Comparison of three breath sampling methods for volatile organic compounds analysis in healthy adults: End-Tidal, Whole, and Continuous

Comparison of three breath sampling methods for volatile organic compounds analysis in healthy adults: End-Tidal, Whole, and Continuous | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparison of three breath sampling methods for volatile organic compounds analysis in healthy adults: End-Tidal, Whole, and Continuous Seiyoung Hwang, Hyunsoo Chung This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8519105/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Breath volatile organic compounds (VOCs) are promising biospecimens to be potentially utilized as non-invasive biomarkers. In this study, we analyzed breath samples from 90 non-smoking healthy adults using three sampling methods—Whole, Continuous, and End-tidal—to compare and evaluate patterns of breath VOCs detected by each. A total of 1,346 VOCs were identified, with 34 consistently detected across all methods, forming a core set of robust compounds. While principal component analysis showed no distinct clustering by method, several VOCs, including methylcyclohexane, toluene, D-Limonene, ethylbenzene and 1,1,2-trichloro-1,2,2-trifluoroethane, displayed method-dependent intensity differences. The Continuous method tended to yield lower values, but overall VOC profiles remained comparable to those from End-tidal samples. Participant surveys indicated that Whole breath was perceived as most convenient, whereas the Continuous method achieved consistent acceptability. These findings suggest that although End-tidal sampling remains the reference method, the Continuous method may serve as a practical and user-friendly alternative, particularly when alveolar air collection is not feasible. By establishing healthy reference VOCs and comparing the feasibility of different approaches, this study provides a foundation for applying breath analysis more flexibly in future clinical and translational research. Health sciences/Biomarkers Physical sciences/Chemistry Health sciences/Medical research Breath analysis Volatile organic compounds Breath sampling methods Gas chromatography-mass spectrometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Breath volatile organic compounds (VOCs) are intensively researched as potential biomarkers, given their non-invasive sampling, collection simplicity and reflection of various physiological and pathological statuses 1 – 3 . Exhaled breath contains hundreds of VOCs originating from endogenous metabolic events or exogenous sources, which provide various information about human body. Breath VOCs have attracted growing interest in both academic and clinical fields, with applications in infectious diseases, metabolic disorders, and various cancers 4 – 9 . These advances are further supported by the development of novel analytical and computational approaches, including sensors, electronic nose (e-nose) devices and machine learning–based algorithms, which enhance pattern recognition and diagnostic accuracy 10 – 12 . As healthcare demands for early disease diagnostics and point-of-care testing continue to rise and expand, the non-invasive nature of breath sampling and the abundance of breath samples make breathomics an attractive approach for both researchers and patients. However, despite these numerous advantages, breath analysis has several limitations to overcome. Since VOCs exist only at trace levels in human breaths, sensitive control of sampling, sophisticated analytical instruments and proper background correction are required 13 – 15 . Breath volatiles can fluctuate depending on factors such as diet, smoking, environmental factors, and behavioral patterns such as sleep or workouts 16 , 17 . Moreover, standardized methods for breath sampling are still in question, which pose challenges for quantifications, reproducibility and broader clinical applications. While debates on the most appropriate breath sampling methods remain, the alveolar fraction of breath is widely recognized for its advantages in accuracy and physiological relevance and thus has been regarded as the most reliable fraction of exhaled air for clinical and translational applications. Because alveolar gases concentrations are approximately close to those present in the actual arterial blood, end-tidal air is known to mirror biological conditions. Moreover, collection of end-tidal air minimizes effect of contamination from external environment and dead space air. To collect the end-tidal fraction, capnography-guided devices such as ReCIVA™ are often used for sampling 18 . Although the advantages of end-tidal sampling are evident, end-tidal collection usually requires costly devices and residual effects from environmental contamination or dead space still remain 19 . In contrast, Whole breath is collecting the entire volume of exhalation. Procedure is easy for subjects to follow, however may lead to dilution of endogenous VOCs due to the presence of dead space air or oral VOCs emissions 20 . The rebreathing (Continuous) approach is the process of repeatedly exhalating into the bag and inhaling from the bag, concentrating alveoli air while maintaing simple procedure. but may cause CO 2 accumulation or patient discomfort 21 . Recent studies have predominantly focused on comparing different breath collection devices or pairwise comparisons of two different sampling methods 19 , 22 . In this study, we aim to compare the End-tidal, Continuous (rebreathing) and Whole methods for breath sampling within same experimental settings to assess stability and feasibility for further applications. Moreover, we sought to propose a practical sampling method which detects meaningful VOCs consistently under less controllable conditions. Methods Participants The study was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2107-148-1237) and was designed as a cross-sectional observational study. All methods were carried out in accordance with relevant guidelines and regulations. Healthy adults aged 20–60 years were recruited through a clinical trial recruitment platform approved by Seoul National University Hospital in South Korea. A total of 90 healthy adults (55 females and 35 males) participated in the study. Only non-smokers or ex-smokers with a smoking cessation of more than 2 years were involved in the study. Exclusion criteria included major systemic diseases, history of gastrointestinal surgery, inability to provide informed consent, or non-compliance with pre-sampling restrictions. Volunteers were asked to fast for more than 8 hours and instructed not to consume alcohol 24 hours prior to the sample collection. Written informed consent was obtained from all participants. Sample Collection After routine blood tests, participants rinsed their mouths with clean water and then rested for more than ten minutes to stabilize breathing, during which they completed paper-based health questionnaires. Each participant was provided with three 3-L PTFE (Polytetrafluoroethylene, Scentroid, Canada) bags and a disposable PTFE mouthpiece (15cm long, inner diameter: 6mm). Before the actual breath sampling, they were trained to inhale for 5 seconds and exhale for 5 seconds and received instructions on three breath sampling methods. For comfort of the subjects, breath collection was performed using a disposable mouthpiece without a nose clip. Participants were guided to inhale through the nose and exhale exclusively through the mouth, and sampling was conducted during stable end-tidal exhalation (Fig. 1 (A) ). 3-liter PTFE bags were cleaned by flushing 5 times with synthetic air (N 2 79%, O 2 21%, Daehan, South Korea). Sampling bags were half-filled with the synthetic air bags, sealed and heated in a drying oven at 80°C for at least 30 min. After heating, the bags were flushed for five times with the synthetic air, sealed with PTFE stoppers, and stored at room temperature until use. The PTFE mouthpieces were wiped with 70% ethanol and flushed several times with the synthetic air. A PTFE tape (1 x 2 cm; Chukoh Chemical Industries, Japan) was cut and prepared for participants to block the end of the mouthpieces with a finger of preference to prevent the collected air inside from leakage or contamination. Breath samples were collected using three different methods. In the time-based end-tidal method, participants inhaled ambient air, released the first 2 seconds of exhalation into room air, and then directly exhaled the following 3 seconds into the PTFE bag to collect the end-tidal fraction. During inhalation of ambient air through the nose, other end of the mouthpiece was covered with the PTFE-tape attached finger. For the whole-breath method, participants inhaled ambient air for 5 seconds and exhaled the entire 5 seconds of breath directly into a PTFE bag. While blocking the end of the mouthpiece with the finger, participants inhaled and exhaled into the sampling bag. In the continuous method, participants inhaled clean synthetic air (21% O₂, 79% N₂, Daehan, South Korea) from the PTFE bag and subsequently exhaled for 5 s into the same bag. While holding the mouthpiece, participants repeated this inhalation-exhalation cycle, and breath sampling was completed after three exhalations for each method (Fig. 1 (B)). To avoid any order-related bias, the sequence of the three sampling methods was randomized. One liter of ambient air was also collected into sorbent tubes as background. After breath sampling, participants were asked to complete a brief questionnaire evaluating the most and second most preferred method among the three breath sampling methods. Sample storage and Analysis One liter of collected breath in each method was adsorbed onto sorbent tubes (Tenax + carbonpack, C2-CAXX-5149, Markes International, UK) with a flow rate of 200mL/min with a GilAir air sampling pump (Sensidyne, USA). The tubes were sealed and stored at -20 ℃ until analysis. All samples were analyzed within 4 weeks after sample collection. Sample analysis was carried out with an Autosampler ULTRA-xr coupled with a Unity-xr (Markes International, UK), which were connected to an Agilent 8890 gas chromatograph and an Agilent G7079B mass spectrometer (Agilent Technologies, USA). Prior to analysis of sample batches, the mass spectrometer was autotuned using the HES (High Efficiency Source) tuning procedure. For thermal desorption, sorbent tubes with breath samples were placed inside the autosampler and desorbed at 280°C for 10 minutes, with a helium trap flow of 50 mL/min. Analytes were cryo-focused on a cold trap at 10°C, then heated to 300°C at 25°C /s. Desorption into the GC column was conducted for 3 mins. The transfer line was kept at 150°C, with a desorb split flow of 4 mL/min (effective split ratio ~ 4:1). Desorbed volatiles were transferred to the column DB-5MS UI (60 m × 0.25 mm i.d., 0.25 µm; Agilent Technologies, USA). Volatiles were separated on the column from initial temperature of 40°C (held for 5 minutes), followed by temperature increase of 7°C/ min before reaching 280°C. The final temperature was held for 3 minutes. Peak deconvolution and compound identification were performed using the Unknowns Analysis software (Agilent Technologies, USA) based on NIST 17 mass spectral library. Match factors of 80 and m/z minimum value of 30 was set as the threshold for tentative compound identification. Peaks detected before 20 mins retention time were included in further analysis. Column-derived compounds (e.g., siloxanes) were excluded. Following data pre-processing, statistical analyses were conducted using MetaboAnalyst (Ver 6.0) and R (Ver 4.5.1). For non-parametric pairwise comparisons of the breath sampling methods, Wilcoxon signed-rank tests were applied. To compare VOCs among the three breath sampling methods (Whole, Continuous and End-tidal), Friedman tests were performed. Multiple testing was controlled by the Benjamini–Hochberg procedure, and a false discovery rate (FDR) threshold of q-value < 0.01 was considered statistically significant. Results Overall VOC Identification and Core Set of Common Compounds After deconvolution and compound matching, a total of 1,346 VOCs in breath were identified across three breath sampling methods. Among all compounds, common breath VOCs detected in more than 50 percents of participants were considered representative and consistent within the cohort. In total, 47 VOCs were identified by combining three breath sampling methods (Fig. 2 , Supplementary Table 1 ). 34 VOCs were commonly observed across all three breath sampling methods, while 13 VOCs were detected by a single method or by a combination of two methods. These 34 VOCs may represent a set of core breath VOCs consistently detected in human breath regardless of sampling method. Method-Specific VOC Detection and Overlap Of all VOCs, a total of 38 VOCs were captured and detected using the End-tidal method. Among the detected compounds, 1-methoxy-2-propanol and butyl isocyanato acetate were detected more frequently by End-tidal method. Two VOCs overlapping with those identified by the Continuous method were 2-methyl-1-pentene and 4,7-dimethylundecane. The Continuous method, which had the largest number of VOCs with frequency greater 50 percent among all methods, identified 41 compounds. Uniquely detecting compounds such as 2,4,4-trimethyl-1-pentene, 2,4-dimethyldecane, methyl vinyl ketone and 4-methyloctane, the Continuous method shared 36 breath VOCs with the End-tidal method. While identifying chlorobenzene with the Continuous method, the Whole method detected total 39 VOCs in human breath. VOCs such as 2-ethylhexanol, carbon tetrachloride, dimethyl disulfide, and heptane were identified solely with the Whole method. 34 common breath VOCs were observed in the Whole and the End-tidal method. Accordingly, the Continuous method shared a greater number of breath VOCs (36 VOCs, 94.73%) than the Whole method (34 VOCs, 89.47%) with the End-tidal gold method which is considered as the gold-standard method. Method-Dependent Differences in VOC Intensities To further evaluate statistical differences in the intensities of individual VOCs, non-parametric statistical analyses were performed. According to Friedman tests for comparing three breath sampling methods, a total of four breath VOCs were found to be statistically different (FDR-adjusted q < 0.01): methylcyclohexane, toluene, D-limonene, and 1,1,2-trichloro-1,2,2-trifluoroethane (Fig. 4 ). The Continuous method showed the lowest median intensity (peak area) of methylcyclohexane compared with the other sampling methods (Fig. 4 (A) ). The median peak area value of Toluene was highest in the Whole method and the lowest in the End-tidal method (Fig. 4 (B) ). Among three methods, the Continuous method exhibited the lowest median level of D-limonene (Fig. 4 (C) ). 1,1,2-trichloro-1,2,2-trifluoroethane was detected with the lowest median intensity in the Continuous method (Fig. 4 (D) ). In the Continuous method, multiple VOCs were present at lower levels than other two methods. However, when comparing the three methods, the observed tendencies of VOCs regarding intensity levels were not consistently reproduced. The results indicate that the overall intensity profiles of VOCs did not follow a uniform trend, highlighting method-dependent variability in VOC detection. Pairwise Comparisons Between Methods Pairwise comparisons between two different sampling methods were performed using Wilcoxon signed-rank tests to identify specific method-dependent differences (Fig. 4 , Supplementary Fig. 1 ), and five VOCs were identified to be statistically significant in pairwise comparison of sampling methods (FDR-adjusted q-value < 0.01). Methylcyclohexane showed statistically significant difference between the Whole and the Continuous method, being detected at the lower level in the Continuous method (Fig. 4 (A) ). Toluene exhibited a statistically significant difference between the Whole and End-tidal methods, with a lower median intensity observed in the End-tidal method (Fig. 4 (B) ). D-limonene showed significant differences in both Whole versus Continuous and End-tidal versus Continuous comparisons, in which the Continuous method consistently yielded lower median levels (Fig. 4 (C) ). Similarly, 1,1,2-trichloro-1,2,2-trifluoroethane demonstrated significant differences when comparing Whole with Continuous and End-tidal with Continuous, again with the Continuous method showing the lower median values (Fig. 4 (D) ). Ethylbenzene presented a significant difference between the Whole and End-tidal methods, where the End-tidal method displayed a lower median intensity ( Supplementary Fig. 1 ). Overall, these results again highlight method-dependent variability across specific VOCs. Chemical Class Distribution of Frequently Detected VOCs To further characterize 47 breath VOCs detected from more than half of participants, each compound was classified into its chemical class (Fig. 5 ). Alkanes were the most abundant group, comprising 32.7% of the total compounds. Aromatic hydrocarbons and halogenated alkanes constituted the next predominant chemical groups, with proportions of 12.2% and 8.2%, respectively. Ketones and organosulfur compounds each represented 6.1% of the total. Alkenes, alcohols, aldehydes and halogenated alkenes were next abundant chemical classes, all contributing equally to the total at 4.1 percent. Although accounting for 2 percent of the total, isocyanate esters, ethers, monoterpenes, esters, hemiterpenes, fluorinated ethers, organosilicon compounds and inorganic compounds were detected. Various chemical classes of breath VOCs in the core set (detection frequency > 50%) indicate that a broad range of chemical classes is present in human breath. Participants’ Preference of Sampling Methods When asked about the most convenient sampling method, the majority of participants selected the whole breath method (n = 55), followed by the continuous method (n = 30), while only a few chose the end-tidal method (n = 5) ( Fig. S1 ). Regarding the second most convenient method, the continuous and end-tidal methods were equally chosen (n = 32, each), whereas the whole breath method was less frequently selected (n = 26). These responses suggest that perceived convenience differed among breath sampling methods, with no single approach being universally preferred. Discussion In this study, breath samples from 90 non-smoking healthy adults were collected, analyzed and compared across the three breath sampling methods: Whole, Continuous and End-tidal. 34 common VOCs out of total 1,346 VOCs were detected by all methods, establishing a core set of breath VOCs which appear robustly regardless of methods. VOCs that are solely detected by specific methods were also observed. Although the PCA plot drawn for multivariate analysis of three breath sampling methods did not demonstrate a clear separation of the three sampling methods, meaningful differences in the intensity levels of commonly detected VOCs were revealed. To our knowledge, no previous study has directly compared these three breath sampling methods within a single cohort, making this work a unique contribution to the field. Previous studies have acknowledged reproducibility and consistency of the End-tidal method since end-tidal breath consists of air exchanged in the alveoli, where gas exchange occurs directly with the blood. Therefore, it contains more VOCs that closely reflect systemic metabolic processes in the body, making it optimal for analyzing endogenous substances related to metabolism 23 24 . Our study found that the Continuous method identified 34 VOCs which overlap with those in the End-tidal method, which was about 5.2% more than the Whole method (Fig. 2 ). Given that the End-tidal method is considered as the reference standard method, the Continuous method exhibits higher consistency with the End-tidal method. VOCs detected consistently (frequency > 50%) differ depending on the sampling method. Moreover, the intensities of VOCs were found to vary across different sampling procedures. When comparing the intensities of individual VOCs, methylcyclohexane, toluene, D-limonene, ethylbenzene and 1,1,2-trichloro-1,2,2-trifluorothane were statistically significant compounds as a result of Friedman tests and paired Wilcoxon rank sum tests (Fig. 4 , Supplementary Fig. 1 ). However, seeing from the box plots of intensities for each VOC, the comparative order between methods were not always consistent. It shows that the trend is compound-dependent which aligns with findings from previous studies 25 , 26 . The pie chart reporting profiles of chemical classes in frequently detected breath VOCs (Fig. 5 ) also support the variability of VOCs intensity among the sampling methods 27 , 28 . Although these VOCs were known to be detected in human breaths, their origins are mostly from exogenous sources such as food derivatives, industrial ingredients or environmental factors 29 – 32 . Exogenous compounds are not to be merely regarded as contaminants, since their interpretation can provide insightful findings in human VOCs analysis. However, endogenous compounds hold greater significance in biological understanding. While acknowledging this, we also considered the feasibility of adopting a more user-friendly sampling procedure that could be more easily applied to patients in clinical or bedside settings. While user preferences varied for the survey on evaluating three sampling methods, the Continuous method was consistently rated acceptable and produced VOC profiles highly similar to those obtained with the End-tidal method ( Supplementary Fig. 2 ). Whole breath, although perceived as the most convenient, may be more influenced by exogenous compounds coming from dead space air 20 , 33 . End-tidal method received relatively few preferences, likely due to additional procedures required for time-based sampling. Beyond the practical concerns of our study design, to eliminate dead space air, sampling end-tidal fraction often requires devices for monitoring CO 2 levels and controlled sampling based on those levels, which may lead to extra expenses and background VOC contamination 34 . Although the End-tidal method is firmly recognized for its enrichment of alveolar air and the whole method is appreciated for its user-friendly procedure, a more practical method that properly captures endogenous compounds in various sampling contexts remains necessary. Through rebreathing (Continuous), endogenous VOCs tend to gradually accumulate inside the sampling bag. This procedure diminishes the effects of dead space air and background contamination, providing breath samples which better represent systematic biological activity 21 , 35 . Moreover, the Continuous method enables the participant to breathe normally, requiring little effort or coordination. However, carbon dioxide levels could rise as the number of rebreathing cycle increases, causing a CO 2 level increase, patient discomfort such as chest tightness or dizziness 24 . To prevent respiratory discomfort, our study was limited to three times of breathing (inhalation and exhalation). Participants first inhaled a clean air source inside the sampling bag to minimize the effect of ambient air contamination. Overall, these considerations led the Continuous method to contain a considerable number of VOCs similar to those obtained with the End-tidal method, while exhibiting practicality and acceptability from the participants. However, several limitations should be acknowledged in this study. Although ambient air was collected prior to breath sampling for removal of background signals, environmental factors were not fully controlled and the contribution of background air to breath VOCs were not precisely determined. In addition, standardized quality control (QC) procedures such as external QC samples or replicate tube analyses were not included. Nevertheless, the primary aim of this work was not to establish an absolute VOC reference library but rather to compare different breath sampling methods under identical analytical conditions. Although 90 participants constitute a relatively large cohort, further validations considering factors such as gender, age and BMI will be necessary. Multi-center validation across laboratories and diverse populations will be essential to confirm reproducibility and generalizability. We plan to further analyze VOCs present in blood and urine to understand correlations with breath VOCs. Although complete elimination of background or environmental contamination remains challenging in the field of study, ongoing efforts toward improving environmental control remain essential for the advancement of breath analysis. Conclusion In conclusion, this study provides one of the first direct comparisons of the Whole, Continuous, and End-tidal breath sampling methods in a large cohort of healthy adults. We identified a core set of 34 VOCs consistently detected across all three methods, emphasizing the robustness of breath analysis while also revealing method-dependent differences in individual VOCs and intensity levels. These findings suggest that no single method can be regarded as universally superior; instead, the choice of sampling strategy should be guided by study context, clinical needs, and the physicochemical properties of target VOCs. While the End-tidal method remains firm as a reference standard, the Continuous method demonstrated a favorable balance between user acceptability and analytical comparability, supporting its potential as a practical alternative, particularly in point-of-care or clinical settings. Our study suggests that the Continuous method may serve as a user-friendly and simple procedure for precise breath sampling, even in elderly or ill patients, supporting the translation of breathomics into clinical applications. Declarations Ethics approval and consent to participate This research does not involve any ethical issues. Competing interests The authors declare no competing interests. Funding The Authors received NO FUNDING for this work Author Contribution Conceptualization, H.C.; Methodology, S.H.; Investigation, S.H.; Data Curation, S.H.; Formal Analysis, S.H.; Writing – Original Draft Preparation, S.H.; Writing – Review & Editing, S.H. and H.C.; Supervision, H.C.All authors reviewed and approved the final manuscript. Acknowledgements We sincerely thank all volunteers for their participation in this study. Data Availability The data that supports the findings of this study are available from the corresponding author, upon reasonable request. References Kim, K. 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Development of a protocol to measure volatile organic compounds in human breath: a comparison of rebreathing and on-line single exhalations using proton transfer reaction mass spectrometry. Physiol. Meas. 29 , 309–330 (2008). Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supplementarycomparisonofthreebreathsamplingmethods.pdf Table1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8519105","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":590148045,"identity":"ec951ec6-e207-4d20-9564-c0cf9daa8885","order_by":0,"name":"Seiyoung Hwang","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Seiyoung","middleName":"","lastName":"Hwang","suffix":""},{"id":590148046,"identity":"4db523c8-b7c4-4ddf-a9a3-e3e85a1e5e10","order_by":1,"name":"Hyunsoo Chung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACAwYGNhAthyRApBZj0rUkNhCtxZyB+dlj3h216fNn9xgw/KhhMDZvIKDFsoHN3Jj3zPHcDXfOGDD2HGMwkzlAyGEHeNikeduO5W6QyDFg4G1gsJEg5DCYlnT5GTkGjH9J0FKTwHAjx4AZaIsZYS2H2cwk57YdMNxwI63gsMwxCWPCWo43P5N421YnLz8jeePDNzU2hjMIaWFgBpOHweQBBgaCdsBBHdEqR8EoGAWjYAQCAE8fNg9xARUuAAAAAElFTkSuQmCC","orcid":"","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Hyunsoo","middleName":"","lastName":"Chung","suffix":""}],"badges":[],"createdAt":"2026-01-05 08:54:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8519105/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8519105/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102598048,"identity":"74059000-efd0-41bb-b4d7-cf16b2228e87","added_by":"auto","created_at":"2026-02-13 12:27:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":512101,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic illustration of breath sampling methods. (A) Schematic diagram of inhalation and exhalation through the mouth into a PTFE sampling bag. (B) Visual depiction of the three breath sampling procedures; End-tidal, Whole, and Continuous. End-tidal method: the participant inhale ambient air for 5s, exhale first two seconds into ambient air and next three seconds of breath into the sampling bag shown in (A). Whole method: the participant inhales ambient air for 5s and exhales whole 5s into the sampling bag. Continuous method: the participant inhales clean air in the PTFE bag and exhales directly into the sampling bag. Then, the participant breathes the collected air inside\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/5b8409e604f4eb8d7bd87ae0.jpeg"},{"id":102598053,"identity":"ea016dd8-9bdc-44b9-8bec-58fa8e068b92","added_by":"auto","created_at":"2026-02-13 12:27:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":183487,"visible":true,"origin":"","legend":"\u003cp\u003eA Venn diagram to illustrate the number of breath volatile organic compounds (VOCs) detected in more than half of the participants for each sampling method, with overlapping areas representing common VOCs across methods (detection frequency \u0026gt; 50%). (Orange: Whole, Green: Continuous, Blue: End-tidal)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/b2ab9dce01a7c7fd9a73eb9c.jpeg"},{"id":102598054,"identity":"aeda2970-db37-4423-96fe-756d496015ad","added_by":"auto","created_at":"2026-02-13 12:27:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125178,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of breath volatile organic compounds(VOCs) profiles obtained using the three breath sampling methods. Each dot represents an individual participant, colored according to sampling method (Green: Continuous, Blue: End-tidal, Orange: Whole breath). The first two principal components (PC1 and PC2) explained 21.8% and 10.6% of the total variance, respectively.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/0f993de50ac008587bec2683.png"},{"id":102598061,"identity":"1c359b1c-e8b4-4c7b-832e-0c0c45230c00","added_by":"auto","created_at":"2026-02-13 12:27:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":155140,"visible":true,"origin":"","legend":"\u003cp\u003eBox plots to display statistically significant volatile organic compounds (VOCs) as a comparison of three breath sampling methods. Statistical significance was evaluated using Friedman tests (False Discovery Rate-adjusted q-values by the Benjamini–Hochberg method) for comparing three breath sampling methods. Pairwise comparisons between two different breath sampling methods were performed with Wilcoxon signed-rank tests, and adjusted p-values are shown above the connecting lines. (A) methylcyclohexane, (B) toluene, (C) D-limonene, and (D) 1,1,2-trichloro-1,2,2-trifluoroethane.Y-axis represents log-transformed peak area value. (Orange: Whole, Green: Continuous, Blue: End-tidal)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/0d07c6e97c2808a3fe3162cf.png"},{"id":102598065,"identity":"7e4da8b2-db5c-470a-a0fc-4658f7f6c62f","added_by":"auto","created_at":"2026-02-13 12:27:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":115327,"visible":true,"origin":"","legend":"\u003cp\u003eA pie chart depicting the chemical group distribution of 47 breath volatile organic compounds detected with frequency higher than 50 percent. Each color of pie chart represents different chemical class (label on the right corner).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/32bbe79e89d4a7ffb9a831f7.png"},{"id":104014108,"identity":"bb58b445-f252-4daa-aff5-8875538bce89","added_by":"auto","created_at":"2026-03-05 16:26:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1703683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/c95ae8a0-9ee6-41ff-aaa3-4597fbd65d9d.pdf"},{"id":102598099,"identity":"b9a55854-1b0c-4809-a312-6f7b6b1657c8","added_by":"auto","created_at":"2026-02-13 12:27:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":320623,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarycomparisonofthreebreathsamplingmethods.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/64182341fc2ce227b76808da.pdf"},{"id":102598058,"identity":"169d4cf5-a019-402d-b42e-a7eed3441743","added_by":"auto","created_at":"2026-02-13 12:27:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27489,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8519105/v1/5fc61553f1e51ea40c5fe123.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of three breath sampling methods for volatile organic compounds analysis in healthy adults: End-Tidal, Whole, and Continuous","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreath volatile organic compounds (VOCs) are intensively researched as potential biomarkers, given their non-invasive sampling, collection simplicity and reflection of various physiological and pathological statuses \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Exhaled breath contains hundreds of VOCs originating from endogenous metabolic events or exogenous sources, which provide various information about human body. Breath VOCs have attracted growing interest in both academic and clinical fields, with applications in infectious diseases, metabolic disorders, and various cancers \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These advances are further supported by the development of novel analytical and computational approaches, including sensors, electronic nose (e-nose) devices and machine learning\u0026ndash;based algorithms, which enhance pattern recognition and diagnostic accuracy \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. As healthcare demands for early disease diagnostics and point-of-care testing continue to rise and expand, the non-invasive nature of breath sampling and the abundance of breath samples make breathomics an attractive approach for both researchers and patients.\u003c/p\u003e \u003cp\u003eHowever, despite these numerous advantages, breath analysis has several limitations to overcome. Since VOCs exist only at trace levels in human breaths, sensitive control of sampling, sophisticated analytical instruments and proper background correction are required \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Breath volatiles can fluctuate depending on factors such as diet, smoking, environmental factors, and behavioral patterns such as sleep or workouts \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, standardized methods for breath sampling are still in question, which pose challenges for quantifications, reproducibility and broader clinical applications.\u003c/p\u003e \u003cp\u003eWhile debates on the most appropriate breath sampling methods remain, the alveolar fraction of breath is widely recognized for its advantages in accuracy and physiological relevance and thus has been regarded as the most reliable fraction of exhaled air for clinical and translational applications. Because alveolar gases concentrations are approximately close to those present in the actual arterial blood, end-tidal air is known to mirror biological conditions. Moreover, collection of end-tidal air minimizes effect of contamination from external environment and dead space air. To collect the end-tidal fraction, capnography-guided devices such as ReCIVA\u0026trade; are often used for sampling \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Although the advantages of end-tidal sampling are evident, end-tidal collection usually requires costly devices and residual effects from environmental contamination or dead space still remain \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In contrast, Whole breath is collecting the entire volume of exhalation. Procedure is easy for subjects to follow, however may lead to dilution of endogenous VOCs due to the presence of dead space air or oral VOCs emissions \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The rebreathing (Continuous) approach is the process of repeatedly exhalating into the bag and inhaling from the bag, concentrating alveoli air while maintaing simple procedure. but may cause CO\u003csub\u003e2\u003c/sub\u003e accumulation or patient discomfort \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent studies have predominantly focused on comparing different breath collection devices or pairwise comparisons of two different sampling methods \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In this study, we aim to compare the End-tidal, Continuous (rebreathing) and Whole methods for breath sampling within same experimental settings to assess stability and feasibility for further applications. Moreover, we sought to propose a practical sampling method which detects meaningful VOCs consistently under less controllable conditions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003e The study was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2107-148-1237) and was designed as a cross-sectional observational study. All methods were carried out in accordance with relevant guidelines and regulations. Healthy adults aged 20\u0026ndash;60 years were recruited through a clinical trial recruitment platform approved by Seoul National University Hospital in South Korea. A total of 90 healthy adults (55 females and 35 males) participated in the study. Only non-smokers or ex-smokers with a smoking cessation of more than 2 years were involved in the study. Exclusion criteria included major systemic diseases, history of gastrointestinal surgery, inability to provide informed consent, or non-compliance with pre-sampling restrictions. Volunteers were asked to fast for more than 8 hours and instructed not to consume alcohol 24 hours prior to the sample collection. Written informed consent was obtained from all participants.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003e After routine blood tests, participants rinsed their mouths with clean water and then rested for more than ten minutes to stabilize breathing, during which they completed paper-based health questionnaires. Each participant was provided with three 3-L PTFE (Polytetrafluoroethylene, Scentroid, Canada) bags and a disposable PTFE mouthpiece (15cm long, inner diameter: 6mm). Before the actual breath sampling, they were trained to inhale for 5 seconds and exhale for 5 seconds and received instructions on three breath sampling methods. For comfort of the subjects, breath collection was performed using a disposable mouthpiece without a nose clip. Participants were guided to inhale through the nose and exhale exclusively through the mouth, and sampling was conducted during stable end-tidal exhalation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e(A)\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3-liter PTFE bags were cleaned by flushing 5 times with synthetic air (N\u003csub\u003e2\u003c/sub\u003e 79%, O\u003csub\u003e2\u003c/sub\u003e 21%, Daehan, South Korea). Sampling bags were half-filled with the synthetic air bags, sealed and heated in a drying oven at 80\u0026deg;C for at least 30 min. After heating, the bags were flushed for five times with the synthetic air, sealed with PTFE stoppers, and stored at room temperature until use. The PTFE mouthpieces were wiped with 70% ethanol and flushed several times with the synthetic air. A PTFE tape (1 x 2 cm; Chukoh Chemical Industries, Japan) was cut and prepared for participants to block the end of the mouthpieces with a finger of preference to prevent the collected air inside from leakage or contamination.\u003c/p\u003e \u003cp\u003eBreath samples were collected using three different methods. In the time-based end-tidal method, participants inhaled ambient air, released the first 2 seconds of exhalation into room air, and then directly exhaled the following 3 seconds into the PTFE bag to collect the end-tidal fraction. During inhalation of ambient air through the nose, other end of the mouthpiece was covered with the PTFE-tape attached finger. For the whole-breath method, participants inhaled ambient air for 5 seconds and exhaled the entire 5 seconds of breath directly into a PTFE bag. While blocking the end of the mouthpiece with the finger, participants inhaled and exhaled into the sampling bag. In the continuous method, participants inhaled clean synthetic air (21% O₂, 79% N₂, Daehan, South Korea) from the PTFE bag and subsequently exhaled for 5 s into the same bag. While holding the mouthpiece, participants repeated this inhalation-exhalation cycle, and breath sampling was completed after three exhalations for each method (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(B)). To avoid any order-related bias, the sequence of the three sampling methods was randomized. One liter of ambient air was also collected into sorbent tubes as background. After breath sampling, participants were asked to complete a brief questionnaire evaluating the most and second most preferred method among the three breath sampling methods.\u003c/p\u003e\n\u003ch3\u003eSample storage and Analysis\u003c/h3\u003e\n\u003cp\u003eOne liter of collected breath in each method was adsorbed onto sorbent tubes (Tenax\u0026thinsp;+\u0026thinsp;carbonpack, C2-CAXX-5149, Markes International, UK) with a flow rate of 200mL/min with a GilAir air sampling pump (Sensidyne, USA). The tubes were sealed and stored at -20 ℃ until analysis. All samples were analyzed within 4 weeks after sample collection.\u003c/p\u003e \u003cp\u003eSample analysis was carried out with an Autosampler ULTRA-xr coupled with a Unity-xr (Markes International, UK), which were connected to an Agilent 8890 gas chromatograph and an Agilent G7079B mass spectrometer (Agilent Technologies, USA). Prior to analysis of sample batches, the mass spectrometer was autotuned using the HES (High Efficiency Source) tuning procedure. For thermal desorption, sorbent tubes with breath samples were placed inside the autosampler and desorbed at 280\u0026deg;C for 10 minutes, with a helium trap flow of 50 mL/min. Analytes were cryo-focused on a cold trap at 10\u0026deg;C, then heated to 300\u0026deg;C at 25\u0026deg;C /s. Desorption into the GC column was conducted for 3 mins. The transfer line was kept at 150\u0026deg;C, with a desorb split flow of 4 mL/min (effective split ratio\u0026thinsp;~\u0026thinsp;4:1). Desorbed volatiles were transferred to the column DB-5MS UI (60 m \u0026times; 0.25 mm i.d., 0.25 \u0026micro;m; Agilent Technologies, USA). Volatiles were separated on the column from initial temperature of 40\u0026deg;C (held for 5 minutes), followed by temperature increase of 7\u0026deg;C/ min before reaching 280\u0026deg;C. The final temperature was held for 3 minutes.\u003c/p\u003e \u003cp\u003ePeak deconvolution and compound identification were performed using the Unknowns Analysis software (Agilent Technologies, USA) based on NIST 17 mass spectral library. Match factors of 80 and m/z minimum value of 30 was set as the threshold for tentative compound identification. Peaks detected before 20 mins retention time were included in further analysis. Column-derived compounds (e.g., siloxanes) were excluded. Following data pre-processing, statistical analyses were conducted using MetaboAnalyst (Ver 6.0) and R (Ver 4.5.1). For non-parametric pairwise comparisons of the breath sampling methods, Wilcoxon signed-rank tests were applied. To compare VOCs among the three breath sampling methods (Whole, Continuous and End-tidal), Friedman tests were performed. Multiple testing was controlled by the Benjamini\u0026ndash;Hochberg procedure, and a false discovery rate (FDR) threshold of q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOverall VOC Identification and Core Set of Common Compounds\u003c/h2\u003e \u003cp\u003eAfter deconvolution and compound matching, a total of 1,346 VOCs in breath were identified across three breath sampling methods. Among all compounds, common breath VOCs detected in more than 50 percents of participants were considered representative and consistent within the cohort. In total, 47 VOCs were identified by combining three breath sampling methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e). 34 VOCs were commonly observed across all three breath sampling methods, while 13 VOCs were detected by a single method or by a combination of two methods. These 34 VOCs may represent a set of core breath VOCs consistently detected in human breath regardless of sampling method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMethod-Specific VOC Detection and Overlap\u003c/h2\u003e \u003cp\u003eOf all VOCs, a total of 38 VOCs were captured and detected using the End-tidal method. Among the detected compounds, 1-methoxy-2-propanol and butyl isocyanato acetate were detected more frequently by End-tidal method. Two VOCs overlapping with those identified by the Continuous method were 2-methyl-1-pentene and 4,7-dimethylundecane. The Continuous method, which had the largest number of VOCs with frequency greater 50 percent among all methods, identified 41 compounds. Uniquely detecting compounds such as 2,4,4-trimethyl-1-pentene, 2,4-dimethyldecane, methyl vinyl ketone and 4-methyloctane, the Continuous method shared 36 breath VOCs with the End-tidal method. While identifying chlorobenzene with the Continuous method, the Whole method detected total 39 VOCs in human breath. VOCs such as 2-ethylhexanol, carbon tetrachloride, dimethyl disulfide, and heptane were identified solely with the Whole method. 34 common breath VOCs were observed in the Whole and the End-tidal method. Accordingly, the Continuous method shared a greater number of breath VOCs (36 VOCs, 94.73%) than the Whole method (34 VOCs, 89.47%) with the End-tidal gold method which is considered as the gold-standard method.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethod-Dependent Differences in VOC Intensities\u003c/h3\u003e\n\u003cp\u003eTo further evaluate statistical differences in the intensities of individual VOCs, non-parametric statistical analyses were performed. According to Friedman tests for comparing three breath sampling methods, a total of four breath VOCs were found to be statistically different (FDR-adjusted q\u0026thinsp;\u0026lt;\u0026thinsp;0.01): methylcyclohexane, toluene, D-limonene, and 1,1,2-trichloro-1,2,2-trifluoroethane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The Continuous method showed the lowest median intensity (peak area) of methylcyclohexane compared with the other sampling methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(A)\u003c/b\u003e). The median peak area value of Toluene was highest in the Whole method and the lowest in the End-tidal method (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(B)\u003c/b\u003e). Among three methods, the Continuous method exhibited the lowest median level of D-limonene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(C)\u003c/b\u003e). 1,1,2-trichloro-1,2,2-trifluoroethane was detected with the lowest median intensity in the Continuous method (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(D)\u003c/b\u003e). In the Continuous method, multiple VOCs were present at lower levels than other two methods. However, when comparing the three methods, the observed tendencies of VOCs regarding intensity levels were not consistently reproduced. The results indicate that the overall intensity profiles of VOCs did not follow a uniform trend, highlighting method-dependent variability in VOC detection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePairwise Comparisons Between Methods\u003c/h2\u003e \u003cp\u003ePairwise comparisons between two different sampling methods were performed using Wilcoxon signed-rank tests to identify specific method-dependent differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e), and five VOCs were identified to be statistically significant in pairwise comparison of sampling methods (FDR-adjusted q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Methylcyclohexane showed statistically significant difference between the Whole and the Continuous method, being detected at the lower level in the Continuous method (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(A)\u003c/b\u003e). Toluene exhibited a statistically significant difference between the Whole and End-tidal methods, with a lower median intensity observed in the End-tidal method (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(B)\u003c/b\u003e). D-limonene showed significant differences in both Whole versus Continuous and End-tidal versus Continuous comparisons, in which the Continuous method consistently yielded lower median levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(C)\u003c/b\u003e). Similarly, 1,1,2-trichloro-1,2,2-trifluoroethane demonstrated significant differences when comparing Whole with Continuous and End-tidal with Continuous, again with the Continuous method showing the lower median values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(D)\u003c/b\u003e). Ethylbenzene presented a significant difference between the Whole and End-tidal methods, where the End-tidal method displayed a lower median intensity (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Overall, these results again highlight method-dependent variability across specific VOCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChemical Class Distribution of Frequently Detected VOCs\u003c/h2\u003e \u003cp\u003e To further characterize 47 breath VOCs detected from more than half of participants, each compound was classified into its chemical class (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Alkanes were the most abundant group, comprising 32.7% of the total compounds. Aromatic hydrocarbons and halogenated alkanes constituted the next predominant chemical groups, with proportions of 12.2% and 8.2%, respectively. Ketones and organosulfur compounds each represented 6.1% of the total. Alkenes, alcohols, aldehydes and halogenated alkenes were next abundant chemical classes, all contributing equally to the total at 4.1 percent. Although accounting for 2 percent of the total, isocyanate esters, ethers, monoterpenes, esters, hemiterpenes, fluorinated ethers, organosilicon compounds and inorganic compounds were detected. Various chemical classes of breath VOCs in the core set (detection frequency\u0026thinsp;\u0026gt;\u0026thinsp;50%) indicate that a broad range of chemical classes is present in human breath.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u0026rsquo; Preference of Sampling Methods\u003c/h2\u003e \u003cp\u003eWhen asked about the most convenient sampling method, the majority of participants selected the whole breath method (n\u0026thinsp;=\u0026thinsp;55), followed by the continuous method (n\u0026thinsp;=\u0026thinsp;30), while only a few chose the end-tidal method (n\u0026thinsp;=\u0026thinsp;5) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Regarding the second most convenient method, the continuous and end-tidal methods were equally chosen (n\u0026thinsp;=\u0026thinsp;32, each), whereas the whole breath method was less frequently selected (n\u0026thinsp;=\u0026thinsp;26). These responses suggest that perceived convenience differed among breath sampling methods, with no single approach being universally preferred.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, breath samples from 90 non-smoking healthy adults were collected, analyzed and compared across the three breath sampling methods: Whole, Continuous and End-tidal. 34 common VOCs out of total 1,346 VOCs were detected by all methods, establishing a core set of breath VOCs which appear robustly regardless of methods. VOCs that are solely detected by specific methods were also observed. Although the PCA plot drawn for multivariate analysis of three breath sampling methods did not demonstrate a clear separation of the three sampling methods, meaningful differences in the intensity levels of commonly detected VOCs were revealed. To our knowledge, no previous study has directly compared these three breath sampling methods within a single cohort, making this work a unique contribution to the field.\u003c/p\u003e \u003cp\u003ePrevious studies have acknowledged reproducibility and consistency of the End-tidal method since end-tidal breath consists of air exchanged in the alveoli, where gas exchange occurs directly with the blood. Therefore, it contains more VOCs that closely reflect systemic metabolic processes in the body, making it optimal for analyzing endogenous substances related to metabolism \u003csup\u003e23 24\u003c/sup\u003e. Our study found that the Continuous method identified 34 VOCs which overlap with those in the End-tidal method, which was about 5.2% more than the Whole method (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Given that the End-tidal method is considered as the reference standard method, the Continuous method exhibits higher consistency with the End-tidal method.\u003c/p\u003e \u003cp\u003eVOCs detected consistently (frequency\u0026thinsp;\u0026gt;\u0026thinsp;50%) differ depending on the sampling method. Moreover, the intensities of VOCs were found to vary across different sampling procedures. When comparing the intensities of individual VOCs, methylcyclohexane, toluene, D-limonene, ethylbenzene and 1,1,2-trichloro-1,2,2-trifluorothane were statistically significant compounds as a result of Friedman tests and paired Wilcoxon rank sum tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). However, seeing from the box plots of intensities for each VOC, the comparative order between methods were not always consistent. It shows that the trend is compound-dependent which aligns with findings from previous studies \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The pie chart reporting profiles of chemical classes in frequently detected breath VOCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) also support the variability of VOCs intensity among the sampling methods \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Although these VOCs were known to be detected in human breaths, their origins are mostly from exogenous sources such as food derivatives, industrial ingredients or environmental factors \u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Exogenous compounds are not to be merely regarded as contaminants, since their interpretation can provide insightful findings in human VOCs analysis. However, endogenous compounds hold greater significance in biological understanding.\u003c/p\u003e \u003cp\u003eWhile acknowledging this, we also considered the feasibility of adopting a more user-friendly sampling procedure that could be more easily applied to patients in clinical or bedside settings. While user preferences varied for the survey on evaluating three sampling methods, the Continuous method was consistently rated acceptable and produced VOC profiles highly similar to those obtained with the End-tidal method (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). Whole breath, although perceived as the most convenient, may be more influenced by exogenous compounds coming from dead space air \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. End-tidal method received relatively few preferences, likely due to additional procedures required for time-based sampling. Beyond the practical concerns of our study design, to eliminate dead space air, sampling end-tidal fraction often requires devices for monitoring CO\u003csub\u003e2\u003c/sub\u003e levels and controlled sampling based on those levels, which may lead to extra expenses and background VOC contamination \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Although the End-tidal method is firmly recognized for its enrichment of alveolar air and the whole method is appreciated for its user-friendly procedure, a more practical method that properly captures endogenous compounds in various sampling contexts remains necessary.\u003c/p\u003e \u003cp\u003eThrough rebreathing (Continuous), endogenous VOCs tend to gradually accumulate inside the sampling bag. This procedure diminishes the effects of dead space air and background contamination, providing breath samples which better represent systematic biological activity \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Moreover, the Continuous method enables the participant to breathe normally, requiring little effort or coordination. However, carbon dioxide levels could rise as the number of rebreathing cycle increases, causing a CO\u003csub\u003e2\u003c/sub\u003e level increase, patient discomfort such as chest tightness or dizziness \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To prevent respiratory discomfort, our study was limited to three times of breathing (inhalation and exhalation). Participants first inhaled a clean air source inside the sampling bag to minimize the effect of ambient air contamination. Overall, these considerations led the Continuous method to contain a considerable number of VOCs similar to those obtained with the End-tidal method, while exhibiting practicality and acceptability from the participants.\u003c/p\u003e \u003cp\u003eHowever, several limitations should be acknowledged in this study. Although ambient air was collected prior to breath sampling for removal of background signals, environmental factors were not fully controlled and the contribution of background air to breath VOCs were not precisely determined. In addition, standardized quality control (QC) procedures such as external QC samples or replicate tube analyses were not included. Nevertheless, the primary aim of this work was not to establish an absolute VOC reference library but rather to compare different breath sampling methods under identical analytical conditions. Although 90 participants constitute a relatively large cohort, further validations considering factors such as gender, age and BMI will be necessary. Multi-center validation across laboratories and diverse populations will be essential to confirm reproducibility and generalizability. We plan to further analyze VOCs present in blood and urine to understand correlations with breath VOCs. Although complete elimination of background or environmental contamination remains challenging in the field of study, ongoing efforts toward improving environmental control remain essential for the advancement of breath analysis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study provides one of the first direct comparisons of the Whole, Continuous, and End-tidal breath sampling methods in a large cohort of healthy adults. We identified a core set of 34 VOCs consistently detected across all three methods, emphasizing the robustness of breath analysis while also revealing method-dependent differences in individual VOCs and intensity levels. These findings suggest that no single method can be regarded as universally superior; instead, the choice of sampling strategy should be guided by study context, clinical needs, and the physicochemical properties of target VOCs. While the End-tidal method remains firm as a reference standard, the Continuous method demonstrated a favorable balance between user acceptability and analytical comparability, supporting its potential as a practical alternative, particularly in point-of-care or clinical settings. Our study suggests that the Continuous method may serve as a user-friendly and simple procedure for precise breath sampling, even in elderly or ill patients, supporting the translation of breathomics into clinical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eThis research does not involve any ethical issues.\u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe Authors received \u003cb\u003eNO FUNDING\u003c/b\u003e for this work\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, H.C.; Methodology, S.H.; Investigation, S.H.; Data Curation, S.H.; Formal Analysis, S.H.; Writing \u0026ndash; Original Draft Preparation, S.H.; Writing \u0026ndash; Review \u0026amp; Editing, S.H. and H.C.; Supervision, H.C.All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe sincerely thank all volunteers for their participation in this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that supports the findings of this study are available from the corresponding author, upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKim, K. 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Critical Review of Volatile Organic Compound Analysis in Breath and In Vitro Cell Culture for Detection of Lung Cancer. \u003cem\u003eMetabolites\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Hara, M. E., O'Hehir, S., Green, S. \u0026amp; Mayhew, C. A. Development of a protocol to measure volatile organic compounds in human breath: a comparison of rebreathing and on-line single exhalations using proton transfer reaction mass spectrometry. \u003cem\u003ePhysiol. Meas.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 309\u0026ndash;330 (2008).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breath analysis, Volatile organic compounds, Breath sampling methods, Gas chromatography-mass spectrometry","lastPublishedDoi":"10.21203/rs.3.rs-8519105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8519105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreath volatile organic compounds (VOCs) are promising biospecimens to be potentially utilized as non-invasive biomarkers. In this study, we analyzed breath samples from 90 non-smoking healthy adults using three sampling methods\u0026mdash;Whole, Continuous, and End-tidal\u0026mdash;to compare and evaluate patterns of breath VOCs detected by each. A total of 1,346 VOCs were identified, with 34 consistently detected across all methods, forming a core set of robust compounds. While principal component analysis showed no distinct clustering by method, several VOCs, including methylcyclohexane, toluene, D-Limonene, ethylbenzene and 1,1,2-trichloro-1,2,2-trifluoroethane, displayed method-dependent intensity differences. The Continuous method tended to yield lower values, but overall VOC profiles remained comparable to those from End-tidal samples. Participant surveys indicated that Whole breath was perceived as most convenient, whereas the Continuous method achieved consistent acceptability. These findings suggest that although End-tidal sampling remains the reference method, the Continuous method may serve as a practical and user-friendly alternative, particularly when alveolar air collection is not feasible. By establishing healthy reference VOCs and comparing the feasibility of different approaches, this study provides a foundation for applying breath analysis more flexibly in future clinical and translational research.\u003c/p\u003e","manuscriptTitle":"Comparison of three breath sampling methods for volatile organic compounds analysis in healthy adults: End-Tidal, Whole, and Continuous","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 12:26:59","doi":"10.21203/rs.3.rs-8519105/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d87a73d-fa66-4b0d-b0eb-24924bda992d","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62794587,"name":"Health sciences/Biomarkers"},{"id":62794588,"name":"Physical sciences/Chemistry"},{"id":62794589,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-03-05T16:26:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 12:26:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8519105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8519105","identity":"rs-8519105","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}