**4. Methods for extraction and determination of VOC emitted by hosts**

Although in many instances scientists do not collect biological samples that they want to analyze, it must be aware about the history of the samples, to procedures for collecting, type of packaging, added preservatives, stability characteristics and sample processing, in addition to the conditions storage and delivery protocol [48]. These aim to maintain the chemical profile of substances and physico-chemical characteristics, avoiding losses and minimizing contam‐ ination risks.

In metabolomic studies involving the diagnosis of dogs infected by *Leishmania* sp. the volatile organic compounds emitted from hair of these animals can be used as biomarkers of infection. Research involving VOC emitted from dog has critical steps such as: i) sampling and sample preparation, ii) choice of the extraction method, and iii) choice of analysis method.

#### **4.1. Sampling and sample storage**

The hair has a large amount of VOC, which can be derived from two sources: endogenous and exogenous. Exogenous VOC adhere to hair according to environmental exposure and the longer the residence time at a given location, the greater the extent of adsorption of these compounds. On other hand, endogenous VOC deriving from internal sources and they are products of host metabolism which when emitted through the skin a fraction is adsorbed by hair. Due to this feature, the hair sample is ideal sample both for outdoor exposure evaluation and for identification of biomarkers of metabolism changes.

When sampling hair it should always be removed from the same anatomical region due to possible variations of VOC profile depending on the area in which the host sample was collected (Figure 3). Sterilized gloves, razors and tweezers should be used in order to avoid sample contamination.

**Figure 3.** Collect of hair in dorse of dog.

Strategies to reduce exogenous compounds are another important aspect since these com‐ pounds can make difficult biomarker identification. For example, dogs can be bathed using mild soap three days before sampling period and the contact of these animals with chemicals must also be avoided.

After collection, the samples should be stored in clean and sterilized plastic bags kept in freezers at -20 º C. As VOC tend to desorb from hair in the course of time, the storage at low temperatures minimizes this process. Anyway, the storage of samples for a long period of time should be avoided.

#### **4.2. Extraction methods**

of packaging, added preservatives, stability characteristics and sample processing, in addition to the conditions storage and delivery protocol [48]. These aim to maintain the chemical profile of substances and physico-chemical characteristics, avoiding losses and minimizing contam‐

In metabolomic studies involving the diagnosis of dogs infected by *Leishmania* sp. the volatile organic compounds emitted from hair of these animals can be used as biomarkers of infection. Research involving VOC emitted from dog has critical steps such as: i) sampling and sample

The hair has a large amount of VOC, which can be derived from two sources: endogenous and exogenous. Exogenous VOC adhere to hair according to environmental exposure and the longer the residence time at a given location, the greater the extent of adsorption of these compounds. On other hand, endogenous VOC deriving from internal sources and they are products of host metabolism which when emitted through the skin a fraction is adsorbed by hair. Due to this feature, the hair sample is ideal sample both for outdoor exposure evaluation

When sampling hair it should always be removed from the same anatomical region due to possible variations of VOC profile depending on the area in which the host sample was collected (Figure 3). Sterilized gloves, razors and tweezers should be used in order to avoid

Strategies to reduce exogenous compounds are another important aspect since these com‐ pounds can make difficult biomarker identification. For example, dogs can be bathed using mild soap three days before sampling period and the contact of these animals with chemicals

preparation, ii) choice of the extraction method, and iii) choice of analysis method.

and for identification of biomarkers of metabolism changes.

ination risks.

**4.1. Sampling and sample storage**

322 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

sample contamination.

**Figure 3.** Collect of hair in dorse of dog.

must also be avoided.

The hair can contain dozens of VOC with different chemical structures and polarities. There‐ fore, the ideal extraction technique should be suitable for sampling VOC with quite different properties and to prevent sample contamination during the whole procedure.

Currently, the most used techniques for sampling VOC from biological materials are solvent extraction, steam distillation (SD) and with simultaneous extraction (SDE), supercritical fluid extraction (SFE) solid phase extraction and solid phase microextraction (SPME).

Solvent extraction, SD and SDE are conventional and widely used methods for sampling VOC from biological samples. These methods always require a long extraction time, the use of large amounts of organic solvents and the performance of many steps. In addition, many unstable volatiles compounds such as alkenes, esters and some unsaturated volatile organic compounds may be thermally decomposed and degraded during extraction or distillation at high temperatures [64].

However, these procedures still are widely applied for the characterization of fragrances and flavors since they are direct and less complex. Recently, SFE techniques, adsorbent extraction and SPME have attracted much attention from analysts due to its green and innovative features for sampling VOC from biological samples.

#### *4.2.1. Solvent extraction*

The solvent extraction is based on the relative solubility of the analyte by an organic solvent. This extraction can be done using a solvent to remove the analytes directly from a solid sample, which typically uses a Soxhlet system.

This extraction technique is perhaps the oldest and has the advantage of being a simple technique with a variety of commercially available pure solvents that provide a wide range of solubility and selectivity. As disadvantages, some of them use large amounts of toxic organic solvents, require a relatively large volume of sample and need ultrapure solvents to avoid sample contamination

The solvent extraction is widely used for sampling of pheromones, because of the lipid nature of many pheromones they are easily dissolved by solvents such as hexane or dichloromethane [65]. When the section of insect that produces pheromone mixture is known, usually a gland may be removed then put in contact with a small volume of solvent for extraction of interesting compounds. However, in this procedure other non-volatile compounds can also be extracted.

#### *4.2.2. Steam distillation and extraction by simultaneous distillation*

The SD is the original sampling method of VOC from plants. VOC are extracted from plants using water vapor steam distillation system followed by solvent extraction. The SDE techni‐ ques couple steam distillation and solvent extraction in order to simplify experimental procedures, to minimize the use of toxic organic solvents and to reduce the losses that occur during the sample transfer [66, 67].

Due to the high temperature during the procedures using SD and SDE, these techniques are not suitable for sampling for VOC from live animals and they can cause thermal decomposition of some thermo-unstable compounds. However, due to the large extraction capacity and the existence of simple SD and SDE experimental devices, they are still considered useful as sampling methods for thermally stable volatile compounds.

Some improvements have been implemented in these conventional sampling techniques to achieve better extraction efficiency. For example, Hashemi et al. [68] combined hydrodistilla‐ tion with solvent microextraction (HD-SME) for extraction of VOC from Artemisia (a type of plant) and obtained better results than those obtained with conventional hydrodistillation technique. It was also developed by Ferhat et al. [69] a microwave assisted SDE method (MW-SDE) for the analysis of volatile compounds form aromatic herbs. This new technique greatly improves the extraction time and amount of organic solvent due to the introduction of microwave energy as a heat source during extraction.

#### *4.2.3. Supercritical fluid extraction*

SFE is a process of separation and extraction by the use of supercritical fluids as the extraction solvents. Supercritical fluids are considered as a clean solvent, less toxic than organic solvents. Carbon dioxide (CO2) is a widely used supercritical fluid to which sometimes a co-solvent is added, such as ethanol or methanol. SFE is a good alternative to liquid-liquid extraction and proved to be an effective technique for sample preparation. Giannuzzo et al. [70] used a mixture of supercritical CO2 with ethanol (15 wt.%) for extraction of flavonoid naringin from citrus paradise, obtaining better results when compared with pure supercritical CO2.

The advantage of carbon dioxide is that it is easy to remove by simply reducing the pressure, which leaves virtually no trace, also being environmentally friendly [71, 72]. However, the SFE has as main drawback the high cost compared with solvent extraction technique. The carbon dioxide used in SFE has limited power for dissolving polar compounds [73]. The study of new supercritical fluid and the development of small scale SFE instruments are required for application of this technique for the study of VOC from biological samples. The SFE is widely used in the extraction of bioactive compounds from vegetables [74].

#### *4.2.4. Extraction with adsorbents*

In the extraction with solid adsorbent a pre-purified inert gas is used as carrier gas that flows continuously through the sample and extracts the VOC. Then, the volatile organic compounds are retained on a solid adsorbent, usually Tenax [75]. Finished the extraction time of VOC, an organic solvent such as hexane is directed through a tube containing the adsorbent for elution of the compounds. Another possibility is to carry out thermal desorption of VOC retained in the adsorbent tube before analysis.

Different types of adsorbents may be used to obtain a more selective sampling, depending on characteristics of VOC present in the sample. The choice of adsorbents must take into account their specific surface area and ability to retain VOC of interest. If thermal desorption mode is used, it should also be assessed the ability to attain complete and rapid desorption of the analytes, the existence of homogeneous and inert surface to prevent the formation of artifacts and irreversible adsorption during sampling, storage of tubes and desorption; low water affinity to avoid hydrolysis reactions and damage to the stationary phase used for chromato‐ graphic analysis, high mechanical and thermal stability [76]. The most used adsorbent materials are sub-classified into activated carbon [77], carbon molecular sieves (Carboxen, Carbosphere e Carbosieve) [78, 79] and porous organic polymers (Tenax, Chromosorb e Porapak) [80, 81].

This technique has great potential for the identification of disease biomarkers. Sporing et al. [82] monitored the release of VOC by a lineage of lung cancer cells using solid adsorbents and analysis by thermal desorption and gas chromatography-mass spectrometry (TD-GC/MS) where several aldehydes and ethers were identified.

### *4.2.5. Solid-phase microextraction (SPME)*

*4.2.2. Steam distillation and extraction by simultaneous distillation*

324 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

sampling methods for thermally stable volatile compounds.

microwave energy as a heat source during extraction.

during the sample transfer [66, 67].

*4.2.3. Supercritical fluid extraction*

*4.2.4. Extraction with adsorbents*

The SD is the original sampling method of VOC from plants. VOC are extracted from plants using water vapor steam distillation system followed by solvent extraction. The SDE techni‐ ques couple steam distillation and solvent extraction in order to simplify experimental procedures, to minimize the use of toxic organic solvents and to reduce the losses that occur

Due to the high temperature during the procedures using SD and SDE, these techniques are not suitable for sampling for VOC from live animals and they can cause thermal decomposition of some thermo-unstable compounds. However, due to the large extraction capacity and the existence of simple SD and SDE experimental devices, they are still considered useful as

Some improvements have been implemented in these conventional sampling techniques to achieve better extraction efficiency. For example, Hashemi et al. [68] combined hydrodistilla‐ tion with solvent microextraction (HD-SME) for extraction of VOC from Artemisia (a type of plant) and obtained better results than those obtained with conventional hydrodistillation technique. It was also developed by Ferhat et al. [69] a microwave assisted SDE method (MW-SDE) for the analysis of volatile compounds form aromatic herbs. This new technique greatly improves the extraction time and amount of organic solvent due to the introduction of

SFE is a process of separation and extraction by the use of supercritical fluids as the extraction solvents. Supercritical fluids are considered as a clean solvent, less toxic than organic solvents. Carbon dioxide (CO2) is a widely used supercritical fluid to which sometimes a co-solvent is added, such as ethanol or methanol. SFE is a good alternative to liquid-liquid extraction and proved to be an effective technique for sample preparation. Giannuzzo et al. [70] used a mixture of supercritical CO2 with ethanol (15 wt.%) for extraction of flavonoid naringin from citrus

The advantage of carbon dioxide is that it is easy to remove by simply reducing the pressure, which leaves virtually no trace, also being environmentally friendly [71, 72]. However, the SFE has as main drawback the high cost compared with solvent extraction technique. The carbon dioxide used in SFE has limited power for dissolving polar compounds [73]. The study of new supercritical fluid and the development of small scale SFE instruments are required for application of this technique for the study of VOC from biological samples. The SFE is widely

In the extraction with solid adsorbent a pre-purified inert gas is used as carrier gas that flows continuously through the sample and extracts the VOC. Then, the volatile organic compounds are retained on a solid adsorbent, usually Tenax [75]. Finished the extraction time of VOC, an

paradise, obtaining better results when compared with pure supercritical CO2.

used in the extraction of bioactive compounds from vegetables [74].

Solid-phase microextraction (SPME) was developed by Arthur e Pawliszyn [83], it has been regarded as one of the most innovative inventions in the field of VOC extraction in the last years. SPME integrates the steps of extraction, concentration and introduction to analysis, reducing sampling time and increasing the sensitivity when compared to other extraction methods.

This is a technique that has been successfully used for the extraction of compounds from environmental samples [84], pharmaceuticals [85], biological [86] and study of fragrances and flavors [87].

Headspace solid-phase microextraction (HS-SPME) is the most widely used form for sampling VOC from different matrices, including biological samples from animals [88-90], vegetables [91-94] e microbes [95, 96].

In recent years, SPME has been applied to the monitoring of compounds in living systems [97]. This application has advantages such as the possibility of sampling compounds without changing the actual condition of the system and to carry out studies about temporal progres‐ sion of a disease of a subject over time without the need to sacrifice one specimen for each sampling point [98]. For example, Bai et al. [99] used SPME technique in vivo for determination of off-flavor compounds in live fish. Geosmin and 2-methylisoborneol (2-MIB), produced by cyanobacteria and actinomycetes, were quantified in fish muscles which give the flavor of "land" and "clay" to fish.

In the area of Leishmaniasis there is only one work reported in literature about VOC extraction. HS-SPME was used by de Oliveira et al. [41] for extraction of VOC from hair of dog healthy and infected by *Leishmania infantum*, followed by GC-MS analysis which detected about 274 compounds, mostly ketones, aldehydes and hydrocarbons

#### **4.3. Methods of analysis**

After extraction VOC from samples it is necessary to identify each component to obtain quantitative and qualitative information about samples. Since the invention and development of gas chromatography technique the analysis of volatile compounds in biological becomes much easier.

GC is an excellent analytical technique for separating compounds which can be volatilized at the temperature applied to the injector device. Therefore, when GC is coupled to a detector it becomes a powerful technique for analysis of VOC in biological samples [100].

Nowadays gas chromatography coupled to flame ionization detector (GC-FID) and GC-MS are the main techniques used for VOC analysis and the GC-MS technique is more powerful due to the great identification ability of mass spectrometry (MS).

In recent years, the development of multi-dimensional CG has significantly improved separation of VOC from complex samples, making easier than before to obtain bio-information related to compounds present in samples [101]. Bean et al. [102] applied comprehensive twodimensional chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) technique to identify the profile of VOC from the metabolism of *Pseudomonas aeruginosa* bacteria. The method enabled better separation and identification of a wider number of compounds compared to the GC-MS technique, enabling the discovery of 28 new VOC characteristic of *P. aeruginosa*.

Another innovative application was proposed by Stadler et al. [103] that used TD × GC-TOF-MS for the identification of VOC released from a pig carcass, which allowed to define of the profile of compounds released during the decomposition of different tissues
