Temporal profiling of human urine VOCs and its ...

2 downloads 28 Views 767KB Size Report
Medical University, Innsbruck, Austria, 3Institute of Nuclear Physics PAN, Kraków, Poland, 4Institute for ...... Journal of Orthomolecular Medicine 15:47–48.
Toxicology Mechanisms and Methods, 2012; 22(7): 502–511 © 2012 Informa Healthcare USA, Inc. ISSN 1537-6516 print/ISSN 1537-6524 online DOI: 10.3109/15376516.2012.682664

RESERACH ARTICLE

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

 emporal profiling of human urine VOCs and its potential T role under the ruins of collapsed buildings Pawel Mochalski1,3, Karin Krapf1,2,4, Clemens Ager1,2, Helmut Wiesenhofer1,2, Agapios Agapiou5, Milt Statheropoulos5, Dietmar Fuchs2,6, Ernst Ellmerer4, Boguslaw Buszewski1,7, and Anton Amann1,2 Breath Research Institute of the Austrian Academy of Sciences, Dornbirn, Austria, 2Univ.-Clinic for Anesthesia, Innsbruck Medical University, Innsbruck, Austria, 3Institute of Nuclear Physics PAN, Kraków, Poland, 4Institute for Organic Chemistry, Leopold-Franzens University Innsbruck, Innsbruck, Austria, 5National Technical University of Athens (NTUA), School of Chemical Engineering, Athens, Greece, 6Division of Biological Chemistry, Biocenter Innsbruck Medical University, Innsbruck, Austria, and 7Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Toruń, Poland 1

Abstract Context: The scent profile of human urine was investigated as potential source of chemical markers of human presence in collapsed buildings after natural or man-made disasters. Objective: The main goals of this study were to build a library of potential biomarkers of human urine to be used for the detection of entrapped victims and to further examine their evolution profile in time. Materials and methods: Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPMEGC-MS) was used to detect and identify the volatile organic compounds (VOCs) spontaneously released from urine of 20 healthy volunteers. Additionally, the evolution of human urine headspace during four days storage at room temperature was investigated. Results: 33 omnipresent species with incidence higher than 80% were selected as potential urine markers. The most represented chemical classes were ketones with 10 representatives, aldehydes (7 species) and sulfur compounds (7 species). The monitoring of the evolution of the urine scent demonstrated an increase in the emission of 26 omnipresent urinary volatiles (rise from 36% to 526%). The highest increase was noted for dimethyldisulfide and dimethyltrisulfide (fivefold increase) and 3-methyl-2-butanone, 4-methyl-2-pentanone and 3-hexanone (fourfold rise). Only three compounds exhibited decreasing trend; dimethylsulfone, octanal and propanal. Conclusion: The ubiquitous urine VOCs identified within this study create a library of potential markers of human urine to be verified in further field studies, involving portable and sensitive instruments, directly applied in the field. Keywords:  Entrapped victims, volatile organic compounds (VOCs), potential markers of human presence, rescue operations

Introduction

20th century (United States Geological Survey, 2003; Centre for Research on the Epidemiology of Disasters, 2003). This is particularly true for overpopulated urban areas with complex street systems and tall blocks of flats. Consequently, the early location of entrapped victims in collapsed buildings is of particular importance for rescue

Natural or man-made disasters (e.g. earthquakes, technical failures, explosions, terrorist attacks) cause frequently high mortality and are responsible for a large number of entrapped people. Earthquakes, for example, induced more than 1,800,000 deaths in the

Address for Correspondence: Boguslaw Buszewski, Chair of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarina St., PL 87-100 Torun, Poland. Tel: +48 56 6114308. E-mail: [email protected] (Received 01 December 2011; revised 27 March 2012; accepted 30 March 2012)

502

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

Role of urine scent profile in USaR applications  503 operations. The use of canines is the method of choice in Urban Search and Rescue (USaR) operations (Ferworn, 2009). Canines with their excellent scenting skills and ability to search relatively large disaster scenes present, however, a number of limitations. The information they provide can be misinterpreted. Their training is timeconsuming and expensive and their working time is relatively short (30 min). Furthermore, during the rescue operations dogs can be easily distracted, get stressed, or even frustrated (Wong and Robinson, 2004). Therefore, a huge demand exists for portable instruments, which could complement, or even replace their work. Various technical devices have been developed to support the work of searching dogs including sound recognition devices (i.e. geophones), telescopic or infrared (IR) cameras, fiber optic devices, sonars, etc. Nevertheless, the canines’ highly developed olfactory sensing system still remains an excellent comprehensive technical search tool. The chemical environment in disasters is considered highly complicated and harsh, especially after massive buildings collapse (e.g. the 9/11 World Trade Center attack) (Dalton, 2003; Dalton et al., 2010). Potentially, it can become extremely hazardous and dangerous able to cause respiratory and toxicological impacts in both humans (i.e. first responders, victims) and animals (i.e. canines) (Gwaltney-Brant et al., 2003). Toxicological agents released might include solids and liquids, particulates and gases; hydrocarbons, polychlorinated biphenyls, hazardous metals, asbestos, various harmful gases (e.g. hydrogen cyanide, hydrogen sulfide, freon, halogenated gases, carbon monoxide), soaps, detergents, acids and alkalis, ethylene glycol, propylene glycol, phenol and alcohols (Murphy et al., 2003). Furthermore, following the recent Japan earthquake, the radioactive danger is also an active threat, as well. In this context, the knowledge of human scent profile and its evolution during entrapment is crucial. The identification of a group of compounds and relevant patterns that is characteristic for human presence in the voids of collapsed buildings appears quite challenging. Volatile species forming the human scent are emitted from different biological fluids (urine, blood, sweat) and tissues (skin, lungs, bowels). Their emission depends on the conditions in the entrapment scene (temperature, humidity, type of collapse), time of entrapment and the medical status of entrapped victims. Moreover, VOCs forming the human scent are spread throughout the ruins, interact with the debris materials (concrete, glass, steel, dust, etc) and mix with environmental contaminants. Consequently, the concentration of human presence markers is low making their identification and detection a really challenging task. Due to the kidneys’ preconcentration capabilities, urine seems to be a promising source of volatile markers. Until now, more than 230 volatiles have been identified in human urine (Wahl et al., 1999; Mills and Walker, 2001; Statheropoulos et al., 2005; Smith et al., 2008). They belong

to numerous chemical classes such as hydrocarbons, aldehydes, ketones, furans, pyrroles, terpenes, sulfur-containing compounds and heterocyclic compounds. In the context of USaR operations, VOCs spontaneously emitted by human urine hold almost an equally important status as compounds emitted by exhaled breath or skin. Currently, Solid-Phase MicroExtraction coupled with Gas Chromatography-Mass Spectrometry (SPMEGC-MS) is the most widely applied method for analyzing the urine headspace (Fustinoni et al., 1999; Mills and Walker, 2001; Statheropoulos et al., 2005; Smith et al., 2008). However, in the majority of the SPME-GC-MS studies, addition of salt, adjustment of pH and heating are applied to improve SPME extraction efficiency. According to our knowledge, relatively few studies investigated the profile of VOCs emitted from non-modified human urine. A preliminary study resulted in the selection of only 11 omnipresent urine volatiles (Statheropoulos et al., 2005). Nevertheless, the time evolution of this profile received limited attention. The possible changes in the urine composition during the entrapment time can significantly modify the scent profile and the usefulness of potential urine markers during rescue operations. In USaR operations, a list of reliably identified compounds ubiquitously appearing in urine, together with information on their concentration time profile is of particular interest. So far, such a list of ubiquitous and reliably identified volatile compounds does not exist. In a previous study (Rudnicka et al., 2010), ion mobility spectrometry (IMS) was employed for the detection of volatiles released from human urine. Despite the excellent sensitivity for some species (ketones, aldehydes), the technique was not very effective for detecting some other important classes of urine-born markers like hydrocarbons, sulfur compounds and aromatic compounds. Therefore, in the present study, detection and identification of urine VOCs relied on a more powerful technique; that of gas chromatography coupled with mass spectrometry (GC-MS), supported by SPME as a pre-concentration method. The main goals of this study were firstly the selection of potential urine-born biomarkers to be used for the detection of entrapped victims and secondly the investigation of their evolution in the urine headspace during urine storage at room temperature over a period of 4 days (the survival rate of victims’ dramatically decreases after the first 72 h).

Methods Test subjects A cohort of 20 healthy normal volunteers (Caucasians from central Europe) was recruited. All volunteers gave informed consent to participate in the study and completed a questionnaire describing their health and smoking status, as well as, their recent food intake. No special dietary regimes were applied. Demographic data for the volunteers are presented in Table 1.

© 2012 Informa Healthcare USA, Inc.

504  P. Mochalski et al. Table 1.  Demographic data of healthy volunteers. Total Smokers Non-smokers Age Age Age median median median (range) (range) n (range) N n Total 20 29.5 7 23 13 32 (19–64) (19–61) (19–64) Female 9 23 5 23 4 22.5 (45%) (19–41) (71%) (19–41) (31%) (19–38) Male 11 40 2 46 9 40 (55%) (26–64) (29%) (31–61) (69%) (26–64)

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

Urine sampling and sample preparation The urine collection was approved by the Ethics Commission of Innsbruck Medical University (study identifier UN4371). Volunteers’ morning urine was collected into 10 ml plastic urine-monovette vessels (Sarstedt, Germany). Prior to the use, the monovettes were thoroughly rinsed with high-purity air at 60°C for 4 h to remove contaminants, which could distort the sample integrity. In addition to this, an effort was made to minimize the storage time of the urine samples in the monovettes to maximum 3 h. After collection, samples were transferred into 10 ml glass vials (9 ml of urine per vial) and frozen at –80°C. Defrosting of the samples was performed at room temperature shortly before experiments. Extraction of VOCs from urine samples was performed in 20 ml headspace vials (Gerstel, Germany) crimped with 1.3 mm butyl/PTFE septa (Macherey-Nagel, Germany). Before crimping, a stirring bar was added to each vial. Vials were evacuated by means of a membrane pump. Next, 3 ml of urine were transferred into each vial using a 5 ml glass syringe (Roth, Germany). Finally, pressure in the vials was balanced with high-purity nitrogen (6.0–99.9999%). Since VOCs spontaneously emitted by human urine were targeted, no additional urine modifications like pH adjustment or mineralization were applied. To investigate the time evolution of the urine headspace, the defrosted samples were transferred into a series of vials, as previously described. The vials were balanced with purified air, stored at room temperature (24°C) and analyzed after certain periods of time. The time instants for measurements were defined as follows: the first sample was analyzed within 1 h after defrosting and the next ones after 12, 24, 48, 72 and 96 h of storage.

Headspace solid-phase microextraction HS-SPME was performed automatically using a multipurpose sampler MPS2 XL (Gerstel, Germany) operating in the SPME mode. Sample vials were incubated and stirred for 10 min at 37°C in the autosampler agitator. Since the purpose was to identify VOCs emitted by human urine being in “contact” with human body, the incubation and extraction steps were carried out at 37°C. CarboxenPDMS fiber was chosen for the SPME as it was reported 

to be the most efficient one in the analyses of urine volatiles (Mills and Walker, 2001). Extraction was achieved by inserting the 75 µm CAR-PDMS SPME fiber (Supelco, Canada) into the vials and exposing it to the headspace gas for 45 min. Subsequently, the fiber was immediately introduced into the inlet of the gas chromatograph where the compounds of interest were thermally desorbed at 290°C in the splitless mode (1 min). The fiber was conditioned at 290°C for 10 min, prior to each analysis.

Chromatographic analysis The analyses were performed using a GC/MS Agilent 7890/5975C (Agilent, Santa Clara, CA). During the desorption, the split/splitless inlet operated in the splitless mode (1 min), followed by the split mode at ratio 1:50. The injector temperature was maintained at 290°C during the whole analysis. The analytes under study were separated using a PoraBond Q column (25 m × 0.32 mm, film thickness 5 µm, Varian, USA) operated at constant flow (helium at 1.7 ml/min). This column was successfully used for the separation of VOCs during previous studies (Bajtarevic et al., 2009; Ligor et al., 2009). The column temperature program was as follows: 90°C for 7 min, increase to 140°C at a rate of 10°C/min, constant temperature of 140°C for 7 min, increase to 260°C at a rate of 15°C/min and 260°C for 12 min. The mass spectrometer worked in full scan mode with an associated scan range from m/z 20 to m/z 200. The quadrupole, ion source and transfer line were kept at 150°C, 230°C and 280°C, respectively. Data were collected with the sampling rate of 3.2 scans/s using the Agilent MSD Chemstation software. The identification of the compounds released from urine samples was performed in two steps. First, the peak spectrum was checked against NIST mass spectral library. Next, NIST spectrum identification was confirmed by the retention times obtained on the basis of standards prepared from pure compounds.

Results Method optimization The present study was focused on species spontaneously emitted by unmodified human urine. Consequently, any sample modification improving the headspace extraction like salt addition, pH adjustment, or sample heating could not be applied. Magnetic stirring (1200 rpm) was the only applied mean accelerating HS extraction. Thus, the optimization of the SPME procedure was restricted to the choice of extraction time and sample volume. Several extraction times (15 min, 30 min, 45 min and 60 min) were investigated. Taking into consideration the analysis time (sample throughput) and the extraction efficiency, 45 min-long extraction was found to be optimal for the majority of substances. This finding is consistent with earlier studies performed under similar conditions (Mills and Walker, 2001). Analogously, several phase ratios (urine volumes) were tested. A sample of 3 ml was

Toxicology Mechanisms and Methods

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

Role of urine scent profile in USaR applications  505 chosen, as it resulted in the same number of detected compounds as larger tested volumes. The stability of the urine VOCs during the storage at −80°C was assessed by analyzing the set of urine samples taken from one healthy volunteer. The analyses were performed as follows: the first one immediately after sampling, next ones after 1, 2, 4 and 7 days of storage. The urine species were found to be stable over the investigated time period with recoveries better than 80%; that is compliant with the results of other authors (Fustinoni et al., 1999; Statheropoulos et al., 2005; Smith et al., 2008). Approximately, 20 blanks were analysed (with deionized water instead of urine) to check the background of the method and to identify the possible contaminants originating from the applied materials. It has been found that the applied plastic monovettes release numerous hydrocarbons during the urine storage. Consequently, a monovettes cleaning procedure has been applied prior to the sampling (see Urine sampling and sample preparation section). The relative standard deviations (RSDs) were calculated on the basis of consecutive analyses of five separate urine samples taken from a single volunteer. The RSD values ranged from 2 to 8% for the majority of compounds. The only exception were sulfur compounds, with RSDs amounting to 10–12%. The limits of detections (LODs) were calculated on the basis of calibration curves obtained from measurements of urine samples spiked with certain aliquots of compounds under study (added mass range 3–50 ng). The correlation coefficients ranged from 0.93 to 0.99. The obtained values of LODs are presented in Table 2.

law constant and the environmental distribution of hydrophobic organic chemicals in biota is related to the octanol–water partition coefficient (Kow), which is an indicator of non-reactive toxicity and solubility of chemical compounds (Statheropoulos et al., 2007). The most represented chemical class within this group were ketones with ten representatives (acetone, 2-butanone, 3-methyl-2-butanone, 3-methyl-2-pentanone, 2-pentanone, 4-methyl-2-pentanone, 2-heptanone, 4-heptanone, 3-hexanone, 3-penten-2-one). The other well represented class was aldehydes comprising seven analytes (propanal, 2-methylpropanal, 2-methyl-butanal, 2-methyl-2-butenal, pentanal, hexanal and octanal). Within the remaining species, there were seven sulfurcontaining compounds (methanethiol, dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), allylisothiocyanate, isothiocyanocyclohexane and dimethyl sulfone (DMSO2)), one aromatic hydrocarbon (toluene), two esters (methyl acetate and ethyl acetate), one terpene (carvone) and four heterocycles (furan, 3-methyl-furan, pyrrole and 1-methyl-pyrrole). Aliphatic hydrocarbons were represented only by isoprene; however, their incidence could be underestimated due to their very low water solubility possibly causing their escape from liquid phase during urinating and sample preparation. Note that 17 compounds were found in all samples and further five substances in all samples except one, as shown in Table 2. Six compounds, namely acetone, 2-butanone, 4-heptanone, toluene, pyrrole and 2-pentanone, were also reported by Statheropoulos et al. (2005) as ubiquitous in human urine samples.

Ubiquitous VOCs in human urine

The main goal of this experiment was to investigate the changes in the emission of VOCs from human urine in the course of 4 days of storage at room temperature. The evolution, expressed as the median percentage change related to the results of the analysis performed immediately after the sample defrosting, is presented in Table 3. The possible trends in the release were evaluated using the bootstrap method (see Andrae, 2010) for a good illustration of the method). First, on the basis of the real data points obtained for each individual compound, 3000 slopes of the trend lines were calculated (linear increase was assumed). The trend was conceded as significant if the number of positive slopes in case of increase, or negative ones in case of decrease, was greater than the chosen confidence interval (0.95). Headspace levels of 26 omnipresent in urine compounds were found to increase in the course of the experiment. After 4 days, the increase ranged from 36% for isothiocyanocyclohexane to 525% for DMTS (median 252%). The highest rise after 96 h was noted for three sulfur species: methylmercaptane (322%), DMDS (499%) and DMTS (525%). Concentrations of 10 ketones were found to grow up with the highest increase observed for

A total of 148 volatile compounds were detected in the headspace of human urine samples. A compound was conceded as detected if the corresponding peak was at least three times higher than the baseline amplitude. More than 80% of analytes were identified not only by NIST spectral library but also by comparison of their retention times with an in-house developed retention time library based on reference standards. A typical SPME-GC-MS chromatogram is shown in Figure 1. The main purpose of this study was to find a set of VOCs that could be used to detect human presence during USaR operations. Consequently, the most ubiquitous VOCs released from human urine were targeted. Thus, the list of potential urine-born markers was restricted to the compounds which were detected in at least 80% of the samples. The threshold of 80% was arbitrarily chosen serving actually the highest ubiquitous possibility. 33 volatile organic compounds met this requirement. Their incidence and some physicochemical properties governing their behavior in the entrapment scene (e.g. absorption, solubility etc.) are presented in Table 2. The exchange rate of a chemical across the air-water interface is correlated by vapor pressure through Henry’s

Evolution of the urinary scent profile during urine storage at room temperature

© 2012 Informa Healthcare USA, Inc.

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

506  P. Mochalski et al. Table 2.  Incidence and selected chemical properties of VOCs released by unmodified human urine. Physicochemical data are from refs {Sangster, 1997 #31;Sander, 1999 #30;Lide, 2010 #23}. Data of two more ketones; cyclopentanone and 3-methylcyclopentanone were included for the reasons described in the text. Henry’s law Octanol-water Incidence n/20 LOD RSD Retention constant (25°C) partition coef. (%) [ng/ml] [%] Possible origin Compound CAS time [min] [mol×dm-3×atm-1] Log Kow (25°C) Methanethiol 74-93-1 4.43# 0.3 0.78* 20/20 (100) 300 10 systemic, bacterial, 0.2 1.34 20/20 (100) 15 2 smoking, dietary Furan 110-00-9 9.82# 13 0.59 19/20 (95) 5 6 systemic, dietary Propanal 123-38-6 9.94# 28 -0.24 20/20 (100) 6 2 systemic, dietary Acetone 67-64-1 10.08# 0.48 0.92* 19/20 (95) 10 11 systemic, dietary Dimethyl sulfide 75-18-3 10.86# 10 0.18 18/20 (90) 14 7 dietary, environmental Methyl acetate 79-20-9 11.6# 0.028 2.42 18/20 (90) 20 4 systemic Isoprene 78-79-5 12.64# 10 0.74* 20/20 (100) 16 2 systemic, dietary 2-Methylpropanal 78-84-2 14.1# 20 0.29 20/20 (100) 4 1 dietary 2-Butanone 78-93-3 15.42# 0.1 1.91* 20/20 (100) 12 7 smoking, dietary 3-Methylfuran 930-27-8 15.71# 7 0.73 16/20 (80) 12 7 dietary, environmental Ethyl acetate 141-78-6 16.84# 10 0.75 19/20 (95) 50 2 systemic, dietary Pyrrole 109-97-7 20.71# 7 0.84 20/20 (100) 4 4 dietary 3-Methyl-2-butanone 563-80-4 21.4# 5 1.23* 20/20 (100) 11 3 systemic, dietary 2-Methylbutanal 96-17-3 21.5# 16 0.91 20/20 (100) 5 2 dietary 2-Pentanone 107-87-9 21.99# 6 1.31* 19/20 (95) 19 4 systemic, dietary Pentanal 110-62-3 22.16# 1-Methyl-pyrrole 96-54-8 22.33 5.15* 1.21 16/20 (80) 8 systemic, dietary 1 1.77 20/20 (100) 4 6 dietary, bacterial Dimethyl disulfide 624-92-0 22.69# 45 1.15* 17/20 (85) 15 8 systemic, dietary 2-Methyl-2-butenal 1115-11-3 22.81# 30 0.52 19/20 (95) 2 5 dietary 3-Penten-2-one 625-33-2 22.89# 100 0.38 15/20 (75) 7 7 dietary Cyclopentanone 120-92-3 23.53# 0.15 2.73 20/20 (100) 2 5 environmental Toluene 108-88-3 23.97# 2.2 1.31 20/20 (100) 2 5 dietary 4-Methyl-2-pentanone 108-10-1 24.23# Allylisothiocyanate 57-06-7 24.28 4.15 2.15* 16/20 (80) 8 dietary 10 1.16* 20/20 (100) 5 3 dietary 3-Methyl-2-pentanone 565-61-7 24.36# 10 1.24* 20/20 (100) 2 2 dietary 3-Hexanone 589-38-8 24.71# 5 1.78 17/20 (85) 14 4 systemic, dietary Hexanal 66-25-1 24.97# Dimethyl sulfone 67-71-0 25.47 50000 -1.41 17/20 (85) 12 environmental 3-Methylcyclopentanone 1757-42-2 25.91 20* 1.05* 13/20 (65) 7 dietary 7 1.73* 20/20 (100) 7 1 environmental, dietary 4-Heptanone 123-19-3 26.45# 7 1.98 20/20 (100) 7 3 environmental 2-Heptanone 110-43-0 26.66# 0.54* 1.87* 18/20 (90) 20 12 dietary Dimethyl trisulfide 3658-80-8 27.24# 2 2.78* 17/20 (85) 6 5 systemic, dietary Octanal 124-13-0 28.51# Isothiocyanocyclohexane 1122-82-3 33.2 0.177* 3.57* 20/20 (100) 8 environmental 13* 2.71 17/20 (85) 15 3 dietary Carvone 2244-16-8 35.03# Values with asterisk are not experimental data, but calculated using EPI Suite software (by U. S. Environmental Protection Agency), # - RT values confirmed by standards.

3-hexanone (383%) and 4-methyl-2-pentanone (365%). Amongst the remaining compounds there were three aldehydes (2-methylpropanal, 2-methyl-butanal and pentanal), three heterocyclic compounds (1-methylpyrrole, furan and 3-methylfuran), carvone and both esters. Incidence of two compounds, not considered on the onset of experiment as the omnipresent species, cyclopentanone and 3-methylcyclopentanone, increased at the end of the investigated period and reached the threshold of 80%. Consequently, these compounds could also be considered as potential markers of human urine. The emission of only three compounds, namely DMSO2, octanal and propanal decreased within 4 days of storage. However, the drop was prominent only for 

DMSO2 (49%). Levels of all remaining compounds (acetone, toluene, DMS, isoprene, pyrrole and allylisothiocyanate), did not fulfill assumed criteria or remained stable. Note that urine samples stored at −80°C over the period of 2 weeks were stable and did not exhibit aforementioned trends. Exemplary trends in the headspace levels of DMDS, 3-hexanone, DMSO2 and acetone within the course of the experiment are presented in Figure 2.

Discussion The different body metabolites contain a wide range of VOCs, producing a variety of odors mainly affected by the different types and amounts of bacteria, oxygen Toxicology Mechanisms and Methods

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

Role of urine scent profile in USaR applications  507

Figure 1.  A typical chromatogram of SPME-GC-MS urine analysis.

availability, types and numbers of skin glands and secretions. Therefore, different groups of chemicals are emanated through the different body parts, creating an “aerial chemical fingerprint” (human scent profile), that may enable the location of entrapped victims under the ruins of collapsed buildings after an earthquake. The 33 omnipresent urine compounds can be considered as an initial library of potential markers to be used for the detection of entrapped persons. However, tracing their dispersion in the ruins of collapsed buildings after an earthquake can be a complicated task. VOCs are excreted in very low concentrations (ppt to ppb level) and their concentration is strongly dependent on entrapment volume, time of entrapment and victims medical condition. Furthermore, human scent is being enriched with volatiles associated to entrapment environment such as kitchen household emissions, dust particles, structure material emissions and with events including gas leaks, sewage emissions, smoldering fires etc. Consequently, it is necessary to select a set of VOCs that are representative for human presence. Health status, diet habit, physical stress and environmental exposure affect the volatile profile of urine samples. The origin of urinary constituents in many cases is still unclear and demands additional studies. On the other hand, ammonia, an omnipresent constituent of human urine, originates from the decomposition of urea. Moreover, many urinary VOCs originate from food and beverages consumption. Sulfur compounds like DMS, DMDS, DMTS and dimethylsulfone are relatively common in human urine (Wahl et al., 1999; Smith et al., 2008). However, their highly

reactive nature, manifested by absorptive and adsorptive features, can lead to significant losses during permeation through the debris. Urine sulfur species can have different origins. Endogenous production is ascribed to the metabolization of the sulfur-containing amino acids methionine and cysteine in the transamination pathway (Miekisch et al., 2001). Methanethiol is produced from methionine by the enzyme l-methionine γ-lyase and from H2S (product of cysteine metabolism) by the enzyme thiol S-methyltransferase. Thiol S-methyltransferase also forms DMS via the methylation of methyl mercaptane (Tangerman, 2009). These processes can be considered as a detoxification mechanism, removing toxic sulfur species (H2S and methanethiol) from tissues. DMS was also found to be the main cause of extra-oral halitosis (Tangerman, 2009). When present in human urine, DMDS can be the product of methanethiol oxidation, whereas dimethyl sulfone can be formed during oxidation of DMS (Tangerman, 2009). Urine dimethyl sulfone can also have exogenous origin. Its elevated concentrations were found in the urine of individuals exposed to dimethyl sulfoxide (DMSO) (Takeuchi et al., 2010). Considerable amounts of methanethiol and DMDS were found to be produced by the colonic flora (Hiele et al., 1991; Furne et al., 2001). Urinary sulfur compounds can also originate from dietary sources. DMDS, DMS and DMTS are present in numerous aliments (vegetables, cheese, fish, meat, baked goods) and beverages (coffee, wine, beer, milk) (Burdock, 2005). For example, significant amounts of methanethiol, DMS, DMDS and dimethyl sulfone were found in human urine after asparagus ingestion (Waring et al., 1987). Also allylisothiocyanate seems to be a food-related compound. It is a constituent of cruciferous vegetables (e.g. mustard,

© 2012 Informa Healthcare USA, Inc.

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

508  P. Mochalski et al. Table 3.  Time evolution of ubiquitous urinary species during storage at room temperature. Example: Ethyl acetate concentration increased by 1% after 12 h, whereas 2-methylpropanal increased by 70% after 12 h. Slope of trend line Relative HS concentration change (median, n = 20) [%] Compound CAS mean STD 12 h 24 h 48 h 72 h 96 h 2-Methylpropanal 78-84-2 2.05 0.35 70 104 140 211 179 2-Methylbutanal 96-17-3 2.68 0.64 81 148 202 278 229 Toluene 108-88-3 −0.06 0.06 10 −8 6 −1 4 3-Methylfuran 930-27-8 0.73 0.08 40 17 57 71 73 Furan 110-00-9 1.08 0.10 64 29 69 97 115 2-Heptanone 110-43-0 3.91 0.18 61 110 212 291 377 Acetone 67-64-1 −0.04 0.04 4 3 6 5 2 2-Butanone 78-93-3 2.43 0.21 62 94 154 182 225 4-Heptanone 123-19-3 3.49 0.12 80 123 217 282 349 3-Hexanone 589-38-8 4.13 0.18 76 112 236 310 383 2-Pentanone 107-87-9 2.74 0.15 71 109 176 209 252 3-Methyl-2-butanone 563-80-4 4.49 0.53 72 139 214 306 334 4-Methyl-2-pentanone 108-10-1 4.02 0.38 82 141 235 280 365 3-Methyl-2-pentanone 565-61-7 2.88 0.12 68 98 172 225 275 Dimethyl disulfide 624-92-0 5.75 0.42 154 193 372 474 499 Methanethiol 74-93-1 3.44 0.32 88 152 263 315 322 Isothiocyanocyclohexane 1122-82-3 0.38 0.06 11 7 18 36 36 Pentanal 110-62-3 1.58 0.29 81 135 148 177 237 Propanal 123-38-6 −0.25 0.20 53 57 63 47 63 Pyrrole 109-97-7 −0.15 0.04 9 6 −2 −7 −14 3-Penten-2-one 625-33-2 2.25 0.15 54 86 140 205 233 Dimethyl sulfide 75-18-3 0.28 0.18 5.13 −21 −18 −28 −21 Methyl acetate 79-20-9 1.78 0.43 23 54 57 44 106 Isoprene 78-79-5 −0.13 0.15 8 −34 −22 −31 −29 Dimethyl trisulfide 3658-80-8 5.35 0.47 85 196 341 515 525 Hexanal 66-25-1 0.51 0.40 50 75 83 88 137 Octanal 124-13-0 −0.41 0.12 15 15 0 −12 −31 2-Methyl-2-butenal 1115-11-3 0.51 0.14 11 23 42 49 56 Dimethyl sulfone 67-71-0 −0.39 0.06 −34 −34 −39 −56 −49 Carvone 99-49-0 2.08 0.45 57 98 191 247 289 Ethyl acetate 141-78-6 0.72 0.25 1 4 10 21 20 1-Methyl-pyrrole 96-54-8 3.14 0.40 58 60 96 175 371 Allylisothiocyanate 57-06-7 0.07 0.16 1 2 3 4 11 Cyclopentanone 120-92-3 1.11 0.39 24 37 60 53 74 3-Methylcyclopentanone 1757-42-2 3.31 0.80 70 136 210 206 337

horseradish) and occurs commonly in the human diet as flavoring agent. Elevated levels of allylisothiocyanate were detected in the urine of individuals after ingestion of mustard (Jiao et al., 1994). The ketones mentioned in Table 2 can originate from decarboxylation of oxo-acids (Mills and Walker, 2001), as well as, from exogenous sources. Acetone (2-propanone) is the major ketone contained in exhaled human breath (Schwarz et al., 2009), blood (Miekisch et al., 2001; O’Hara et al., 2009) and urine (Mills and Walker, 2001; Smith et al., 2008). It is formed in hepatocytes from acetoacetate during decarboxylation of excess Acetyl–CoA (Schwarz et al., 2009). Urine 4-heptanone is most probably of exogenous origin. It is supposed to be a product of β-oxidation of 2-ethylhexanoic acid, a metabolic product of the plasticizer di-(2-ethylhexyl)-phthalate (Walker and Mills, 2001; Wahl et al., 2004). 2-heptanone was detected in urine of employees exposed to heptane in shoe and tire 

factories and was proposed to be one of the n-heptane metabolites (Perbellini et al., 1986). All detected ketones are omnipresent in beverages (beer, wine, rum, whisky, coffee, tea) (Tressl et al., 1978; Yannai 2004; Burdock, 2005), numerous food categories and particularly in fruit, vegetables, cheese, milk, meat and bread (Yannai 2004; Burdock, 2005). They are also used in small amounts as flavoring ingredients (Ziegler, 2007). Urine aldehydes can be both of endogenous and exogenous origin. In biological systems, they are produced during lipid peroxidation by the so-called β-cleavage reaction of lipid alkoxyl radicals (Kim et al., 1999; Draper et al., 2000). In animal tissues (and also in food), numerous alkanals and alkenals are generated during the oxidation of ω-6 and ω-3 polyunsaturated fatty acids (e.g. hexanal from ω-6 PUFA) (Kim et al., 1999). Aldehydes are also common constituents of natural flavors of numerous foods and beverages (Yannai 2004; Burdock, 2005). Toxicology Mechanisms and Methods

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

Role of urine scent profile in USaR applications  509

Figure 2.  Evolution of the emission of dimethyldisulfide, 3-hexanone, DMSO2 and acetone.

Hexanal, for instance, was found in more than 300 food products including vegetables, fruits, cheeses, coffee, milk, bread, butter and meats (Burdock, 2005). Lowered aldehydes levels in urine of fasting rats suggest that at least part of urine aldehydes is of dietary origin (Kim et al., 1999). Found in all samples, furan and its derivative 3-methylfuran, seem to be of exogenous origin. These heterocyclic compounds can originate from cigarette smoke and are commonly present in the breath of smokers and passive smokers (Buszewski et al., 2008). Significant amounts of furan are also present in jarred and canned food, dry products (e.g. snacks, bread crust) and roasted coffee beans (Zoller, 2007). Coffee drinking seems to be of particular importance as the furan levels in coffee brews are especially high. How pyrroles are produced in human body and appear in the urine is still unclear. The endogenous pyrroles can come from metabolization of aminosugars and N-acetylneurominic acid in the central nervous system. They can also be a by-product in the synthesis of porphyrins, bile pigment, or an oxidation product of hemopyrrole and bilirubin (Jackson et al., 1997; Jackson et al., 2000). Urine pyrroles can also have exogenous origin. They are present in numerous beverages (coffee, tea, beer) and thermally treated meat (boiled, roasted and fried chicken, beef, pork) (Burdock, 2005). Isoprene (2-methyl-1,3-butadiene) is the most abundant hydrocarbon present in exhaled breath (Buszewski et al., 2008; Kushch et al., 2008; King et al., 2009; Ligor et al., 2009; King et al., 2010) and blood (Miekisch et al., 2001; O’Hara et al., 2009). Isoprene present in human tissues is of endogenous origin; however, its metabolic

pathways are still uncertain. It is commonly accepted that isoprene is a by-product of cholesterol biosynthesis (Kushch et al., 2008). Two omnipresent esters, methyl acetate and ethyl acetate, are constituents of natural aromas and ubiquitous in fruit, vegetables, beverages (beer, coffee, tea) and cheese (Burdock, 2005). Both esters are also used as solvents in glues, paints and perfumes, thus indoor or urban air cannot be excluded as exogenous source (Kostiainen, 1994). Toluene and isothiocyano-cyclohexane are of exogenous origin. Isothiocyano-cyclohexane is commonly used by car industry and in insulation materials and occurs in urban and industrial air (Gallego et al., 2007). Toluene is also used as solvent in the paint industry and is present in paints and varnishes (Tsujimoto et al., 2005). Consequently, they are environmental contaminants present in indoor air (Kostiainen, 1994). Human urine scent profile appears to be changing over time. This is probably the result of a number of factors, mainly including intense bacterial activity and metabolism, pH changing, modifications in urine characteristics (affecting for example the VOCs solubility, presence of photo-chemically degradable VOCs) and decomposition of urine constituents (like in case of ammonia produced during urea decay). Moreover, the production of some VOCs, as a result of reactions between urine constituents, cannot be excluded. Regardless for the reason of this increase, it can be considered as a favorable effect improving the VOCs detection. In this context, ketones (e.g. 3-hexanone, 2-heptanone, 4-heptanone, 4-methyl2-pentanone), showing the highest increase in emission during storage, seem to be the most promising markers of human urine. However, in real situations, the presence of water, oxygen and light might interact with the evolved

© 2012 Informa Healthcare USA, Inc.

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

510  P. Mochalski et al. VOCs, affecting the stability, reactivity and in general their chemical evolution in time. In the present study, the identification of potential urine “markers” of human presence was performed using HS-SPME-GC-MS analysis, a powerful, reliable and selective technique. On the other hand, this method is considered time- and effort-consuming and can usually be performed in laboratory conditions by well-trained personnel. Consequently, further investigation is necessary prior selecting reliable and selective field chemical methods for the early location of entrapped victims. In the near future, nanomaterials and microfabrication, are expected to play a fundamental role in the advancements of gas sensors. Driving elements in the field, are the development of improved components such as micro electro mechanical systems (MEMS) and silicon components, micro gas chromatography (micro GC), micro mass spectrometers, micro IMS, micro spectrophotometers, quartz micro balance (QMB), surface and bulk acoustic wave (SAW and BAW) devices, sensor arrays and electronic noses (Hodgkinson et al., 2006). Therefore, gas sensing technology is expected to play a dominant role in the on-line identification of human scent profile in the disaster zone.

Conclusion The main goal of this study was to select a preliminary set of VOCs spontaneously released from human urine, suitable as markers for the presence of entrapped persons. The ideal urine marker should be relatively volatile, non-reactive and omnipresent at relatively high concentrations to ensure its certain detection in the proximity of entrapped persons. A total number of 33 species were found to be ubiquitous in the headspace of unmodified human urine at human body temperature. Their incidence in the individual urine samples was higher than 80%. This group should be considered as a basis for further field studies involving portable, highly sensitive and selective instruments. Additionally, the influence of storage time on urine VOCs samples was investigated at room temperature. A considerable increase in the headspace levels of 26 compounds was noted. Only 3 species exhibited significant drop of the release. The increase in the secretion of omnipresent urine VOCs can be regarded as a favorable phenomenon improving their detection after longer storage times. Further investigations involving portable, rapid, sensitive and selective detection techniques are necessary to confirm the usefulness of the proposed volatiles for the early location of entrapped victims. These studies should be also focus on the interactions with the debris materials (e.g. dust, cement, wood, plastic, glass) and the mixing of scent profile with environmental contaminants.



Declaration of interest The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007-13) under grant agreement No. 217967 (“SGL for USaR” project, Second Generation Locator for Urban Search and Rescue Operations, www.sgl-eu.org). We appreciate funding from the Austrian Federal Ministry for Transport, Innovation and Technology (BMVIT/BMWA, project 818803, KIRAS). We greatly appreciate the generous support of the government of Vorarlberg and its governor Landeshauptmann Dr. Herbert Sausgruber. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References Andrae R. 2010. Error estimation in astronomy. A guide, http://arxiv. org/abs/1009.2755. Bajtarevic A, Ager C, Pienz M, Klieber M, Schwarz K, Ligor M, Ligor T, Filipiak W, Denz H, Fiegl M, Hilbe W, Weiss W, Lukas P, Jamnig H, Hackl M, Haidenberger A, Buszewski B, Miekisch W, Schubert J, Amann A. 2009. Noninvasive detection of lung cancer by analysis of exhaled breath. BMC Cancer 9:348. Burdock GA. 2005. Fenaroli’s handbook of flavor ingredients, Boca Raton, London, New York Washington DC, CRC Press. Buszewski B, Ulanowska A, Ligor T, Denderz N, Amann A. 2008. Analysis of exhaled breath from smokers, passive smokers and non-smokers by solidphase microextraction gas chromatography mass spectrometry. Biomedical Chromatography 23:551–556. Centre for research on the epidemiology of disasters 2003. Dalton L. 2003. Chemical analysis of a disaster. Chemical & Engineering News 81:26–30. Dalton PH, Opiekun RE, Gould M, McDermott R, Wilson T, Maute C, Ozdener MH, Zhao K, Emmett E, Lees PS, Herbert R, Moline J. 2010. Chemosensory loss: functional consequences of the world trade center disaster. Environ Health Perspect 118:1251–1256. Draper HH, Csallany AS, Hadley M. 2000. Urinary aldehydes as indicators of lipid peroxidation in vivo. Free Radic Biol Med 29:1071–1077. Ferworn A. 2009. Canine augmentation technology for Urban Search and Rescue. In: Helton WS. (ed.) Canine Ergonomics: The Science of Working Dogs. Boca Raton: CRC Press. Furne J, Springfield J, Koenig T, DeMaster E, Levitt MD. 2001. Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa. Biochem Pharmacol 62:255–259. Fustinoni S, Giampiccolo R, Pulvirenti S, Buratti M, Colombi A. 1999. Headspace solid-phase microextraction for the determination of benzene, toluene, ethylbenzene and xylenes in urine. J Chromatogr B Biomed Sci Appl 723:105–115. Gallego E, Roca FX , Perales F , Ribes A , Carrera G , Guardino X, Berenguer MJ. 2007. Isocyanatocyclohexane and isothiocyanatocyclohexane levels in urban and industrial areas and possible emission-related activities. Atmospheric Environment 41:8228–8240. Gwaltney-Brant SM, Murphy LA, Wismer TA, Albretsen JC. 2003. General toxicologic hazards and risks for search-and-rescue dogs responding to urban disasters. J Am Vet Med Assoc 222:292–295. Hiele M, Ghoos Y, Rutgeerts P, Vantrappen G, Schoorens D. 1991. Influence of nutritional substrates on the formation of volatiles by the fecal flora. Gastroenterology 100:1597–1602.

Toxicology Mechanisms and Methods

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by ETH Zuerich on 08/23/12 For personal use only.

Role of urine scent profile in USaR applications  511 Hodgkinson J, Saffell J, Luff J, Shaw J, Ramsden J, Huggins C, Bogue R, Carline R. 2006. MNT Gas Sensor Roadmap, Ver.3.0. MNT Gas Sensor Forum. Jackson JA, Riordan HD, Neathery SS, Mayer K. 2000. Urine Pyrrolles Revisited. Journal of Orthomolecular Medicine 15:47–48. Jackson JA, Riordan HD, Neathery SS, Riordan NH . 1997. Urinary Pyrrole in Health and Disease.Journal of Orthomolecular Medicine 12:96–98. Jiao D, Ho CT, Foiles P, Chung FL. 1994. Identification and quantification of the N-acetylcysteine conjugate of allyl isothiocyanate in human urine after ingestion of mustard. Cancer Epidemiol Biomarkers Prev 3:487–492. Kim SS, Gallaher DD, Csallany AS. 1999. Lipophilic aldehydes and related carbonyl compounds in rat and human urine. Lipids 34:489–496. King J, Koc H, Unterkofler K, Mochalski P, Kupferthaler A, Teschl G, Teschl S, Hinterhuber H, Amann A. 2010. Physiological modeling of isoprene dynamics in exhaled breath. J Theor Biol 267:626–637. King J, Kupferthaler A, Unterkofler K, Koc H, Teschl S, Teschl G, Miekisch W, Schubert J, Hinterhuber H, Amann A. 2009. Isoprene and acetone concentration profiles during exercise on an ergometer. J Breath Res 3:027006. Kostiainen R. 1994. Volatile Organic Compounds in the Indoor Air of Normal and Sick Houses. Atmospheric Environment 29:693–702. Kushch I, Arendacká B, Stolc S, Mochalski P, Filipiak W, Schwarz K, Schwentner L, Schmid A, Dzien A, Lechleitner M, Witkovský V, Miekisch W, Schubert J, Unterkofler K, Amann A. 2008. Breath isoprene–aspects of normal physiology related to age, gender and cholesterol profile as determined in a proton transfer reaction mass spectrometry study. Clin Chem Lab Med 46:1011–1018. Lide DR. (ed.) 2010. CRC Handbook of Chemistry and Physics: Taylor & Francis. Ligor M, Ligor T, Bajtarevic A, Ager C, Pienz M, Klieber M, Denz H, Fiegl M, Hilbe W, Weiss W, Lukas P, Jamnig H, Hackl M, Buszewski B, Miekisch W, Schubert J, Amann A. 2009. Determination of volatile organic compounds in exhaled breath of patients with lung cancer using solid phase microextraction and gas chromatography mass spectrometry. Clin Chem Lab Med 47:550–560. Miekisch W, Schubert JK, Vagts DA, Geiger K. 2001. Analysis of volatile disease markers in blood. Clin Chem 47:1053–1060. Mills GA, Walker V. 2001. Headspace solid-phase microextraction profiling of volatile compounds in urine: application to metabolic investigations. J Chromatogr B Biomed Sci Appl 753:259–268. Murphy LA, Gwaltney-Brant SM, Albretsen JC, Wismer TA. 2003. Toxicologic agents of concern for search-and-rescue dogs responding to urban disasters. J Am Vet Med Assoc 222:296–304. O’Hara ME, Clutton-Brock TH, Green S, Mayhew CA. 2009. Endogenous volatile organic compounds in breath and blood of healthy volunteers: examining breath analysis as a surrogate for blood measurements. J Breath Res 3:027005. Perbellini L, Brugnone F, Cocheo V, De Rosa E, Bartolucci GB. 1986. Identification of the n-heptane metabolites in rat and human urine. Arch Toxicol 58:229–234. Rudnicka J, Mochalski P, Agapiou A, Statheropoulos M, Amann A, Buszewski B. 2010. Application of ion mobility spectrometry for the detection of human urine. Anal Bioanal Chem 398:2031–2038. Sander R, 1999. Compilation of Henry’s Law Constants for Inorganic and Organic Species of Potential Importance in Environmental Chemistry (Version 3). http://www.henrys-law.org Sangster J. 1997. Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, Chichester, New York, Weinheim, Brisbane, Singapore, Toronto, John Wiley & Sons Ltd. Schwarz K, Pizzini A, Arendacká B, Zerlauth K, Filipiak W, Schmid A, Dzien A, Neuner S, Lechleitner M, Scholl-Bürgi S, Miekisch

W, Schubert J, Unterkofler K, Witkovský V, Gastl G, Amann A. 2009. Breath acetone-aspects of normal physiology related to age and gender as determined in a PTR-MS study. J Breath Res 3:027003. Smith S, Burden H, Persad R, Whittington K, de Lacy Costello B, Ratcliffe NM, Probert CS. 2008. A comparative study of the analysis of human urine headspace using gas chromatography-mass spectrometry. J Breath Res 2:037022. Statheropoulos M, Agapiou A, Spiliopoulou C, Pallis GC, Sianos E. 2007. Environmental aspects of VOCs evolved in the early stages of human decomposition. Sci Total Environ 385:221–227. Statheropoulos M, Sianos E, Agapiou A, Georgiadou A, Pappa A, Tzamtzis N, Giotaki H, Papageorgiou C, Kolostoumbis D. 2005. Preliminary investigation of using volatile organic compounds from human expired air, blood and urine for locating entrapped people in earthquakes. J Chromatogr B Analyt Technol Biomed Life Sci 822:112–117. Takeuchi A, Yamamoto S, Narai R, Nishida M, Yashiki M, Sakui N, Namera A. 2010. Determination of dimethyl sulfoxide and dimethyl sulfone in urine by gas chromatography-mass spectrometry after preparation using 2,2-dimethoxypropane. Biomed Chromatogr 24:465–471. Tangerman A. 2009. Measurement and biological significance of the volatile sulfur compounds hydrogen sulfide, methanethiol and dimethyl sulfide in various biological matrices. J Chromatogr B Analyt Technol Biomed Life Sci 877:3366–3377. Tressl R, Friese L, Fendesack F, Koppler H. 1978 . Gas ChromatogrephicMass Spectrometric Investigation of Hop Aroma Constituents in Beer. J Agric Food Chem 26:1422–1426. Tsujimoto Y, Munehiro W, Nam VD, Noda T, Shimizu M, Yamaguchi Y, Moriwaki H, Morimoto T, Kakiuchi K, Maeda Y, Tanaka M. 2005. Determination of Urinary Phenolic Metabolites from Rats Treated with 1,2,3 and 1,3,5- Trimethylbenzenes. J Occup Health 47:337–339. United States geological survey. 2003. United States Geological Survey. Available: www.usgs.gov. Wahl HG, Hoffmann A, Luft D, Liebich HM. 1999. Analysis of volatile organic compounds in human urine by headspace gas chromatography-mass spectrometry with a multipurpose sampler. J Chromatogr A 847:117–125. Wahl HG, Hong Q, Hildenbrand S, Risler T, Luft D, Liebich H. 2004. 4-Heptanone is a metabolite of the plasticizer di(2-ethylhexyl) phthalate (DEHP) in haemodialysis patients. Nephrol Dial Transplant 19:2576–2583. Walker V, Mills GA. 2001. Urine 4-heptanone: a beta-oxidation product of 2-ethylhexanoic acid from plasticisers. Clin Chim Acta 306:51–61. Waring RH, Mitchell SC, Fenwick GR. 1987. The chemical nature of the urinary odour produced by man after asparagus ingestion. Xenobiotica 17:1363–1371. Wong J, Robinson C. 2004. Urban search and rescue technology needs: identification of needs. Federal Emergency Management Agency (FEMA) and the National Institute of Justice (NIJ). Document number 207771. Yannai S. (ed.) 2004. Dictionary of food compounds with CD-ROM. Additives, Flavors, and Ingredients: Chapman & Hall/CRC. Ziegler H. (ed.) 2007. Flavourings. Production, Composition, Applications, Regulations., Weinheim: WILEY-VCH Verlag GmbH & Co. KGaA. Zoller O, Sager F, Reinhard H. 2007. Furan in food: headspace method and product survey. Food Addit Contam 24 Suppl 1:91–107.

© 2012 Informa Healthcare USA, Inc.