same; however, their quantities vary between them. This can be due to intra and/or interindividual variations.

50 Chromatography – The Most Versatile Method of Chemical Analysis

filtered through a 0.45 µm syringe driven HPLC filter.

our objectives.

acetonitrile.

**8.4. Results and discussion** 

unidentified peaks.

We used a HPLC Thermo Finnigan made up of a gradient quaternary pump, on-line degassifier, a thermostatic module for the column and an UV-Vis detector with double wavelength, set at 200 and 210 nm. Analyses were performed on a reversed-phase C-18 column and a similar pre-column. We adapted the HPLC experimental protocol from [25] to

Standard bile acids were CA, DCA, DHCA, CDCA, LCA, UDCA, GCA, sodium glicocholate, GDCA, GCDCA, TCA, TDCA, TLCA and CME, and CHOL. All were prepared in methanol at a concentration of 0.1%; and, when it was necessary, at 0.2% and 0.4%, and

As mobile phase we used a solution of 0.3% ammonium carbonate in water/acetonitrile. The analyses were performed under a linear gradient constituted as follows: 73:27 v/v for 10 minutes, 68:32 v/v for 10 minutes, 50:50 v/v for 10 minutes and from 50 to 100% acetonitrile until 60 minutes. The flow rate was 0.8 ml/min. Column temperature was set at 25-27°C, and the sample volume injected was 20 µl. For the most retained compounds, solvent gradient was modified, gradually increasing from the minute 30 to the minute 60 from 50% to 100%

We run a mixture of all standards before each series of samples, so as to compensate any possible variation in retention times due to column efficiency loss or environmental conditions. Sample peaks were tentatively assigned comparing their retention times with

For purposes of quantification, calibration curves for all standards in methanol were constructed. Regression equations were obtained through linear regression analysis and

In each chromatogram we measured the retention time (Rt) expressed in minutes which is defined as the time that each compound spends in eluting from the column, the peak area (A) expressed in absorbance units (mAU), which determines the quantity of each compound

HPLC has been applied in a great variety of studies with different aims; however it has never been used as an ecological tool for differentiating species through fecal bile acids. In our work, we could identify the fecal bile acid pattern for all Xenarthra studied species, allowing the differentiation of wild-collected feces. No differences were observed between males and females, or between captive and wild individuals of the same species. All the species presented DHCA, TCA, TLCA and LCA, besides from CHOL, and three

When we calculated the percentage composition of each bile acid in samples from different individuals of the same species, we could observe that the compounds were the same;

those of standard solutions, previously injected in the same conditions.

applied to the peak area of each bile acid as a function of concentration.

present in the sample and the peak width measured at the base line (W).

Variations in the composition of bile acids were reported for other biological samples. In [71] authors found differences in the hepatic composition of bile acids among different individuals of rats. Moreover, in [92] they recently reported diurnal variations, between morning and afternoon, in liver and plasma bile acids in laboratory mice. Bile acid circadian rhythm has been recognized for several years by various authors in different biological materials such as plasma, liver, gallbladder and intestinal contents. Our results coincide with those studies in which they did not report variations in the bile acid pattern of the species but only in the quantity of some of them [92].

Individual retention times for all 15 standard bile acids and CHOL, were obtained. A typical chromatogram of the mixture of standards shown in Fig. 4. Free bile acids, taurineconjugated and glycine-conjugated bile acids were resolved in less than 32 minutes, achieving also the resolution of DCA and GDCA acids which could not be separated in previous works [25, 80].

**Figure 4.** A typical HPLC chromatogram of the mixture of all the standards used. CA, GCA, TCA, CME, DHCA, DCA, CDCA, GDCA, GCDCA, UDCA, TDCA, TCDCA, LCA, TLCA, CHOL.

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

Use of Chromatography in Animal Ecology 53

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

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

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

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

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

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

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

species from fecal material, avoiding capture and manipulation of animals.

or partially mixed with the substrate.

shorter analysis times and low costs.

sample at the time.

technique to identify unknown wild-collected feces.

precise resolution and confirmation of complex bile acid patterns.

detection in complex biological samples, such as tissues and feces [66].

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 absorbance values than their corresponding glyco-conjugated ones.

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

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 reflected in the peak sharpness.

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

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.
