Volatolomics: A broad area of experimentation.

Chemical analysis (detection and monitoring) of compounds associated with the metabolic activities of an organism is at the cutting edge of science. Volatile metabolomics (volatolomics) are applied in a broad range of applications including: biomedical research (e.g. disease diagnostic tools, personalized healthcare and nutrition, etc.), toxicological analysis (e.g. exposure tool to environmental pollutants, toxic and hazardous chemical environments, industrial accidents, etc.), molecular communications, forensics, safety and security (e.g. search and rescue operations). In the present review paper, an overview of recent advances and applications of volatolomics will be given. The main focus will be on volatile organic compounds (VOCs) originating from biological secretions of various organisms (e.g. microorganisms, insects, plants, humans) and resulting fusion of chemical information. Bench-top and portable or field-deployable technologies-systems will also be presented and discussed.

communications, forensics, safety and security (e.g. search and rescue operations).
In the present review paper, an overview of recent advances and applications of the volatolomics will be given. The main focus will be on volatile organic compounds (VOCs) originating from biological secretions of various organisms (e.g. microorganisms, insects, plants, humans) and resulting fusion of chemical information. Bench-top and portable or field-deployable technologies-systems will also be presented and discussed.

Metabolomics
Recent advances in molecular sensing technologies allow the development of analytical approaches and tools that can screen, analyse and decode phenotype variations. The outcomes of such analyses can elaborate the details of processes evolved within the metabolism of an organism. Metabolomics describes these biological processes by utilizing low molecular weight (<2000 Da) compounds that are produced by cells as chemical tools during metabolism [1,2]. Metabolomics enables the characterization and screening of specific markers and their patterns to obtain sufficient information of the physiological health status of an organism, as well as during environmental and genetic changes or under stress conditions [1].
Gold standard instrumentation available for metabolomics is stand-alone or hyphenated mass spectrometry (MS), ion mobility spectrometry (IMS), nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), gas chromatography (GC) and Fourier transform infrared spectroscopy (FT-IR). These analytical techniques are complementary allowing qualitative, quantitative, structural and time evolving information about the metabolites [3]. 3 The Human Metabolome Database (HMDB) [4] is considered to be the most complete collection available of human metabolites found in urine, blood and cerebrospinal fluid samples. It is an open access collection of all the available chemical, physical and biological data associated with human metabolites (>40,000) including spectral information, quantitative information and their classification.
The combined knowledge of the metabolome, genome and proteome can describe an organism both chemically and biologically. However, due to past and recent climatic events, environmental and pollution factors, diet, human lifestyle and medication, the human metabolome and all the processes occurring within it may be influenced. Thus, the metabolome is a synergy of endogenously generated and exogenously introduced compounds, some of which interfere [5]. This review paper focuses on an essential part of metabolomics: the volatile-omics (volatolomics) which are mainly associated with the study of volatile emissions of final stage metabolites [6,7]. This allows a holistic approach to the volatolome from the microworld (microorganisms e.g. bacteria) to the macro-world (e.g. insects, plants, humans). It identifies highly promising current application areas with rapid growth potential and considers a possible future orientation towards field chemical analysis with miniaturized and portable analytical systems for real-time in-vivo metabolomic and volatolomic research ( Figure 1). 4 Figure 1: Workflow illustration summarizing selective applications for volatolomics.

Volatolomics
Olfaction is one of the most important senses and processes for chemical and biological communication and interaction between humans, animals and plants.
Volatolomics is a recently introduced non-invasive approach that constitutes the study of the volatile organic compounds (VOCs) that are emitted by the metabolome [6]. VOCs are low molecular weight organic compounds (< 350 amu) which can transmit from the liquid phase into the gaseous phase at room temperature (25 o C) and pressure of 760 mmHg. They can be categorised in a wide range of groups depending their volatility (e.g. very VOCs, VOCs, semi VOCs, etc.), their origin (e.g. biogenic or anthropogenic, endogenous or exogenous, etc.) and their emission source (e.g. outdoor, indoor, industrial, material, building, etc.) [8]. Traditionally, 5 volatolomics focuses on VOCs originating from human body secretions (e.g. breath, sweat, urine, feces, saliva, etc.) and carry information related to medical diagnostics. However, as it will be discussed below, volatolomics can cover all living organisms and is therefore a useful tool in a wide range of applications [9][10][11][12][13].

a) Humans
Within the human body, a complex interaction of biochemical processes continuously occurs. These processes result in volatile metabolites which are emitted in human bodily fluids and tissues (i.e. lungs, skin, etc.). When abnormalities or changes occur, the conventional final volatile metabolic products are altered, and/or new VOCs are generated. Variations in the volatile profile of the human biochemistry can be identified in the headspace area above human skin -sweat, urine, blood, saliva, cells and directly in the exhaled air [9][10][11][12][13].
The human volatolome constitutes of a matrix of compounds contained within human body odor (breath and skin VOCs) which can provide diagnostic information of various diseases in an efficient and non-invasive way with the right analytical tools [14]. Due to the nature of human VOC samples, highly sensitive (low limits of detection -ppq or ppt) and accurate, highly resolving approaches-methodologies are required for sample collection and analysis.

b) Microorganisms
VOCs play a major role in intra-and inter-kingdom microbial communication [15,16].
Microorganisms can release specific VOCs that allow above ground and below ground short-distance and long-distance interactions among themselves or with 6 plants and insects [15,17]. These microbial VOCs are responsible for the generation of beneficial or harmful effects on other organisms, as they allow accurate communication (chemical signalling followed by message transmission and subsequent decoding) [15,16,[18][19][20][21]. Microbial molecular communication using VOCs occurs between bacteria, fungi, bacteria-fungi, bacteria-fungi-plants, etc [15].
Headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) has been utilised extensively to study volatile metabolites of biocontrol fungi [22]. In addition to SPME, thermal desorption (TD) has been used for sample collection. Proton-transfer-reaction mass spectrometry (PTR-MS) has also been employed for on-line sniffing of volatiles produced from microbial cultures (Escherichia coli, Shigella flexneri, Salmonella enterica, and Candida tropicalis) [16].
Bohm et al. give an extensive overview of the role of VOCs in the micro-world [15].
It is noticeable that VOCs produced by bacteria can be used as biocontrol agents against plant pathogens, can boost the inhibitory activity against specific bacterial diseases, can enhance the growth of neighbouring bacteria, or even can modulate the behaviour of other bacteria against certain conditions. In the world of fungi, VOCs such as 1-octen-3-ol can modulate the development of filamentous fungi [15].
Other fungal VOCs can regulate the growth and the establishment of their population [15]. Some other fungal species can generate VOCs with strong effect against their competitors or other antagonistic fungal species. VOCs play a major role in the fungi-bacteria communication for partner identification or to modulate the growth of a population or even to repel competitors [15]. Other microorganisms such as the nematodes Caenorhabditis Elegans have highly developed chemo-7 sensory reception systems and can be used for the detection of VOCs for environmental monitoring and/or security related operations (e.g. detection of explosive-related chemicals) [23].

c) Plants
Plants are constantly exchanging information using chemical signals (biogenic VOCs) concerning their surrounding environment, including the generation of defence alarms in the case of external enemies. This can result in the activation of specific mechanisms for survival. Plant communication is performed either underground (by chemicals released by their roots and spread through the soil) or in the ambient air by specific VOCs [24][25][26]. Plant communication above soil is a well explored area.
However, little is known about under soil communication between plants and micro and macro organisms. This is important for providing the right nutrition for optimal plant growth.
Other technologies, appropriate for field chemical analysis of VOCs emitted by plants are the electronic nose (e-nose or EN), which has been reported for successful application in agriculture [43][44][45][46][47][48][49][50][51][52]. ENs are intelligent chemical sensor array systems, typically consisting of two major components: (a) a gas sensor array, and (b) a Airsense Analytics Inc., Germany, consisting of an array of 10 MOS sensors, to measure the profile of VOCs released from wheat damaged with age and insects [48]. The authors were able to classify the different categories of wheat grains.
Spinelli et al. evaluated a near infrared and EN system to detect fire blight in pear plants [49]. It was reported that the EN system was able to provide a distinct olfactory signature required to identify that disease. Markom et al. used an EN to detect stem rot disease in oil palm plantation during field experiments [50]. The novel EOS 835 EN (Sacmi Imola s.c.a.r.l., Italy) based on an array of six thin film MOS sensors was used for the determination of the fingerprint of peeled tomatoes, which have been artificially spoiled with bacteria and fungi [51]. The same EN was applied to detect spoilage of maize cultivation by fungi [52].
Moreover, IMS and specifically field asymmetric -IMS (FAIMS) has been used on a tomato greenhouse for health monitoring of tomato plants [53]. Linear ion trap mass spectrometry (LIT-MS) has also been previously used by Soparawalla et al. for in situ analysis of agrochemicals residues on apples using ambient ionization and results concluded in clear distinction between organic and non-organic apples [54].

d) Insects
As mentioned above, human body odor results from the combined complex interaction of skin glands and the secreting organic compounds from the colonised bacteria of the human skin. These bacteria metabolise and transform the odorless sweat to an odorous liquid comprising of some hundreds of VOCs that disperse in the surrounding environment and mediate the attraction of insects [8,[55][56][57][58]. 10 Human detection by insects is usually a matter of time, distance and is affected by the surrounding environmental conditions, e.g. relative humidity, temperature, etc.
Mosquitoes are a worldwide threat both for human public health, as well as to plant cultivation and also industrial livestock production. Malaria, yellow fever, leishmaniasis, plague, dengue fever and typhus are representative of diseases that mosquitoes are responsible for causing and spreading. In Africa, the risk to public health is huge with more than 700,000 deaths per annum caused by mosquito bites [59]. Anthropophilic mosquitoes using physical and chemical cues can sniff VOCs produced by the human body and to bite. Along with dogs, rats and bees, they are considered ideal biological detectors. Previous work using GC-MS has proposed potential chemical compounds found in human body odor which attract mosquitoes ( Table 1). The role of the volatolomics in insects' world is further discussed throughout the paper at various points, such as in the agricultural and forensic applications that are followed.  [60,61]. After exposure to extreme environments, metabolomics can be used to perform rapid toxicological analysis of affected humans [62,63]. Blood and urine samples can be collected for analysis to determine possible inflammations by measuring the chemicals themselves or their by-products or final metabolites. Inhaled toxic VOCs such as pneumotoxic metals, organometallic compounds, halogenated hydrocarbons and/or aerosols and particles and their spread in the human organs can be measured in human breath using trace-level analytical techniques (e.g. MS, IMS, etc.) [64][65][66][67][68][69][70][71].
Exhaled breath testing has been recently implemented as a safety health status monitoring tool in occupational hazard applications such as wildfires, in dangerous industrial areas and the military to protect personnel from exogenous chemical threats [62]. Chronic exposure to harmful compounds can also be measured via breath monitoring and characterization. This allows the development of toxicokinetic 12 models, chemical toxicity prioritization, prediction models for inflammatory responses for medical doctors and first responders [70].

Commonly available instrumentation for off-line elemental analysis is based on
inductively coupled plasma -mass spectrometry (ICP-MS) or electrothermal atomic absorption spectroscopy (ETAAS) [72,73]. Direct injection ICP-MS results on the total amount of an element. In some cases, speciation is required which can be obtain by hyphenated ICP-MS techniques such as LC-ICP-MS. These techniques despite their advantageous characteristics, lack portability and time-resolving capability which is usually more than 1 hour excluding sample transportation time. These generate the requirement for portable or even wearable sensing systems allowing in-situ detection capabilities of trace elements in a qualitative and quantitative way.

Disease diagnostic tool
VOCs emitted from human biological sources (e.g. breath, sweat, urine, faeces, etc.) have been used for a wide range of applications including diagnostic purposes in medicine, toxicological analysis, doping control, etc. [74,75]. Exhaled breath has been extensively studied for decades and widely used for a number of health-related issues (e.g., early diagnosis of diseases, pharmacokinetic studies, substance metabolism monitoring). VOCs in human exhaled breath can provide pivotal characteristic information of human health status, of metabolic processes, of various pathological conditions (e.g. oxidative stress, lung cancer, breast cancer, prostate cancer, etc.) and disorders [76][77][78][79][80][81]. Despite the usefulness of the VOCs and the potential for reliable on-site measurements, further investigation both qualitatively 13 and quantitatively, are still required to provide clinically established single or multicomponent volatile signature/alarm patterns.
Volatile biomarkers from human secretions (e.g. breath, urine) have the potential to distinguish patients with asthma from patients with other lung diseases [76,77,80,81,82]. Increased levels of ethane, pentane and aldehydes have been identified in the exhaled breath of patients with asthma as possible breath markers [83].
Preliminary research has been undertaken in this direction with positive outcomes, which still requires further investigation and evaluation. However, due to the small patient sample size considered, further research is required. VOCs from human secretions have also been investigated as potential biomarkers for the detection of various types of cancer. Researchers at the University of Liverpool perform pioneer research on bowel, prostate and liver cancer [84][85][86]. They have developed an electronic nose (Odoreader) based on GC combined with advanced mathematical models able to sniff VOCs from biological samples and to diagnose malignancies with high accuracy. VOCs generated from different fecal microbiotas of children with celiac disease before and after they followed a gluten free diet and comparison with healthy children using solid-phase microextraction SPME-GC-MS has been investigated [87]. The dental community is currently using gas-phase analytical technologies to assess, and cure halitosis produced by oral bacteria that produce volatile sulfur compounds (VSCs) such as hydrogen sulfide, methyl mercaptan, and dimethyl sulfide [88]. Research has also been done in the correlation of VSCs with the progression of periodontal disease [89]. The Zurich Exhalomics [90] project combines three non-invasive technological approaches: secondary electrospray 14 ionization (SESI)-MS, quantum cascade laser-based vibrational spectroscopy and chemical sensors with aim to detect and monitor characteristic volatile biomarkers in as many as possible breath detectable diseases. Table 2

Personalised nutrition and healthcare
The process of nutrition involves the consumption, assimilation and bioprocessing of food resources for energy production, growth and repairing processes of the human body. Nutrition balance also involves the processes of catabolism and excretion. Existing monitoring methodologies for food products' biotransformation available in the market include demanding and time-consuming clinical studies using mostly blood and urine analysis. Alternatively, on-line exhaled breath analysis could be used as a noninvasive tool for the determination of volatile breath markers associated with the metabolism of existing or new food products (e.g. 3D printed products enriched with bioactive ingredients). Expired air could allow the real time monitoring of targeted food properties or the synergism of various food-stuffs or even diets enriched with specific groups of compounds (e.g. diet high in protein consumption) [103][104][105][106]. Research in this field is still limited; however, these characteristic breath markers could potentially be associated with short-and long-term health effects, beneficial actions or drawbacks of these products. The chemical analysis of expired air with state-of-the-art analytical instrumentation, novel sampling methodologies and advanced signal processing approaches such as chemometrics, could allow the chemical profiling of breath variations when a particular diet is followed, or a natural or synthetic substance is taken by an individual.
In personalized healthcare, breath analysis has been demonstrated by clinicians to allow determination of the smoking habits of an individual [107][108][109][110]. Currently, blood and urine analysis of cotinine (a primary metabolic product of nicotine) was used as a diagnostic tool. However, recent research on breath samples of smokers 18 and non-smokers using SESI-high resolution MS showed that some breath markers (e.g. hydroxy-2,4-hexadienal a benzene metabolite which is related to tobacco smoke) could provide, with high precision, information on the smoking conditions of a human [107]. Similarly, the early stage metabolites (11-hydroxy-delta-9tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol) of (-)-trans-Δ9tetrahydrocannabinol (THC) was shown that can be detected in human exhaled breath using field asymmetric IMS-MS. The above allows the differentiation of people who use medical and recreational marijuana from non-users (within a few hours after consumption) and provides data for real-time pharmacokinetic measurements [111][112][113][114][115][116].
In personalized nutrition and healthcare, exhaled air analysis is advantageous compared to conventional blood or urine analysis. This is due to its non-invasive nature, the unlimited sample quantity and requires no sample preparation or pretreatment. Breath analysis can be supported by demonstrated field analytical chemistry technologies for real-time on-site analysis.

Search and Rescue
The most critical task during crisis management operations in any scale of a disaster is the search, early detection and localization of human survivors. Equally important is the localization of dead bodies for delivery to their families. In the recent years,  found in human urine with common building materials (i.e. quartz sand) were also investigated with ion mobility spectrometry (IMS) [127].

As a continuation of the SGL for USaR project, the Deployable SAR Integrated
Chain with Unmanned Systems (DARIUS) project [118] [128]. The developed sensor was tested with nine volunteers placed within a plethysmographic chamber, and its performance was evaluated with selective reagent ionization time-of-flight mass spectrometry (SRI-TOF-MS) [128].
The small size and weight alongside its high sensitivity (low ppb), specificity and response times (<3 min) allows integration onto handheld devices of rescuers or on unmanned aerial vehicles (UAV) for remote sensing. Table 3 summarizes representative VOCs emitted from various human body sources. composition can affect decay process and thus the metabolic processes happening onto the corpse [139,140]. Laboratory-based or on-site detection and characterization of VOCs related to human body decay in crime scenes is interrelated to thanatochemistry (the chemistry of death) and can allow the description of the decomposition process at different stages. Qualitative and quantitative data on specific VOCs can provide information on the exact time of death or can even describe a forensic event or a murder case. Decomposition is a multistage process which evolves from autolysis of individual cells to a more complex tissue breakdown.
Briefly it unfolds from the fresh phase to the bloated stage, then to the active decay followed by the advanced decay and eventually to the skeletonization. Large biomolecules breakdown into simple organic matter molecules and release VOCs with characteristic odours. These VOCs attract specific groups of insects, which can be used by forensic entomologists to estimate the postmortem period [139][140][141][142][143][144][145]. Table 4  Technologies for underwater (e.g. a river or a lake) detection of human corpses need also to be developed. Monitoring of characteristic VOCs emitted from a dead human body hidden under water could be very useful for on-site investigations. There is still a huge gap in knowledge for such environments. Technologies such as membrane inlet MS (MIMS) [146] could be used to screen water sources using membrane sampling probes which allow the selective detection and screening of VOCs of interest.

Lab-based systems for volatolomics
GC-MS is the most widely used traditional analytical technique for volatolomics [9,11,100,133]. It is considered to be the gold standard in the chemical analysis of volatile and semi-volatile organic compounds, combining characteristics that allow the qualitative identification and quantification of trace-level components evolved from complex sample matrices. A gas chromatograph (GC) is a precise pneumatic and temperature-controlled oven that acts as a pre-separation device, which allows sample molecules to travel through a mobile gas phase over a stationary liquid or solid phase and to be separated according to their retention time prior to the MS introduction and analysis [8]. The MS can have a single, dual or triple quadrupole mass analyser, a time-of-flight (ToF), an ion trap, an orbitrap, etc [147,148]. Samples 27 can be introduced into the GC injection port by a micro syringe containing sample molecules dissolved in a solvent or with a solid-phase microextraction (SPME) fiber coated with a liquid or a solid extracting phase. Sample VOCs and semi-VOCs collection can also be performed by using stainless steel or glass tubes filled with absorptive materials. The tubes can be then introduced in a thermal desorption unit (TDU) for heating and sample extraction within the GC oven [123,126].   (Table 6) for volatolomics studies have limitations and improvements are still needed. The disadvantages that they present are mainly connected with their size, weight, power consumption, as well as the mass range that they can detect and the speed of analysis. Even when designed as fieldable instruments, their dimensions render them to be not user friendly, difficult for transportation and for on-site operation. They usually weight between 14.5-37 kg with power consumption between 120-600 W, slow response times (sec. to min.) and a limited small mass range of compounds that they can detect.
Portable Fourier transform infrared (FTIR) spectroscopy and e-noses have also the potential for on-site analysis of VOCs emitted from biological samples. The IRSpirit 33 from SHIMADZU [150] is a compact and lightweight FTIR spectrophotometer able to perform reliable detection and quantification tests. A portable electronic nose called PEN from AIRSENSE Analytics [172] is a small and robust system able to analyse gas mixtures up to 10 components. It can provide quantitative analysis and can be used for in-field applications of volatolomics. Cyranose® 320 [173] is also a handheld chemical sensor (array of nanocomposite sensors and advanced pattern recognition algorithms) that can measure vapours and VOCs in low concentrations. Cyranose® 320 has already been utilised in medical diagnostics for breath metabolomics for pneumonia, lung cancer, and pulmonary diseases as well as in colorectal cancer and toxic exposures. It has investigated also in micro-organism volatolomics, e.g.
bacterial classification or bacteria identification in blood and urine. The portable zNose [174] is based on miniaturised gas chromatography that can provide accurate chemical analysis of complex mixtures within 60 sec. Due to its technical characteristics zNose can perform on-site analysis of pathological samples and VOCs at low ppt concentrations.

Conclusions
Metabolomics is a broad area of experimentation targeting the analysis and comprehension of the metabolic processes occurring within an organism. A subset of the metabolomics is volatolomics, which focuses on the chemical investigation