Search

Background: There is a need to develop accurate and reliable non-invasive methods to evaluate chronic kidney disease (CKD) status and assess disease progression. Given it is recognized that dysregulation in metabolic pathways occur from early CKD, there is a basis in utilizing metabolomic biomarkers to monitor CKD progression. Volatile Organic Compounds (VOCs), a form of metabolomic biomarker, are gaseous products of metabolic processes in organisms which are typically released with greater abundance in disease conditions when there is dysregulation in metabolism. How urinary VOCs reflect the abnormal metabolic profile of patients with CKD status is unknown. Our study aimed to explore this., Methods: Individuals aged 18-75 years undergoing kidney biopsy were included. Pre-biopsy urine samples were collected. All biopsy samples had an interstitial fibrosis and tubular atrophy (IFTA) grade scored by standardized assessment. Urine supernatant was extracted from residue and sampled for stir bar sorptive extraction followed by Gas chromatography-mass spectrometry (GC-MS) analysis. Post-processing of GC-MS data separated complex mixtures of VOCs based on their volatility and polarity. Mass-to-charge ratios and fragment patterns were measured for individual VOCs identification and quantification. Linear discriminant analysis (LDA) was performed to assess the ability of urinary VOCs in discriminating between IFTA 0 ('no or minimal IFTA' i.e. <10%, IFTA), IFTA 1 ('mild IFTA' i.e. 10-25% IFTA) and IFTA ≥ 2 ('moderate or severe IFTA' i.e. >25% IFTA). Linear regression analysis adjusting for age, sex, estimated glomerular filtration rate, diabetes mellitus (DM) status, and albuminuria was conducted to determine significantly regulated urinary VOCs amongst the groups., Results: 64 study participants (22 individuals IFTA 0, 15 individuals IFTA 1, 27 individuals IFTA ≥ 2) were included. There were 34 VOCs identified from GC-MS which were statistically associated with correct classification between the IFTA groups, and LDA demonstrated individuals with IFTA 0, IFTA 1 and IFTA ≥ 2 could be significantly separated by their urinary VOCs profile (p < 0.001). Multivariate linear regression analysis reported 4 VOCs significantly upregulated in the IFTA 1 compared to the IFTA 0 group, and 2 VOCs significantly upregulated in the IFTA ≥ 2 compared to the IFTA 1 group (p < 0.05). Significantly upregulated urinary VOCs belonged to one of four functional groups - aldehydes, ketones, hydrocarbons, or alcohols., Conclusions: We report novel links between urinary VOCs and tubulointerstitial histopathology. Our findings suggest the application of urinary VOCs as a metabolomic biomarker may have a useful clinical role to non-invasively assess CKD status during disease progression., (© 2024. Crown.)

With increasing prevalence rate of depression by years, more attention has been paid to the influence of environmental pollutants on depression, but relationship between exposure to volatile organic compounds (VOCs) and depression is rarely studied. Therefore, this cross-sectional study use the National Center for Health Statistics (NHANES) database (2013-2016 years) to explore association between exposure to multiple VOCs and depression in general population. Multiple linear and logistic regression models were used to analyze the association between urinary VOC metabolism (mVOCs) and depression. To further analyze effect of multiple mVOCs mixed exposure, Bayesian kernel machine regression (BKMR) models were performed. A total of 3240 participants and 16 mVOCs were included in the analysis. Results showed that 10 mVOCs exposure were positively correlated with depression by multiple linear and logistic regression models, especially CYMA and MHBMA3, which also showed significant positive association with depression in BKMR model. Mixed exposure of multiple mVOCs was significantly positively correlated with depression. Gender differences were existed in effects of some VOCs concentrations on depression. AAMA, CYMA and MA had significant positive correlations with depression by women, and DHBMA had significant positive correlations with depression by men. Hence, this study showed that exposing to VOCs might have negative impacts on depression, and impact of CYMA and MHBMA3 on depression may be more evident, which provide new ideas for prevention and control of depression. But further research and exploration are needed to clarify the mechanism and influence factors of this relationship, to demonstrate the reliability of these relationship., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

While evidence indicates that exposure to oil spill incidents can affect mental health, it is unclear whether the mental health effects result from the incident itself or from exposure to associated chemicals. Oil contains chemicals that can impact mental health and these chemicals may have long-term effects due to their persistence in the environment. To address the gap in current knowledge, we conducted cross-sectional and prospective analyses of data from adults who participated in the Health Effects of the Hebei Spirit Oil Spill study. To assess chemical exposure from oil spills, we used indirect exposure indicators such as distance from the contaminated oil band to residences and duration of clean-up work, along with direct exposure indicators such as urine metabolite concentrations of volatile organic compounds and polycyclic aromatic hydrocarbons. Mental health assessments covered posttraumatic stress disorder (PTSD), depression, state anxiety, and trait anxiety. In the cross-sectional analyses, all four mental health issues were found to be associated with proximity to the oil band (p-value<0.05) and showed a positive association with clean-up work duration (p-value<0.05). Cox regression analysis revealed that higher urinary t, t-muconic acid levels were associated with an increased risk of depression (Hazard Ratio [HR] = 1.55, 95 % Confidence Interval [CI] = 1.05-2.28), and elevated 1-hydroxypyrene levels increased the risk of PTSD (HR = 1.60, 95 % CI = 1.03-2.48). Additionally, higher urinary 2-naphthol levels were associated with increased state anxiety (HR = 1.39, 95 % CI = 1.00-1.93) and trait anxiety (HR = 1.64, 95 % CI = 1.15-2.32). These associations persisted even after controlling for distance and duration variables related to psychosocial exposure. Our findings suggest that environmental disaster response plans should prioritize minimizing chemical exposure while also considering the duration and nature of the mental health impacts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Epidemiological evidence emphasizes air pollutants' role in chronic kidney disease (CKD). Volatile organic compounds (VOCs) contribute to air pollution, yet research on VOCs and kidney damage, especially gender disparities, is limited., Methods: This study analyzed NHANES data to explore associations between urinary VOC metabolite mixtures (VOCMs) and key kidney-related parameters: estimated glomerular filtration rate (eGFR), albumin-to-creatinine ratio (ACR), chronic kidney disease (CKD), and albuminuria. Mediation analyses assessed the potential mediating roles of biological aging (BA) and serum albumin in VOCM mixtures' effects on kidney damage. Sensitivity analyses were also conducted., Results: The mixture analysis unveiled a noteworthy positive association between VOCM mixtures and the risk of developing CKD, coupled with a significant negative correlation with eGFR within the overall participant cohort. These findings remained consistent when examining the female subgroup. However, among male participants, no significant link emerged between VOCM mixtures and CKD or eGFR. Furthermore, in both the overall and female participant groups, there was an absence of a significant correlation between VOCM mixtures and either ACR or albuminuria. On the other hand, in male participants, while no significant correlation was detected with albuminuria, a significant positive correlation was observed with ACR. Pollutant analysis identified potential links between kidney damage and 1,3-butadiene, toluene, ethylbenzene, styrene, xylene, acrolein, crotonaldehyde and propylene oxide. Mediation analyses suggested that BA might partially mediate the relationship between VOCM mixtures and kidney damage., Conclusion: The current findings highlight the widespread exposure to VOCs among the general U.S. adult population and indicate a potential correlation between exposure to VOC mixtures and compromised renal function parameters, with notable gender disparities. Females appear to exhibit greater sensitivity to impaired renal function resulting from VOCs exposure. Anti-aging treatments may offer some mitigation against kidney damage due to VOCs exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nitrosamines and semi-volatile organic compounds (SVOCs) are carcinogenic contaminants in water and biological matrices. Conventional analytical methods often struggle to detect trace concentrations due to poor extraction efficacies. This study presents a novel, low-cost, in-syringe-assisted fast extraction cum cleanup technique coupled with GC-FID for monitoring four nitrosamines and two SVOCs in drinking water and human urine samples to measure the contamination and exposure levels. This extraction protocol combines a novel green in-syringe liquid-liquid extraction step using dimethyl carbonate as the green extraction solvent, coupled with a semi-automated solid-phase extraction cleanup process. Then, the final extractant is analyzed using gas chromatography-flame ionization detection (GC-FID) for monitoring. The method demonstrated excellent linearity (R 2  > 0.998) between 1.5 and 500 ng mL⁻ 1 for all six target compounds. Detection limits ranged from 1.0 to 2.0 ng mL⁻ 1 . Extraction recoveries were between 87 and 105% for both urine samples and water samples. Intra-day and inter-day precision were below 9% RSD. The blue applicability grade index evaluation scored 70.0, indicating good practical applicability. The developed analytical protocol offers a sensitive, accurate, low-cost, rapid, and environmentally friendly method for simultaneously quantifying multiple nitrosamines and SVOCs in environmental and human samples. Its performance characteristics and sustainability metrics suggest the potential for broad application in monitoring and exposure studies., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Phenylketonuria (PKU) is one of the most common genetic metabolic diseases, especially among newborns. Traditional clinical examination of newborn blood samples for PKU is invasive, laborious, and limited to hospitals and healthcare facilities. We reported herein a SERS-based sensor array with three thiophenolic nanoreceptors built on a patterned nanorod vertical array for rapid and inexpensive detection of characteristic volatile biomarkers indicative of PKU in the urine and accurate classification of newborn baby patients all performed on a hand-held SERS spectrophotometer. The well-ordered array was generated from the volatility-driven assembly of gold nanorods (AuNRs) into an upright and closely packed hexagonal configuration. The uniformly distributed nanowells between AuNRs offered an intense and aspect-ratio-dependent plasmonic field for the molecular enhancement of SERS outputs. The SERS-based detector was integrated into a test chip for regular monitoring of volatile phenylketone bodies in the spiked solution or patients' urine within 5 min, allowing the quantification of a wide variety of normal or abnormal metabolites at their physiologically relevant concentration range. The detection limits for common biomarkers of PKU, including phenylpyruvic acid, 4-hydroxyphenylacetic acid, and phenylacetic acid, were at a few μM and well below the diagnostic thresholds. Moreover, the volatile headspace mixtures from a given urine sample could be fingerprinted by the sensor array and discriminated using machine-learning algorithms. Ultimately, the discrimination of baby patients among 26 cases of mild and classic PKU phenotypes and 17 cases of healthy volunteers could be realized with an overall accuracy of 97%. This hand-held SERS platform plays a pivotal role in advancing healthcare applications in quick screening of neonatal PKU through a facile urinary vapor test.

Northeastern British Columbia is a region of prolific unconventional oil and gas (UOG) activity. UOG activity can release volatile organic compounds (VOCs) which can elevate oxidative stress and disrupt antioxidant activity in exposed pregnant individuals, potentially increasing the risk of adverse pregnancy outcomes. This study measured biomarkers of oxidative stress and antioxidant activity in pooled urine samples of 85 pregnant individuals living in Northeastern British Columbia, to analyze associations between indoor air VOCs, oil and gas well density and proximity metrics, and biomarker concentrations. Concentrations of catalase, superoxide dismutase (SOD), glutathione S-transferase, total antioxidant capacity, 6-hydroxymelatonin sulfate (aMT6s), malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and 8-isoprostane were measured using assay kits. Associations between exposure metrics and biomarker concentrations were determined using multiple linear regression models adjusted for biomarker-specific covariables. UOG proximity was associated with decreased SOD and 8-OHdG. Decreased 8-OHdG was associated with increased proximity to all wells. Decreased aMT6s were observed with increased indoor air hexanal concentrations. MDA was negatively associated with indoor air 1,4-dioxane concentrations. No statistically significant associations were found between other biomarkers and exposure metrics. Although some associations linked oil and gas activity to altered oxidative stress and antioxidant activity, the possibility of chance findings due to the large number of tests cannot be discounted. This study shows that living near UOG wells may alter oxidative stress and antioxidant activity in pregnant individuals. More research is needed to elucidate underlying mechanisms and to what degree UOG activity affects oxidative stress and antioxidant activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology.)

Introduction: Metal carpentry includes a wide range of work activities such as welding and cutting metallic components, use of solvents and paints. Therefore, the employees in these types of activities are mainly exposed to welding fumes and volatile organic solvents. Here, we present an NMR-based metabolomic approach for assessing urinary profiles of workers in the same company that are exposed to two different risk factors., Methods: The study enrolled 40 male subjects exposed to welding fumes, 13 male subjects exposed to volatile organic compounds of a metal carpentry company, and 24 healthy volunteers. All samples were collected, in the middle of the working week at fast. Thirty-five urinary metabolites belonging to different chemical classes such as amino acids, organic acids and amines were identified and quantified. Results were processed by multivariate statistical analysis for identifying significant metabolites for each working group examined, compared to controls., Results: Workers exposed to welding fumes displayed urinary increase in glutamine, tyrosine, taurine, creatine, methylguanidine and pseudouridine associated to oxidative impairment, while workers exposed to volatile organic compounds showed higher urinary levels of branched chain aminoacids., Conclusion: Our work identified specific urinary profile related to each occupational exposure, even if it is below the threshold limit values., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 De Rosa, Giampaoli, Sciubba, Marini, Tranfo, Sisto, Miccheli, Tricarico, Fetoni and Spagnoli.)

Background: We investigated the correlation between urine VOC metabolites and airway function in children exposed to anthropogenic volatile organic compounds (VOCs), notable pollutants impacting respiratory health., Methods: Out of 157 respondents, 141 completed skin prick tests, spirometry, IOS, and provided urine samples following the International Study of Asthma and Allergies in Childhood (ISAAC)-related questions. Allergic sensitization was assessed through skin prick tests, and airway functions were evaluated using spirometry and impulse oscillometry (IOS). Forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) was recorded and FEV1/FVC ratio was calculated. Airway mechanics parameters including respiratory resistance at 5 Hz (Rrs5) mean respiratory resistance between 5 Hz and 20 Hz (Rrs5-20), were also recorded. Urine concentrations of metabolites of benzene, ethylbenzene, toluene, xylene, styrene, formaldehyde, carbon-disulfide were analyzed by gas chromatography/tandem mass spectroscopy., Results: The median age at study participation was 7.1 (SD 0.3) years. Muconic acid (benzene metabolites) and o-methyl-hippuric acid (xylene metabolites) above medians were associated with a significant increase in Rrs5 (muconic acid: aβ = 0.150, p  = .002; o-methyl-hippuric acid: aβ = 0.143, p  = .023) and a decrease in FEV1/FVC (o-methyl-hippuric acid: aβ = 0.054, p  = .028) compared to those below median. No associations were observed for Rrs5-20 and FEV1 between the groups categorized as above and below the median (all parameter p values > .05)., Conclusions: Elevated levels of benzene and xylene metabolites were associated with a significant increase in Rrs5 and a decrease in FEV1/FVC, related to increased resistance and restrictive lung conditions compared to individuals with concentrations below the median.

Evidence on the association of volatile organic compounds (VOCs) with chronic bronchitis (CB) and emphysema is spare and defective. To evaluate the relationship between urinary metabolites of VOCs (mVOCs) with CB and emphysema, and to identify the potential mVOC of paramount importance, data from NHANES 2011-2014 waves were utilized. Logistic regression was conducted to estimate the independent association of mVOCs with respiratory outcomes. Least absolute shrinkage and selection operator (LASSO) regression was performed to screen a parsimonious set of CB- and emphysema-relevant mVOCs that were used for further co-exposure analyses of weighted quantile sum (WQS) regression and Bayesian kernel machine regression (BKMR). Mediation analysis was employed to detect the mediating role of inflammatory makers in such associations. In single exposure analytic model, nine mVOCs were individually and positively associated with CB, while four mVOCs were with emphysema. In WQS regression, positive association between LASSO selected mVOCs and CB was identified (OR = 1.82, 95% CI: 1.25 to 2.69), and N-acetyl-S-(4-hydroxy-2-butenyl)-l-cysteine (MHBMA3) weighted the highest. Results from BKMR further validated such combined association and the significance of MHBMA3. As for emphysema, significantly positive overall trend of mVOCs was only observed in BKMR model and N-acetyl-S-(N-methylcarbamoyl)-l-cysteine (AMCC) contributed most to the mixed effect. White blood cell count (WBC) and lymphocyte number (LYM) were mediators in the positive pattern of mVOCs mixture with CB, while association between mVOCs mixture and emphysema was significantly mediated by LYM and segmented neutrophils num (NEO). This study demonstrated that exposure to VOCs was associated with CB and emphysema independently and combinedly, which might be partly speculated that VOCs were linked to activated inflammations. Our findings shed novel light on VOCs related respiratory illness, and provide a new basis for the contribution of certain VOCs to the risk of CB and emphysema, which has potential public health implications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Volatile organic compounds (VOCs) present in human urine are promising biomarkers for various health conditions and environmental exposures. However, their reliable detection is challenging due to the complexity of urinary matrices and the low concentrations of VOCs. Moreover, untargeted approaches present considerable challenges in terms of data interpretation, increasing the complexity of method development. Here we address these challenges by developing a new method that combines solid-phase microextraction (SPME) Arrow with gas chromatography-high resolution mass spectrometry (GC-HRMS), using a design of experiments (DOE) approach for targeted and untargeted compounds. This methodology, specifically tailored for SPME Arrow, represents a significant advancement in untargeted urinary analysis., Results: The method was developed based on targeted and untargeted outcomes, were ranking results focus on the highest response area of 11 spiked target VOCs representative of urinary volatilomics, and on identifying the maximum untargeted number of VOCs. The method was developed focusing on the highest response area of 11 spiked target VOCs representative of urinary volatilomics and identifying the maximum number of VOCs. A univariate method determined the optimal coating type, urine volume, and salt addition. Subsequently, a central composite design (CCD) DOE was used to determine ideal temperature, extraction, and incubation times. The best method obtained has an extraction time of 60 min at a temperature of 53 °C, with an SPME Arrow CAR/PDMS using 2 mL of urine, with 0.25 % w/v of NaCl and a pH of 2. Compared to conventional SPME fibers, the SPME Arrow showed improved extraction efficiency, detecting more VOCs. Finally, the enhanced method was successfully applied to urine samples from children exposed and non-exposed to tobacco smoke, identifying specific VOCs, like p-cymene and p-isopropenyl toluene related to tobacco exposure., Significance: By integrating both targeted and untargeted approaches, the developed method comprehensively captures the complexity of urinary metabolomics. This dual strategy ensures the precise identification of known compounds and the discovery of novel biomarkers, thereby providing a more complete metabolic profile. Such an approach is crucial for advancing in non-invasive diagnostics and environmental health studies, as it offers deeper insights into the intricate relationships between metabolic processes and various health conditions., Competing Interests: Declaration of competing interest Declaration of competing interest no competing interest to be disclosed., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In the context of the evolving landscape of nicotine consumption, the assessment of biomarkers plays a crucial role in understanding the health impact of different product categories. Exhaled breath (EB) emerges as a promising, non-invasive matrix for biomarker analysis, complementary to conventional urine and plasma data. This study explores distinctive EB biomarker profiles among users of combustible cigarettes (CC), heated tobacco products (HTP), electronic cigarettes (EC), smokeless/oral tobacco (OT), and oral/dermal nicotine products (NRT). We have successfully developed and validated a non-targeted GC-TOF-MS method for the analysis of EB samples across the aforementioned product categories. A total of 66 compounds were identified, with significantly elevated levels in at least one study group. The study found that CC users had higher levels of established VOCs associated with smoking, which supports the proof-of-concept of the method. Breathomic analysis identified increased levels of p-cymene and α-pinene in EC users, while HTP users showed potential biomarker candidates like γ-butyrolactone. This study underscores the utility of EB biomarkers for a comprehensive evaluation of diverse nicotine products. The unique advantages offered by EB analysis position it as a valuable tool for understanding the relationship between exposure and health outcomes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Max Scherer reports financial support was provided by Foundation for a Smokefree-World. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study was to estimate the associations of volatile organic compounds (VOCs) exposure with the prevalence of total and specific cardiovascular disease (CVD) among the general adult population. This cross-sectional study analyzed 15 urinary VOC metabolites in the general population using the 2011-2016 National Health and Nutrition Examination Survey ( n  = 5,213). The weighted study population with 47.0 years median age, was primarily female (51.2%). The prevalence of total CVD in the overall population was 7.9%. The single-exposure analyzes of AAMA, ATCA, CEMA, CYMA, DHBMA, 3HPMA, and 3MHA +4MHA were significantly associated with increased prevalence of total CVD. Qgcomp regression consistently showed that urinary VOCs-mixed exposure was positively correlated with the prevalence of total and specific CVDs (chronic heart failure, angina, and stroke), and highlighted each VOCs metabolite weights and direction. The similar results were observed for the WQS regression using mixed analysis methods. In conclusion, exposure to VOCs increases CVD prevalence and advances the identification of risk factors for CVD for environmental study.

While volatile organic compounds (VOCs) impair various organs, their influence on hearing loss (HL) has not been extensively researched. We aimed to identify the association between VOCs and HL or high-frequency hearing loss (HFHL). We extracted data on age, sex, pure tone audiometry, hypertension, occupational noise exposure, and creatinine-corrected urine VOC metabolite concentrations from the eighth Korea National Health and Nutrition Survey. Among the VOC metabolites, N-acetyl-S-(benzyl)-L-cysteine (BMA, P = 0.004), N-acetyl-S-(phenyl)-L-cysteine (SPMA, P = 0.027), and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA, P < 0.001) showed associations with HL. Additionally, HFHL exhibited significant associations with BMA (P = 0.005), 3- and 4-methylhippuric acid (3, 4 MHA, P = 0.049), mandelic acid (MA, P = 0.015), SPMA (P < 0.001), N-acetyl-S-(3-hydroxypropyl)-L-cysteine (3-HPMA, P < 0.001), and DHBMA (P < 0.001). After controlling other factors, DHBMA were associated with HL (P = 0.021) and HFHL (P = 0.014) and exhibited a linear association with the mean hearing level (β = 0.054, P = 0.024) and high-frequency hearing level (β = 0.045, P = 0.037). Since 1,3-butadiene may act as an ototoxic material, early screening for workers exposed to 1,3-butadiene and reducing exposure to 1,3-butadiene in everyday life may be helpful to prevent further HL., (© 2024. The Author(s).)

Health risks to humans after "fume and smell events", short-term incidents on aircrafts that are accompanied by unpleasant odour or visible smoke, remain a subject of controversy. We assessed exposure to volatile organic compounds (VOC) and organophosphorus compounds (OPC) by biomonitoring in 375 aircrew members after self-reported "fume and smell events" and in 88 persons of the general population. A total of 20 parameters were analysed in blood and urine by gas chromatography and mass spectrometry. Median levels of acetone in blood and urine and 2-propanol in blood were elevated in aircrews compared to controls (p < 0.0001). Additionally, elevated peak exposures, best estimated by the 95th percentiles, were observed in aircrews for n-heptane and n-octane in blood, and acetone, 2,5-hexanedione and o-cresol in urine. Only the maximum observed levels of 2,5-hexandione in urine (768 μg/L) and toluene in blood (77 μg/L) in aircrew members were higher than the current biological exposure indices (BEI® levels) (500 and 20 μg/L, respectively) of the American Conference of Governmental Industrial Hygienists (US-ACGIH) for workers occupationally exposed to n-hexane and toluene, two well-accepted human neurotoxicants. Low-level exposures to n-hexane and toluene could be also observed in controls. The majority of OPC parameters in urine, including those of neurotoxic ortho-isomers of tricresylphosphate, were below the limit of quantitation in both aircrews and controls. Our comparative VOC and OPC analyses in biological samples of a large number of aircrew members and controls suggest that exposures are similar in both groups and generally low., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Many diseases leave behind specific metabolites which can be detected from breath and urine as volatile organic compounds (VOC). Our group previously described VOC-based methods for the detection of bladder cancer and urinary tract infections. This study investigated whether prostate cancer can be diagnosed from VOCs in urine headspace., Methods: For this pilot study, mid-stream urine samples were collected from 56 patients with histologically confirmed prostate cancer. A control group was formed with 53 healthy male volunteers matched for age who had recently undergone a negative screening by prostate-specific antigen (PSA) and digital rectal exam. Headspace measurements were performed with the electronic nose Cyranose 320 TM . Statistical comparison was performed using principal component analysis, calculating Mahalanobis distance, and linear discriminant analysis. Further measurements were carried out with ion mobility spectrometry (IMS) to compare detection accuracy and to identify potential individual analytes. Bonferroni correction was applied for multiple testing., Results: The electronic nose yielded a sensitivity of 77% and specificity of 62%. Mahalanobis distance was 0.964, which is indicative of limited group separation. IMS identified a total of 38 individual analytical peaks, two of which showed significant differences between groups (p < 0.05). To discriminate between tumor and controls, a decision tree with nine steps was generated. This model led to a sensitivity of 98% and specificity of 100%., Conclusions: VOC-based detection of prostate cancer seems feasible in principle. While the first results with an electronic nose show some limitations, the approach can compete with other urine-based marker systems. However, it seems less reliable than PSA testing. IMS is more accurate than the electronic nose with promising sensitivity and specificity, which warrants further research. The individual relevant metabolites identified by IMS should further be characterized using gas chromatography/mass spectrometry to facilitate potential targeted rapid testing., (© 2024 The Authors. The Prostate published by Wiley Periodicals LLC.)

Purpose: Despite many efforts, no reliable urinary marker system has so far shown the potential to substitute cystoscopy. Measuring volatile organic compounds (VOCs) from urine is a promising alternative. VOCs are metabolic products which can be measured from the headspace of urine samples. Previous studies confirmed that the urine of bladder tumor patients has a different VOC profile than healthy controls. In this pilot study, the feasibility of discriminating VOCs from urine of bladder cancer patients from that of healthy control subjects was investigated. Aim of this study was to investigate whether VOC-based diagnosis of bladder cancer from urine samples is feasible using multicapillary column ion mobility spectrometry (MCC/IMS) and to identify potential molecular correlates to the relevant analytes., Methods: Headspace measurements of urine samples of 30 patients with confirmed transitional cell carcinoma (TCC) and 30 healthy controls were performed using MCC/IMS. In the results of the measurements, peaks showing significant differences between both groups were identified and implemented into a decision tree with respect to achieve group separation. Molecular correlates were predicted using a pre-defined dataset., Results: Eight peaks with significantly differing intensity were identified, 5 of which were highly significant. Using a six-step decision tree, MCC/IMS showed a sensitivity of 90% and specificity of 100% in group separation., Conclusion: VOC-based detection of bladder cancer is feasible. MCC/IMS is a suitable method for urine-based diagnosis and should be further validated. The molecular characteristics and metabolic background of the analytes require further workup., (© 2024. The Author(s).)

Volatile organic compounds (VOCs) are a group of ubiquitous environment pollutants especially released into the workplace. Assessment of VOCs exposure in occupational populations is therefore a crucial issue for occupational health. However, simultaneous biomonitoring of a variety of VOCs is less studied. In this study, a simple and sensitive method was developed for the simultaneous determination of 51 prototype VOCs in urine by headspace-thermal desorption coupled to gas chromatography-mass spectrometry (HS-TD-GC-MS). The urinary sample was pretreated with only adding 0.50 g of sodium chloride to 2 mL of urine and 51 VOCs should be determined with limits of detection (LODs) between 13.6 ng/L and 24.5 ng/L. The method linearity ranged from 0.005 to 10 μg/L with correlation coefficients (r) of 0.991 to 0.999. The precision for intraday and inter-day, measured by the variation coefficient (CV) at three levels of concentration, was below 15 %, except for 4-isopropyl toluene, dichloromethane, and trichloromethane at low concentration. For medium and high levels, recoveries of all target VOCs were within the standard range, but 1,1-dichloropropene and styrene, which were slightly under 80 % at low levels. In addition, the proposed method has been used to determine urine samples collected in three times (before, during and after working) from 152 workers at four different factories. 41 types of prototype VOCs were detected in workers urine. Significant differences (Kruskal-Wallis chi-squared = 117.18, df = 1, P < 0.05) in the concentration levels of VOCs between the exposed and unexposed groups were observed, but not between the three sampling times (Kruskal-Wallis chi-squared = 3.39, df = 2, P = 0.183). The present study provides an alternative method for biomonitoring and assessing mixed exposures to VOCs in occupational populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Volatile organic compounds (VOCs) encompass hundreds of high production volume chemicals and have been reported to be associated with adverse respiratory outcomes such as chronic obstructive pulmonary disease (COPD). However, research on the combined toxic effects of exposure to various VOCs on COPD is lacking. We aimed to assess the effect of VOC metabolite mixture on COPD risk in a large population sample., Methods: We assessed the effect of VOC metabolite mixture on COPD risk in 5997 adults from the National Health and Nutrition Examination Survey (NHANES) from 2011 to 2020 (pre-pandemic) using multivariate logistic regression, Bayesian weighted quantile sum regression (BWQS), quantile-based g-Computation method (Qgcomp), and Bayesian kernel machine regression (BKMR). We explored whether these associations were mediated by white blood cell (WBC) count and total bilirubin., Results: In the logistic regression model, we observed a significantly increased risk of COPD associated with 9 VOC metabolites. Conversely, N-acetyl-S-(benzyl)-L-cysteine (BMA) and N-acetyl-S-(n-propyl)-L-cysteine (BPMA) showed insignificant negative correlations with COPD risk. The overall mixture exposure demonstrated a significant positive relationship with COPD in both the BWQS model (adjusted odds ratio (OR) = 1.30, 95% confidence interval (CI): 1.06, 1.58) and BKMR model, and with marginal significance in the Qgcomp model (adjusted OR = 1.22, 95% CI: 0.98, 1.52). All three models indicated a significant effect of the VOC metabolite mixture on COPD in non-current smokers. WBC count mediated 7.1% of the VOC mixture associated-COPD in non-current smokers., Conclusions: Our findings provide novel evidence suggesting that VOCs may have adverse associations with COPD in the general population, with N, N- Dimethylformamide and 1,3-Butadiene contributing most. These findings underscore the significance of understanding the potential health risks associated with VOC mixture and emphasize the need for targeted interventions to mitigate the adverse effects on COPD risk., (© 2024. The Author(s).)

Background: Markers of airway inflammation can be helpful in the management of childhood asthma. Residential activities, such as intensive asthma camps at alpine altitude climate (AAC), can help reduce bronchial inflammation in patients who fail to achieve optimal control of the disease. Analysis of volatile organic compounds (VOCs) can be obtained using electronic devices such as e-Noses. We aimed to identify alterations in urinary e-Nose sensors among children with asthma participating in an intensive camp at AAC and to investigate associations between urinary e-Nose analysis and airway inflammation., Methods: We analyzed data collected in children with asthma recruited between July and September 2020. All children were born and resided at altitudes below 600 m asl. Urinary VOCs (measured using the Cyranose 320® VOC analyzer), Fractional exhaled Nitric Oxide (FeNO) and spirometry were evaluated upon children's arrival at the Istituto Pio XII, Misurina (BL), Italy, at 1756 m asl (T0), and after 7 (T1) and 15 days (T2) of stay., Results: Twenty-two patients (68.2% males; median age: 14.5 years) were enrolled. From T0 to T1 and T2, the negative trend for FeNO was significant (p < .001). Significant associations were observed between e-Nose sensors S7 (p = .002), S12 (p = .013), S16 (p = .027), S17 (p = .017), S22 (p = .029), S29 (p = .021), S31 (p = .009) and ΔFeNO at T0-T1. ΔFeNO at T0-T2 was significantly associated with S17 (p = .015), S19 (p = .004), S21 (p = .020), S24 (p = .012), S25 (p = .018), S26 (p = .008), S27 (p = .002), S29 (p = .007), S30 (p = .013)., Conclusions: We showed that a decrease in FeNO levels after a short sojourn at AAC is associated with behaviors of individual urinary e-Nose sensors in children with asthma., (© 2024 Wiley Periodicals LLC.)

In mammals, especially rodents, social behaviours, such as parenting, territoriality or mate attraction, are largely based on olfactory communication through chemosignals. These behaviours are mediated by species-specific chemosignals, including small organic molecules and proteins that are secreted in the urine or in various fluids from exocrine glands. Chemosignal detection is mainly ensured by olfactory neurons in two specific sensory organs, the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). This study aimed to characterise the olfactory communication in the fossorial ecotype of the water voles, Arvicola terrestris. We first measured the olfactory investigation of urine and lateral scent gland secretions from conspecifics. Our results showed that water voles can discriminate the sex of conspecifics based on the smell of urine, and that urinary male odour is attractive for female voles. Then, we demonstrated the ability of the VNO and MOE to detect volatile organic compounds (VOCs) found in water vole secretions using live-cell calcium imaging in dissociated cells. Finally, we evaluated the attractiveness of two mixtures of VOCs from urine or lateral scent glands in the field during a cyclical outbreak of vole populations., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: During the use of electronic cigarettes (e-cigarettes), users are still exposed to carcinogens similar to those found in tobacco products. Since these carcinogens are metabolized and excreted in urine, they may have carcinogenic effects on the bladder urinary tract epithelium. This meta-analysis aimed to compare bladder cancer carcinogens in the urine of tobacco users and e-cigarette users using a large number of samples., Methods: A systematic meta-analysis was performed using data obtained from several scientific databases (up to November 2023). This cumulative analysis was performed following the Preferred Reporting Items for Systematic Evaluation and Meta-Analysis (PRISMA) and Assessing the Methodological Quality of Systematic Evaluations (AMSTAR) guidelines, according to a protocol registered with PROSPERO. This study was registered on PROSPERO and obtained the unique number: CRD42023455600., Results: The analysis included 10 high-quality studies that considered polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOCs) and tobacco-specific nitrosamines (TSNAs). Statistical indicators show that there is a difference between the tobacco user group and the e-cigarette user group in terms of 1-Hydroxynaphthalene (1-NAP) [weighted mean difference (WMD)10.14, 95% confidence interval (CI) (8.41 to 11.88), p < 0.05], 1-Hydroxyphenanthrene (1-PHE) [WMD 0.08, 95% CI (-0.14 to 0.31), p > 0.05], 1-Hydroxypyrene (1-PYR) [WMD 0.16, 95% CI (0.12 to 0.20), p < 0.05], 2-Hydroxyfluorene (2-FLU) [WMD 0.69, 95% CI (0.58 to 0.80), p < 0.05], 2-Hydroxynaphthalene (2-NAP) [WMD 7.48, 95% CI (4.15 to 10.80), p < 0.05], 3-Hydroxyfluorene (3-FLU) [WMD 0.57, 95% CI (0.48 to 0.66), p < 0.05], 2-Carbamoylethylmercapturic acid (AAMA) [WMD 66.47, 95% CI (27.49 to 105.46), p < 0.05], 4-Hydroxy-2-buten-1-yl-mercapturic acid (MHBMA) [WMD 287.79, 95% CI (-54.47 to 630.04), p > 0.05], 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNAL) [WMD 189.37, 95% CI (78.45 to 300.29), p < 0.05], or N0-nitrosonornicotine (NNN) [WMD 11.66, 95% CI (7.32 to 16.00), p < 0.05]., Conclusion: Urinary bladder cancer markers were significantly higher in traditional tobacco users than in e-cigarette users. Systematic review registration : PROSPERO (CRD42023455600: https://www.crd.york.ac.uk/PROSPERO/)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Feng, Huang, Peng, Liang, Deng, Zhang, Li and Wu.)

The analysis of volatile organic compounds (VOCs) present in various biological samples holds immense potential for non-invasive disease diagnostics and metabolic profiling. One of the biological fluids that are suitable for use in clinical practice is urine. Given the limited quantity of VOCs in the urine headspace, it's imperative to enhance their extraction into the gaseous phase and prevent any degradation of VOCs during the thawing process. The study aimed to test several key parameters (incubation time, temperature, and thawing) that can influence urine volatilome and monitor selected VOCs for their stability. The analysis in this study was performed using a BreathSpec® (G.A.S., Dortmund, Germany) device consisting of a gas chromatograph (GC) coupled with an ion mobility spectrometer (IMS). Testing three different temperatures and incubation times yielded a low number of VOCs (9 out of 34) that exhibited statistically significant differences. However, examining three thawing conditions revealed no VOCs with statistically significant changes. Thus, we conclude that urine composition remains relatively stable despite exposure to various thermal stresses.

Background: The gradual evolution of the detection and quantification of volatile organic compounds (VOCs) has been instrumental in cancer diagnosis. The primary objective of this study was to assess the diagnostic potential of exhaled breath and urinary VOCs in cancer detection. As VOCs are indicative of tumor and human metabolism, our work also sought to investigate the metabolic pathways linked to the development of cancerous tumors., Materials and Methods: An electronic search was performed in the PubMed database. Original studies on VOCs within exhaled breath and urine for cancer detection with a control group were included. A meta-analysis was conducted using a bivariate model to assess the sensitivity and specificity of the VOCs for cancer detection. Fagan's nomogram was designed to leverage the findings from our diagnostic analysis for the purpose of estimating the likelihood of cancer in patients. Ultimately, MetOrigin was employed to conduct an analysis of the metabolic pathways associated with VOCs in relation to both human and/or microbiota., Results: The pooled sensitivity, specificity and the area under the curve for cancer screening utilizing exhaled breath and urinary VOCs were determined to be 0.89, 0.88, and 0.95, respectively. A pretest probability of 51% can be considered as the threshold for diagnosing cancers with VOCs. As the estimated pretest probability of cancer exceeds 51%, it becomes more appropriate to emphasize the 'ruling in' approach. Conversely, when the estimated pretest probability of cancer falls below 51%, it is more suitable to emphasize the 'ruling out' approach. A total of 14, 14, 6, and 7 microbiota-related VOCs were identified in relation to lung, colorectal, breast, and liver cancers, respectively. The enrichment analysis of volatile metabolites revealed a significant enrichment of butanoate metabolism in the aforementioned tumor types., Conclusions: The analysis of exhaled breath and urinary VOCs showed promise for cancer screening. In addition, the enrichment analysis of volatile metabolites revealed a significant enrichment of butanoate metabolism in four tumor types, namely lung, colorectum, breast and liver. These findings hold significant implications for the prospective clinical application of multiomics correlation in disease management and the exploration of potential therapeutic targets., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Volatile organic compounds (VOCs) are ubiquitous environmental pollutants and have been implicated in adverse health outcomes. In this study, concentrations of 11 VOC metabolites (mVOCs) and three oxidative stress biomarkers (8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-guanosine, and dityrosine) were determined in 205 urine samples collected from 12 cities across mainland China. Urinary ∑ 11 mVOC concentrations ranged from 498 to 1660 ng/mL, with a geometric mean (GM) value of 1070 ng/mL. The factorial analysis revealed that cooking, solvents, and vehicle emissions were the three primary sources of VOC exposure. A significant regional variation was clearly found in ∑ 11 mVOC concentrations across four regions in China, with high urine VOC concentrations found in North and South China (GM: 1450 and 1340 ng/mL). The multiple linear regression model revealed that most mVOCs were significantly positively correlated with three oxidative stress markers (β range: 0.06-0.22). Mixture effect regression showed that isoprene, crotonaldehyde, acrolein, and benzene were the strongest contributors to oxidative stress. Approximately 80% of the participants have HQ values greater than 1.0 for 1,3-butadiene and benzene, suggesting that their exposure doses were close to potential adverse health effects. Our findings provide comprehensive information on human exposure and potential health risks of VOCs in China.

Purpose of Review: Urine volatile organic compound (VOC) testing for early detection of urological cancers is a minimally invasive and promising method. The objective of this review was to present the results of recently published work on this subject., Recent Findings: Organic volatile compounds are produced through oxidative stress and peroxidation of cell membranes, and they are eliminated through feces, urine, and sweat. Studies looking for VOCs in urine for the diagnosis of urological cancers have mostly focused on bladder and prostate cancers. However, the number of patients included in the studies was small. The electronic nose was the most widely used means of detecting VOCs in urine for the detection of urological cancers. MOS sensors and pattern recognition machine learning were more used for the composition of electronic noses. Early detection of urological cancers by detection of VOCs in urine is a method with encouraging results with sensitivities ranging from 27 to 100% and specificities ranging from 72 to 94%., Summary: The olfactory signature of urine from patients with urological cancers is a promising biomarker for the early diagnosis of urological cancers. The electronic nose with its ability to recognize complex odors is an excellent alterative to canine diagnosis and analytical techniques. Nevertheless, additional research improving the technology of Enoses and the methodology of the studies is necessary for its implementation in daily clinical practice., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection is essential to achieving a better outcome and prognosis. Volatile organic compounds (VOCs) reflect alterations in the pathophysiology and body metabolism processes, as shown in various types of cancers. The biosensor platform (BSP) urine test uses animals' unique, proficient, and accurate ability to scent lung cancer VOCs. The BSP is a testing platform for the binary (negative/positive) recognition of the signature VOCs of lung cancer by trained and qualified Long-Evans rats as biosensors (BSs). The results of the current double-blind study show high accuracy in lung cancer VOC recognition, with 93% sensitivity and 91% specificity. The BSP test is safe, rapid, objective and can be performed repetitively, enabling periodic cancer monitoring as well as an aid to existing diagnostic methods. The future implementation of such urine tests as routine screening and monitoring tools has the potential to significantly increase detection rate as well as curability rates with lower healthcare expenditure. This paper offers a first instructive clinical platform utilizing VOC's in urine for detection of lung cancer using the innovative BSP to deal with the pressing need for an early lung cancer detection test tool.

Volatile organic compounds (VOCs) are a group of pollutants pervasive in daily life with identified adverse health effects. However, no study has investigated the variability in VOC metabolites during pregnancy and their relationships with oxidative stress biomarkers in pregnant women. In the present study, the variability of 21 selected VOC metabolites was examined and their relationships with three selected oxidative stress biomarkers measured in spot urine samples at three trimesters of 1094 pregnant women were analyzed. Nineteen VOC metabolites were ubiquitous in the urine samples with detection rates ranging from 75.9% to 100%. Monohydroxybutenyl mercapturic acid (MHBMA) and s-phenyl mercapturic acid (PMA) had detection rates lower than 1.00%. Intraclass correlation coefficients (ICCs) of the detected analytes at three trimesters ranged 0.07-0.24, and the concentrations were highest in the first trimester. Higher concentrations of some VOC metabolites were related with participant characteristics including higher pre-pregnancy body mass index (BMI), lower education level, unemployment during pregnancy, multiparity, and sampling season of summer or winter. In repeated cross-sectional analyses, interquartile range (IQR) increases in the 19 detected VOC metabolites were positively related with 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), and 4-hydroxy nonenal mercapturic acid (HNEMA) with the estimates ranging from 9.00% to 204%. The mixture effect of the VOC metabolites on the oxidative stress biomarkers was further assessed using weighted quantile sum regression (WQS) models and the results showed that the WQS index of VOC metabolite mixture was significantly associated with 8-OHdG (β: 0.37, 0,32, and 0.39 at the 1st, 2nd, and 3rd trimester, respectively), 8-OHG (0.38, 0.32, and 0.39) and HNEMA (1.21, 1.08, and 1.10). Glycidamide mercapturic acid (GAMA), and trans,trans-muconic acid (MU) were the strongest contributors of the mixture effect on 8-OHdG, 8-OHG, and HNEMA, respectively. Overall, urinary concentrations of the VOC metabolites during pregnancy were strongly associated with the oxidative stress biomarkers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent liver diseases among adolescents. Several animal studies have suggested that volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) increase NAFLD risk. However, few epidemiological studies have confirmed the association between VOCs, PAHs and NAFLD in the general adolescent population. Therefore, we analyzed 798 adolescents from the Korean National Environmental Health Survey (KoNEHS), 2015-2017, to examine the associations of urinary metabolites of VOCs and PAHs with serum alanine aminotransferase (ALT) activity and NAFLD prevalence. We performed linear regression, logistic regression, and Bayesian kernel machine regression (BKMR) to evaluate the association of urinary VOCs and PAHs metabolites with ALT levels and NAFLD prevalence. After adjusting for all covariates, urinary benzylmercapturic acid and 2-hydroxyfluorene levels were found to increase ALT activity and NAFLD prevalence. Additionally, the BKMR analyses showed a significantly positive overall effect on ALT activity and NAFLD prevalence with urinary concentrations of VOCs and PAHs metabolites, with 2-hydroxyfluorene as the biggest contributor. Our study suggests that exposure to low-level VOCs and PAHs may have a detrimental effect on NAFLD risk in adolescents. Given the increasing prevalence of NAFLD in adolescents, future cohort studies are confirmed to comprehend the effect of these chemicals on NAFLD risk., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aim: Breast cancer (BC) is the leading cause of cancer-related death in females. The development of non-invasive methods for the early diagnosis of BC still remains challenge. Here, we aimed to discover the urinary volatile organic compounds (VOCs) pattern of BC patients and identify potential VOC biomarkers for BC diagnosis., Methods: Urine samples were analyzed by headspace-solid phase microextraction (HS-SPME) combined with gas chromatography-high resolution mass spectrometry (GC-HRMS). To assure reliable analysis, the factors influencing HS-SPME extraction efficiency were comprehensively investigated and optimized by combing the Plackett-Burman design (PBD) with the central composite design (CCD). The established HS-SPME/GC-HRMS method was validated and applied to analyze urine samples from BC patients (n = 80) and healthy controls (n = 88)., Results: A total number of 134 VOCs belonging to distinct chemical classes were identified by GC-HRMS. BC patients demonstrated unique urinary VOCs pattern. Orthogonal partial least squares-discriminant analysis (OPLS-DA) showed a clear separation between BC patients and healthy controls. Eight potential VOC biomarkers were identified using multivariate and univariate statistical analysis. The predictive ability of candidate VOC biomarkers was further investigated by the random forest (RF) algorithm. The candidate VOC biomarkers yielded 76.3% sensitivity and 85.4% specificity on the training set, and achieved 76.0% sensitivity and 92.3% specificity on the validation set., Conclusions: Overall, this work not only established a standardized HS-SPME/GC-HRMS approach for urinary VOCs analysis, but also highlighted the value of urinary VOCs for BC diagnosis. The knowledge gained from this study paves the way for early diagnosis of BC using urine in a non-invasive manner., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: As neonates are susceptible for many diseases, establishing noninvasive diagnostic methods is desirable. We hypothesized that volatile organic compounds (VOCs) could be successfully measured in diaper samples., Methods: We performed a feasibility study to investigate whether ambient air-independent headspace measurements of the VOC profiles of diapers from premature infants can be conducted using ion mobility spectrometer coupled with multi-capillary columns (B & S Analytik GmbH)., Results: We analysed 39 diapers filled with stool (n = 10) or urine (n = 20) respectively, using empty diapers as a control (n = 9). A total of 158 different VOCs were identified, and we classified the content of the diapers (urine or stool) according to their VOC profiles with a significance level of p < 0.05., Conclusions: We have developed a novel method to study headspace VOC profiles of biosamples using ion mobility spectrometry coupled with multi-capillary columns. Using this method, we have characterized the VOC profiles of stool and urine of preterm neonates. Future studies are warranted to characterize specific VOC profiles in infections and other diseases of the preterm neonate, thus establishing quick and noninvasive diagnostics in the routine care of the highly vulnerable preterm and term neonates., (© 2022 The Authors. European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

Background: The diagnosis and surveillance of urothelial bladder cancer (UBC) require cystoscopy. There is a need for biomarkers to reduce the frequency of cystoscopy in surveillance; urinary volatile organic compound (VOC) analysis could fulfil this role. This cross-sectional study compared the VOC profiles of patients with and without UBC, to investigate metabolomic signatures as biomarkers., Methods: Urine samples were collected from haematuria clinic patients undergoing diagnostic cystoscopy and UBC patients undergoing surveillance. Urinary headspace sampling utilised solid-phase microextraction and VOC analysis applied gas chromatography-mass spectrometry; the output underwent metabolomic analysis., Results: The median participant age was 70 years, 66.2% were male. Of the haematuria patients, 21 had a new UBC diagnosis, 125 had no cancer. In the surveillance group, 75 had recurrent UBC, 84 were recurrence-free. A distinctive VOC profile was observed in UBC patients compared with controls. Ten VOCs had statistically significant abundances useful to classify patients (false discovery rate range 1.9 × 10 -7 -2.8 × 10 -2 ). Two prediction models were evaluated using internal validation. An eight-VOC diagnostic biomarker panel achieved AUROC 0.77 (sensitivity 0.71, specificity 0.72). A six-VOC surveillance biomarker panel obtained AUROC 0.80 (sensitivity 0.71 and specificity 0.80)., Conclusions: Urinary VOC analysis could aid the diagnosis and surveillance of UBC., (© 2022. The Author(s).)

Trained detection dogs distinguish between urine samples from healthy organisms and organisms with malignant tumors, suggesting that the volatile urine metabolome contains information about tumor progression. The aim of this study was to determine whether the stage of tumor growth affects the chemical differences in the urine of mice and to what extent the "olfactory image of disease" perceived by dogs coincides with the "image of disease" recorded by the mass spectrometer. We used a novel laser ionization mass spectrometry method and propose a mass spectrometric analysis without detailed interpretation of the spectrum of volatile metabolomes in urine. The mass spectrometer we use works without sample preparation and registers volatile organic compounds in air at room temperature without changing the pH of the sample, i.e. under conditions similar to those in which dogs solve the same problem. The experimental cancer models were male BDF-f1 hybrid mice transplanted with hepatocarcinoma tissue, and similar mice transplanted with healthy liver tissue were used as controls. Our data show that both dogs and our proposed laser mass spectrometry method are able to detect both the entire spectrum of volatile organic compounds associated with the disease and minor changes in this spectrum during its course., (© 2022. The Author(s).)

More than one million new cases of prostate cancer (PCa) were reported worldwide in 2020, and a significant increase of PCa incidence up to 2040 is estimated. Despite potential treatability in early stages, PCa diagnosis is challenging because of late symptoms' onset and limits of current screening procedures. It has been now accepted that cell transformation leads to release of volatile organic compounds in biologic fluids, including urine. Thus, several studies proposed the possibility to develop new diagnostic tools based on urine analysis. Among these, electronic noses (eNoses) represent one of the most promising devices, because of their potential to provide a non-invasive diagnosis. Here we describe the approach aimed at defining the experimental protocol for eNose application for PCa diagnosis. Our research investigates effects of sample preparation and analysis on eNose responses and repeatability. The dependence of eNose diagnostic performance on urine portion analysed, techniques involved for extracting urine volatiles and conditioning temperature were analysed. 192 subjects (132 PCa patients and 60 controls) were involved. The developed experimental protocol has resulted in accuracy, sensitivity and specificity of 83% (CI 95% 77-89), 82% (CI 95% 73-88) and 87% (CI 95% 75-94), respectively. Our findings define eNoses as valuable diagnostic tool allowing rapid and non-invasive PCa diagnosis., (© 2021. The Author(s).)

Safe and noninvasive methods for breast cancer screening with improved accuracy are urgently needed. Volatile organic compounds (VOCs) in biological samples such as breath and blood have been investigated as noninvasive novel markers of cancer. We investigated volatile organic compounds in urine to assess their potential for the detection of breast cancer. One hundred and ten women with biopsy-proven breast cancer and 177 healthy volunteers were enrolled. The subjects were divided into two groups: a training set and an external validation set. Urine samples were collected and analyzed by gas chromatography and mass spectrometry. A predictive model was constructed by multivariate analysis, and the sensitivity and specificity of the model were confirmed using both a training set and an external set with reproducibility tests. The training set included 60 breast cancer patients (age 34-88 years, mean 60.3) and 60 healthy controls (age 34-81 years, mean 58.7). The external validation set included 50 breast cancer patients (age 35-85 years, mean 58.8) and 117 healthy controls (age 18-84 years, mean 51.2). One hundred and ninety-one compounds detected in at least 80% of the samples from the training set were used for further analysis. The predictive model that best-detected breast cancer at various clinical stages was constructed using a combination of two of the compounds, 2-propanol and 2-butanone. The sensitivity and specificity in the training set were 93.3% and 83.3%, respectively. Triplicated reproducibility tests were performed by randomly choosing ten samples from each group, and the results showed a matching rate of 100% for the breast cancer patient group and 90% for the healthy control group. Our prediction model using two VOCs is a useful complement to the current diagnostic tools. Further studies inclusive of benign tumors and non-breast malignancies are warranted., (© 2021. The Author(s).)

Objective: Many individuals with bladder cancer have undergone a surgical urostomy and often complain of being self-conscious of the unpleasant smell of their own urine. The focus of this study was to test the efficacy of a pouch cover made of a carbon and zeolite containing polyester material to inhibit the smell of urine by comparing two trained dogs' response time in detecting volatile organic compounds (VOCs) in urine, with and without the fabric covering the samples., Methods: This study used a randomized, blinded experimental design to evaluate the efficacy of a fabric to interfere with two highly trained dogs' ability to detect specific VOCs present in the urine of prostate cancer patient. Ninety urine samples were analyzed in this study., Results: Prior to the experiment, both dogs accurately detected VOCs in the uncovered test urine samples of men with prostate cancer with a sensitivity and specificity of nearly 100%. Both dogs recognized the "uncovered" urine samples of men with prostate cancer within two seconds. When the test sample was covered with the study fabric, the test urine samples were detected within 30-40 seconds and in some instances the dogs were not able to identify the covered samples, whatsoever., Conclusion: The findings of this study demonstrate that the carbon and zeolite containing polyester fabric did significantly interfere with the ability of the dogs to detect VOCs in urine of men with prostate cancer. The fabric may show promise as a pouch cover in controlling offensive urine odor which many ostomates experience.

Background: Lung cancer is the most common oncological cause of death in the Western world. Early diagnosis is critical for successful treatment. However, no effective screening methods exist. A promising approach could be the use of volatile organic compounds as diagnostic biomarkers. To date there are several studies, in which dogs were trained to discriminate cancer samples from controls. In this study we evaluated the abilities of specifically trained dogs to distinguish samples derived from lung cancer patients of various tumor stages from matched healthy controls., Methods: This single center, double-blind clinical trial was approved by the local ethics committee, project no FF20/2016. The dog was conditioned with urine and breath samples of 36 cancer patients and 150 controls; afterwards, further 246 patients were included: 41 lung cancer patients comprising all stages and 205 healthy controls. From each patient two breath and urine samples were collected and shock frozen. Only samples from new subjects were presented to the dog during study phase randomized, double-blinded. This resulted in a specific conditioned reaction pointing to the cancer sample., Results: Using a combination of urine and breath samples, the dog correctly predicted 40 out of 41 cancer samples, corresponding to an overall detection rate of cancer samples of 97.6% (95% CI [87.1, 99.9%]). Using urine samples only the dog achieved a detection rate of 87.8% (95% CI [73.8, 95.9%]). With breath samples, the dog correctly identified cancer in 32 of 41 samples, resulting in a detection rate of 78% (95% CI [62.4, 89.4%])., Conclusions: It is known from current literature that breath and urine samples carry VOCs pointing to cancer growth. We conclude that olfactory detection of lung cancer by specifically trained dogs is highly suggestive to be a simple and non-invasive tool to detect lung cancer. To translate this approach into practice further target compounds need to be identified., (© 2021. The Author(s).)

Hepatocellular carcinoma (HCC) biomarkers are lacking in clinical practice. We therefore explored the pattern and composition of urinary volatile organic compounds (VOCs) in HCC patients. This was done in order to assess the feasibility of a potential non-invasive test for HCC, and to enhance our understanding of the disease. This pilot study recruited 58 participants, of whom 20 were HCC cases and 38 were non-HCC cases. The non-HCC cases included healthy individuals and patients with various stages of non-alcoholic fatty liver disease (NAFLD), including those with and without fibrosis. Urine was analysed using gas chromatography-ion mobility spectrometry (GC-IMS) and gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). GC-IMS was able to separate HCC from fibrotic cases with an area under the curve (AUC) of 0.97 (0.91-1.00), and from non-fibrotic cases with an AUC of 0.62 (0.48-0.76). For GC-TOF-MS, a subset of samples was analysed in which seven chemicals were identified and tentatively linked with HCC. These include 4-methyl-2,4- bis ( p -hydroxyphenyl)pent-1-ene (2TMS derivative), 2-butanone, 2-hexanone, benzene, 1-ethyl-2-methyl-, 3-butene-1,2-diol, 1-(2-furanyl)-, bicyclo(4.1.0)heptane, 3,7,7-trimethyl-, [1S-(1a,3β,6a)]-, and sulpiride. Urinary VOC analysis using both GC-IMS and GC-TOF-MS proved to be a feasible method of identifying HCC cases, and was also able to enhance our understanding of HCC pathogenesis.

The red fox is a highly adaptable mammal that has established itself world-wide in many different environments. Contributing to its success is a social structure based on chemical signalling between individuals. Urine scent marking behaviour has long been known in foxes, but there has not been a recent study of the chemical composition of fox urine. We have used solid-phase microextraction and gas chromatography-mass spectrometry to analyze the urinary volatiles in 15 free-ranging wild foxes (2 female) living in farmlands and bush in Victoria, Australia. Foxes here are routinely culled as feral pests, and the urine was collected by bladder puncture soon after death. Compounds were identified from their mass spectra and Kovats retention indices. There were 53 possible endogenous scent compounds, 10 plant-derived compounds and 5 anthropogenic xenobiotics. Among the plant chemicals were several aromatic apocarotenoids previously found in greater abundance in the fox tail gland. They reflect the dietary consumption of carotenoids, essential for optimal health. One third of all the endogenous volatiles were sulfur compounds, a highly odiferous group which included thiols, methylsulfides and polysulfides. Five of the sulfur compounds (3-isopentenyl thiol, 1- and 2-phenylethyl methyl sulfide, octanethiol and benzyl methyl sulfide) have only been found in foxes, and four others (isopentyl methyl sulfide, 3-isopentenyl methyl sulfide, and 1- and 2-phenylethane thiol) only in some canid, mink and skunk species. This indicates that they are not normal mammalian metabolites and have evolved to serve a specific role. This role is for defence in musteloids and most likely for chemical communication in canids. The total production of sulfur compounds varied greatly between foxes (median 1.2, range 0.4-32.3 μg 'acetophenone equivalents'/mg creatinine) as did the relative abundance of different chemical types. The urinary scent chemistry may represent a highly evolved system of semiochemicals for communication between foxes., Competing Interests: The authors have declared that no competing interests exist.

Current methods for non-invasive prostate cancer (PrCa) detection have a high false-positive rate and often result in unnecessary biopsies. Previous work has suggested that urinary volatile organic compound (VOC) biomarkers may be able to distinguish PrCa cases from benign disease. The behavior of the nematode Caenorhabditis elegans has been proposed as a tool to take advantage of these potential VOC profiles. To test the ability of C. elegans Bristol N2 to distinguish PrCa cases from controls, we performed chemotaxis assays using human urine samples collected from men screened for PrCa. Behavioral response of nematodes towards diluted urine from PrCa cases was compared to response to samples from cancer-free controls. Overall, we observed a significant attraction of young adult-stage C. elegans nematodes to 1:100 diluted urine from confirmed PrCa cases and repulsion of C. elegans to urine from controls. When C. elegans chemotaxis index was considered alongside prostate-specific antigen levels for distinguishing cancer from cancer-free controls, the accuracy of patient classification was 81%. We also observed behavioral attraction of C. elegans to two previously reported VOCs to be increased in PrCa patient urine. We conclude nematode behavior distinguishes PrCa case urine from controls in a dilution-dependent manner., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Volatile organic compounds (VOCs) exiting in urine are potential biomarkers of chronic kidney diseases. Headspace solid phase microextraction (HS-SPME) was applied for extraction VOCs over the urine samples. Volatile metabolites were separated and identified by means of two-dimensional gas chromatography and time of flight mass spectrometry (GC × GC TOF MS). Patients with glomerular diseases ( n = 27) and healthy controls ( n = 20) were recruited in the study. Different VOCs profiles were obtained from patients and control. Developed methodology offers the opportunity to examine the metabolic profile associated with glomerulopathy. Four compounds found in elevated amounts in the patients group, i.e., methyl hexadecanoate; 9-hexadecen-1-ol; 6,10-dimethyl-5,9-undecadien-2-one and 2-pentanone were proposed as markers of glomerular diseases.

Volatile organic compounds (VOCs) are important and ubiquitous air pollutants, which may lead to a significant increase in the prevalence of respiratory diseases. To investigate the relationships between VOCs exposure and childhood asthma, 252 asthmatic children and 69 healthy children were recruited. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of oxidative DNA damage), trans-3'-hydroxycotinine (OH-Cot, a biomarker of passive smoking) and 27 VOC metabolites were simultaneously determined by an ultra-high-performance liquid chromatography-tandem mass spectrometer. Results showed that levels of 8-OHdG and most VOC metabolites in asthmatic children were significantly higher than those in healthy children. More than half of the VOC metabolites were significantly and positively associated with OH-Cot with maximal β coefficient of 0.169, suggesting that second-hand smoking is one important source of VOCs exposure for children in Guangzhou. Significant dose-response relationships between most VOC metabolites and 8-OHdG were observed. Each unit increase in ln-transformed VOC metabolite levels was significantly associated with 5.5-32% increase in ln-transformed 8-OHdG level. Moreover, each unit increase in ln-transformed 8-OHdG level was associated with an 896% increased odd ratios (OR) of asthma in children (OR = 9.96, 95% confidence intervals (CI): 4.75, 20.9), indicating that oxidative stress induced by VOCs exposure may have a significant impact on childhood asthma. Urinary 3-&4-Methylhippuric acid (3-&4-MHA, OR: 5.78, 95% CI: 3.50, 9.54), rac 2-Aminothiazoline-4-carboxylic acid (ATCA, OR: 2.90, 95% CI: 1.69, 4.99) and N-Acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA, OR: 2.76, 95% CI: 1.73, 4.43) which may derive from m/p-xylene, cyanide and 1,3-butadiene exposure, respectively, could significantly and maximally increase the odds of asthma. Interestingly, they also had the strongest associations with 8-OHdG among all investigated VOC metabolites. Moreover, DHBMA strongly correlated with most VOC metabolites. Hence, DHBMA is a suitable biomarker to indicate not only VOCs exposure profile, but also the DNA damage-mediated asthma induced by VOCs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Prostate cancer is the second leading cause of cancer death in men in the developed world. A more sensitive and specific detection strategy for lethal prostate cancer beyond serum prostate specific antigen (PSA) population screening is urgently needed. Diagnosis by canine olfaction, using dogs trained to detect cancer by smell, has been shown to be both specific and sensitive. While dogs themselves are impractical as scalable diagnostic sensors, machine olfaction for cancer detection is testable. However, studies bridging the divide between clinical diagnostic techniques, artificial intelligence, and molecular analysis remains difficult due to the significant divide between these disciplines. We tested the clinical feasibility of a cross-disciplinary, integrative approach to early prostate cancer biosensing in urine using trained canine olfaction, volatile organic compound (VOC) analysis by gas chromatography-mass spectroscopy (GC-MS) artificial neural network (ANN)-assisted examination, and microbial profiling in a double-blinded pilot study. Two dogs were trained to detect Gleason 9 prostate cancer in urine collected from biopsy-confirmed patients. Biopsy-negative controls were used to assess canine specificity as prostate cancer biodetectors. Urine samples were simultaneously analyzed for their VOC content in headspace via GC-MS and urinary microbiota content via 16S rDNA Illumina sequencing. In addition, the dogs' diagnoses were used to train an ANN to detect significant peaks in the GC-MS data. The canine olfaction system was 71% sensitive and between 70-76% specific at detecting Gleason 9 prostate cancer. We have also confirmed VOC differences by GC-MS and microbiota differences by 16S rDNA sequencing between cancer positive and biopsy-negative controls. Furthermore, the trained ANN identified regions of interest in the GC-MS data, informed by the canine diagnoses. Methodology and feasibility are established to inform larger-scale studies using canine olfaction, urinary VOCs, and urinary microbiota profiling to develop machine olfaction diagnostic tools. Scalable multi-disciplinary tools may then be compared to PSA screening for earlier, non-invasive, more specific and sensitive detection of clinically aggressive prostate cancers in urine samples., Competing Interests: The authors have read the journal’s policy and have the following competing interests: ST is a paid employee of Imagination Engines, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Background: In the human body, volatile organic compounds (VOCs) are produced by different tissues then secreted in different body fluids and subsequently excreted. Here we explore a non-invasive method for the detection of liver, prostate and bladder cancers., Methods: We recruited 140 cases. There were 31 hepatocellular carcinomas (HCC), 62 prostate carcinomas, 29 bladder carcinomas and 18 non-cancer cases. Male to female ratio was 5:1 and mean age was 72 years. Urinary VOCs were detected by applying solid-phase microextraction (SPME) technique., Results: The sensitivity for detection of HCC with normal alpha fetoprotein (AFP) was 68% (SE 0.06, 95% CI 0.54 to 0.81 and P < 0.005). The VOCs sensitivity in the detection of HCC cases with raised AFP was 83%. (SE 0.05, 95% CI 0.73 to 0.93 and P < 0.0001). The VOCs sensitivity for prostate cancer detection was 70% (SE 0.049, 95% CI 0.60 to 0.79 and P < 0.0002) and sensitivity for bladder cancer detection was 81% (SE 0.052, 95% CI 0.70 to 0.91 and P < 0.0001)., Conclusions: SPME urinary VOCs analysis was able to differentiate between controls and each of hepatocellular, prostate and bladder cancers. This suggests that urinary VOCs are cancer specific and could potentially be used as a diagnostic method.

Metabolomics is a potential tool for the discovery of new biomarkers in the early diagnosis of diseases. An ultra-fast gas chromatography system equipped to an electronic nose detector (FGC eNose) was used to identify the metabolomic profile of Volatile Organic Compounds (VOCs) in type 2 diabetes (T2D) urine from Mexican population. A cross-sectional, comparative, and clinical study with translational approach was performed. We recruited twenty T2D patients and twenty-one healthy subjects. Urine samples were taken and analyzed by FGC eNose. Eighty-eight compounds were identified through Kovats's indexes. A natural variation of 30% between the metabolites, expressed by study groups, was observed in Principal Component 1 and 2 with a significant difference (p < 0.001). The model, performed through a Canonical Analysis of Principal coordinated (CAP), allowed a correct classification of 84.6% between healthy and T2D patients, with a 15.4% error. The metabolites 2-propenal, 2-propanol, butane- 2,3-dione and 2-methylpropanal, were increased in patients with T2D, and they were strongly correlated with discrimination between clinically healthy people and T2D patients. This study identified metabolites in urine through FGC eNose that can be used as biomarkers in the identification of T2D patients. However, more studies are needed for its implementation in clinical practice., (© 2020 John Wiley & Sons, Ltd.)

Bile acid diarrhoea (BAD) is a common disorder resulting from increased loss of bile acids (BAs), overlapping irritable bowel syndrome with diarrhoea (IBS-D). The gut microbiota metabolises primary BAs to secondary BAs, with differing impacts on metabolism and homeostasis. The aim of this study was to profile the microbiome, metabolic products and bile acids in BAD. Patients with BAD diagnosed by SeHCAT testing, were compared with other IBS-D patients, and healthy controls. Faecal 16S ribosomal RNA gene analysis was undertaken. Faecal short chain fatty acid (SCFA) and urinary volatile organic compounds (VOCs) were measured. BAs were quantified in serum and faeces. Faecal bacterial diversity was significantly reduced in patients with BAD. Several taxa were enriched compared to IBS-D. SCFA amounts differed in BAD, controls and IBS-D, with significantly more propionate in BAD. Separation of VOC profiles was evident, but the greatest discrimination was between IBS-D and controls. Unconjugated and primary BA in serum and faeces were significantly higher in BAD. The faecal percentage primary BA was inversely related to SeHCAT. BAD produces dysbiosis, with metabolite differences, including VOC, SCFA and primary BAs when compared to IBS-D. These findings provide new mechanistic insights into the pathophysiology of BAD.

The development of screening methods for various types of cancer is of utmost importance as the early diagnostics of these diseases significantly increases the chances for patient's successful medical treatment and recovery. In this study we have developed the procedure for chromatographic profiling of urine samples based on solid-phase microextraction and GC-MS. 50 urine samples (20 from the patients with biopsy conformed prostate cancer and 30 from control group) were studied in the optimized experimental conditions. Application of chemometric classification algorithms such as k-nearest neighbors and partial least squares-discriminant analysis allowed construction of predictive models yielding very high sensitivity, specificity and accuracy values all close to 100%. This gives a good promise for further validation of this approach with a broader sample sets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Urinary volatile compounds (VCs) have been recently assessed for disease diagnoses. They belong to very diverse chemical classes, and they are characterized by different volatilities, polarities and concentrations, complicating their analysis via a single analytical procedure. There remains a need for better, lower-cost methods for VC biomarker discovery. Thus, there is a strong need for alternative methods, enabling the detection of a broader range of VCs. Therefore, the main aim of this study was to optimize a simple and reliable liquid-liquid extraction (LLE) procedure for the analysis of VCs in urine using gas chromatography-mass spectrometry (GC-MS), in order to obtain the maximum number of responses. Extraction parameters such as pH, type of solvent and ionic strength were optimized. Moreover, the same extracts were analyzed using Proton Nuclear Magnetic Resonance Spectroscopy ( 1 H-NMR), to evaluate the applicability of a single urine extraction for multiplatform purposes. After the evaluation of experimental conditions, an LLE protocol using 2 mL of urine in the presence of 2 mL of 1 M sulfuric acid and sodium sulphate extracted with dichloromethane was found to be optimal. The optimized method was validated with the external standards and was found to be precise and linear, and allowed for detection of >400 peaks in a single run present in at least 50% of six samples-considerably more than the number of peaks detected by solid-phase microextracton fiber pre-concentration-GC-MS (328 ± 6 vs. 234 ± 4). 1 H-NMR spectroscopy of the polar and non-polar extracts extended the range to >40 more (mainly low volatility compounds) metabolites (non-destructively), the majority of which were different from GC-MS. The more peaks detectable, the greater the opportunity of assessing a fingerprint of several compounds to aid biomarker discovery. In summary, we have successfully demonstrated the potential of LLE as a cheap and simple alternative for the analysis of VCs in urine, and for the first time the applicability of a single urine solvent extraction procedure for detecting a wide range of analytes using both GC-MS and 1 H-NMR analysis to enhance putative biomarker detection. The proposed method will simplify the transport between laboratories and storage of samples, as compared to intact urine samples.