Influence of Ethanol on Breath Acetone Measurements Using ... - MDPI

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Influence of Ethanol on Breath Acetone Measurements Using an External Cavity Quantum Cascade Laser Raymund Centeno, Julien Mandon, Frans J. M. Harren and Simona M. Cristescu * Department of Molecular & Laser Physics, IMM, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands; [email protected] (R.C.); [email protected] (J.M.); [email protected] (F.J.M.H.) * Correspondence: [email protected]; Tel.: +31-024-365-2277 Received: 31 March 2016; Accepted: 24 April 2016; Published: 27 April 2016

Abstract: Broadly tunable external cavity quantum cascade lasers (EC-QCLs) in combination with off-axis integrated cavity enhanced spectroscopy (OA-ICOS) provide high molecular gas sensitivity and selectivity. We used an EC-QCL in the region of 1150–1300 cm´1 in both broadband scan mode, as well as narrow scanning mode around 1216 cm´1 , respectively, for detection of acetone in exhaled breath. This wavelength region is essential for accurate determination of breath acetone due to the relative low spectral influence of other endogenous molecules like water, carbon dioxide or methane. We demonstrated that ethanol has a strong spectroscopic influence on the acetone concentration in exhaled breath, an important detail that has been overlooked so far. An ethanol correction is proposed and validated with the reference measurements from a proton-transfer reaction mass spectrometer (PTR-MS) for the same breath samples from ten persons. With the ethanol correction, both broadband and narrowband molecular spectroscopy represent an attractive way to accurately assess the exhaled breath acetone. The importance of considering spectroscopic ethanol influence is essential, especially for the narrowband scans, (e.g., 1216 cm´1 ), for which the error in determining the acetone concentrations can rise up to 39% if it is not considered. Keywords: external cavity quantum cascade lasers (EC-QCLs); off-axis integrated cavity enhanced spectroscopy (OA-ICOS); broadband scan; narrowband scan; exhaled acetone; ethanol

1. Introduction Accurate determination of concentration levels of metabolites in human breath offers a valuable non-invasive way for disease diagnosis and metabolic status monitoring [1]. Normal human breath contains common atmospheric gases such as H2 O, CO2 , N2 and O2 in relatively high concentrations, and hundreds of volatile organic compounds (VOCs), among which acetone and ethanol are two examples of the most abundant ones with levels varying from tens of parts-per-billion volume (ppbv) to parts-per-million volume (ppmv) [1,2]. Acetone may represent a promising alternative for metabolic monitoring, providing real-time information on fat burning [3]. Elevated breath acetone concentrations have been reported in patients presenting with diabetes [4], persons dieting (i.e., Atkins Diet) or fasting [5,6], persons after exercising [7] or in patients after cardiovascular surgery due to metabolic stress [8]. Turner et al. [9] performed longitudinal studies of acetone and endogenous ethanol production in the breath of 30 healthy volunteers over a six-month period using selected ion flow tube mass spectrometry (SIFT-MS). The acetone concentration ranged from 100 ppbv to more than 1 ppmv, while ethanol ranged from 0 to 380 ppbv. Generally, the reported acetone concentrations in breath in literature vary from 0.15 to 2.7 ppmv with an overall mean of 0.5 ppmv [9,10] and for ethanol from 0.01 to 1.2 ppmv and with a mean of ~0.2 ppmv [11]. Photonics 2016, 3, 22; doi:10.3390/photonics3020022

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Laser-based absorption spectroscopy has advanced over the last few decades such that it allows highly sensitive, selective, real-time, and accurate measurements of compounds in breath for clinical testing [2,12]. It can be performed either via narrow frequency scanning or broadband tuning. When performing the former, external cavity lasers are used in the same manner as distributed feedback (DFB) lasers: the laser frequency scans over a single spectral absorption line of the molecule of interest within a limited wavelength range (~2–5 cm´1 ). The use of external cavity lasers (ECL) in such a narrow-scan mode offers some advantages over DFB lasers; as the overall tuning range of the ECL (>150 cm´1 ) is much larger than of a single DFB laser the selection of molecular interference-free regions is easier. In addition, detection of multi-species absorbers is possible by changing the ECL center wavelength. Overall, external cavity quantum cascade lasers (EC-QCLs) [13–15] represents a light source with high power, broad tuning range, narrow linewidth, and rapid wavelength tuning for the sensitive and accurate detection of trace gases like in breath analysis [16,17]. Examples of applications using EC-QCLs in narrowband absorption include detection of exhaled acetone [18,19], ammonia [20] and nitric oxide [21]. Broadband tuning allows the detection of larger molecules with dense ro-vibrational transitions as well as complex shape and broader spectral features. In these cases, a measurement of the detailed shape of the absorption spectrum over a wider wavelength range provides the ability to distinguish between different molecules based on comparison to reference library spectra. Combining QCLs with an external cavity offers a substantially enlarged tuning range compared to DFB-QCLs and facilitates the evaluation of broader absorption features, thereby minimizing the effect of line-broadening during the quantitative data evaluation. While the tuning range of a single EC-QCL is much smaller than a Fourier transform infrared (FT-IR) spectrometer, it is still sufficient to capture enough detail in the absorption spectra for reliable identification of large molecules in the gas-phase or many condensed-phase materials. Examples of EC-QCLs measuring broadband absorption spectra include the detection of ethane using wavelength modulation spectroscopy with a sensitivity of 100 ppbv [22], Freon 125 with quartz enhanced photoacoustic spectroscopy (QEPAS) [23] and fluorocarbons at low ppbv levels in real-time [24]. In both near-IR and mid-IR wavelength regions, acetone has strong absorption bands which overlap with those of ethanol. Interestingly, ethanol has not been included so far in the spectroscopic analysis of breath acetone. In consequence, this motivates the aim of the present study to investigate the effect of ethanol on the quantification of acetone levels in exhaled breath. The present work combines a continuous-wave (cw) external cavity quantum cascade laser, described in detail elsewhere [17], operating between 1120 and 1450 cm´1 with off-axis integrated cavity output spectroscopy (OA-ICOS) [25–27]. This region is well suited for monitoring breath acetone given the relative low spectral influence of other substances in exhaled human breath, such as carbon dioxide, water vapor, methane, and ethanol at endogenous levels. The EC-QCL has been used in a broadband-scan mode for the region of 1150–1300 cm´1 , as well as in narrowband-scan mode around 1216 cm´1 . 2. Experimental Setup The experimental setup has been described in details previously [17]. In short, a home-made widely tunable EC-QCL (1129–1432 cm´1 ) with a maximum power output of 200 mW was combined with Off-Axis Integrated Cavity Output Spectroscopy (OA-ICOS) (Figure 1) [21,28]. The optical cavity had a length of 30 cm with two high reflective mirrors (radius of curvature 1 m, diameter 25 mm, reflectivity 99.97%, CRD Optics, Fort Bragg, CA, USA), which resulted in an effective path length of 1 km. A sinusoidal current modulation of 116 kHz (amplitude of 100 mV) was applied to the QCL. The light after the cavity was collected with a ZnSe lens (f = 5 cm, diameter 25 mm) and directed onto a sensitive infrared detector (PVI-4TE-10.6, VIGO System SA, Vigo, Poland, bandwidth 100 MHz, sensitivity 1.8 ˆ 1010 cm¨ Hz1/2 /W).

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Figure1.1. A A schematic schematic view view of of the the external external cavity cavity quantum quantum cascade Figure cascade laser laser (EC-QCL) (EC-QCL) showing showingthe the arrangement of the laser components including grating, mirror, and step motors. The arrangement of the laser components including grating, mirror, and step motors. Thelaser laserwas was mountedvertically verticallyon onaacopper copper block block due due to to polarization, polarization, in mounted in front front of of which which aalens lensin inaa3D-positioning 3D-positioning system was installed. The light exited at the front and was guided via two focusing lenses system was installed. The light exited at the front and was guided via two focusing lenses (L1,(L1, L2) L2) and A schematic of onto the external cavity quantum cooled cascadedetector. laser (EC-QCL) showing the and Figure a high 1. finesse off-axisview cavity the thermo-electrically a high finesse off-axis cavity onto the thermo-electrically cooled detector. arrangement of the laser components including grating, mirror, and step motors. The laser was mounted vertically on a copper block due to polarization, in front of which a lens in a 3D-positioning Broadband absorption spectra were obtained by rapid coarse tuning of the grating with a DC system was installed. The light exited the front and guided via two focusing lenses (L1, L2) Broadband absorption spectra wereatobtained by was rapid tuning of the grating a DC motor, covering the acetone and ethanol absorption bandscoarse between 1150–1300 cm−1·in with 3 s. Each and a high finesse off-axis cavity onto the thermo-electrically cooled detector. ´1 in 3 s. Each motor, covering the acetone and ethanol absorption bands between 1150–1300 cm spectrum was scanned 10 times and the detector signal was sent via a lock-in amplifier (τ = 100 ms) spectrum was scannedcard 10 times and the detector signal was sent via a lock-in amplifier = 100 ms) to to a dataBroadband acquisition (NIspectra PCI-6259, Austin, USA). The(τ narrowband absorption wereNational obtained Instruments, by rapid coarse tuningTX, of the grating with a DC aspectra datamotor, acquisition card (NI Instruments, Austin, TX, 1150–1300 USA).covering Thecm narrowband spectra −1·in were measured byPCI-6259, a 50and HzNational scan ofabsorption the grating piezo of the laser 0.33 cm for an covering the acetone ethanol bands between s. −1 Each ´1 for an integration were measured by a 50 Hz scan of the grating piezo of the laser covering 0.3 cm spectrum wasof scanned integration time 1 s. 10 times and the detector signal was sent via a lock-in amplifier (τ = 100 ms) time of s. acquisition card (NI PCI-6259, National Instruments, Austin, TX, USA). The narrowband to a1 data spectra were measured by aLimit 50 Hz scan of the grating piezo of the laser covering 0.3 cm−1 for an 3. Calibration and Detection 3. Calibration and Detection Limit integration time of 1 s. For system calibration, a reference homemade gas mixture of 17 ± 0.06 ppmv acetone in nitrogen system a reference homemade gas mixture of 17 ˘ 0.06 ppmv acetone in nitrogen (N2)For used.calibration, Background signals 3.was Calibration and Detection Limit were established by flushing the cell with nitrogen gas and (N used. Background flushing cell with gas and 2 ) was concentrations various were signals appliedwere with established a dynamic by flow system the of two mass nitrogen flow controllers For system calibration, a reference homemade gas mixture of 17 ±two 0.06mass ppmvflow acetone in nitrogen various concentrations were applied with a dynamic flow system of controllers (Brooks (Brooks Instruments, Ede, The Netherlands). (N2) was used. Background signals were established by flushing the cell with nitrogen gas and Instruments, Ede, The Netherlands). Acetoneconcentrations concentrationwere wasapplied determined scanningflow the laser over the spectral region from 1150 various with by a dynamic system of two mass flow controllers Acetone concentration was determined by scanning the laser over the spectral region from 1150 −1 to 1300 cm Instruments, and applying a linear fit to the measured signal versus the reference signal corresponding (Brooks Ede, The Netherlands). ´1 and applying a linear fit to the measured signal versus the reference signal corresponding toto1300 cm 17 ppmv acetone in N2 (both background Thelaser unknown retrieved Acetone concentration was determinedsubtracted). by scanning the over theconcentration spectral regionisfrom 1150 by tomultiplying 17 to ppmv acetone in N2 (both background subtracted). unknown concentration is retrieved by −1 and the reference concentration by measured the first-order coefficient of the linear 1300 cm applying a linear fit to the signalThe versus the reference signalfit. corresponding multiplying the acetone reference concentration by the first-order coefficient ofconcentration the linear fit. to 17 ppmv in N 2 (bothcurve background subtracted). The unknown is retrieved by The resulting calibration for acetone is shown in Figure 2, where the measured The resulting calibration curve for acetone is shown in Figure 2, where the measured multiplying the reference concentration by the first-order coefficient of the linear fit. concentrations are plotted against the calculated one from the mixing ratio with N2. The linear fit The resulting calibration curve for acetone isresults, shown in system Figure 2,ratio where theNmeasured concentrations are plotted against theBased calculated one from the the mixingshows with Thea wide linear yields a Pearson coefficient of r = 0.95. on these linearity 2 .over concentrations are plotted against the calculated one from the mixing ratio with N 2 . Thelinearity linear fit over fitrange yields Pearson coefficient r = 0.95. limit Based results, the system shows of aconcentrations and theofdetection of on the these system was estimated at ~30 ppbv acetone for yields a Pearson coefficient of r = the 0.95.detection Based on limit theseof results, the system shows linearity over a wide a30 wide range of concentrations and the system was estimated at ~30 ppbv acetone s integration time. range of concentrations and the detection limit of the system was estimated at ~30 ppbv acetone for for 30 s integration time.

30 s integration time.

Figure2.2. Measurements Measurements of of different different acetone concentrations at 200 mbar plotted as aa function of Figure 200 mbar plotted function Figure 2. Measurements of differentacetone acetone concentrations concentrations atat200 mbar plotted as aas function of of calculated acetone concentrations from the ratio with nitrogen averaged scans inin calculated acetone concentrationsfrom fromthe the mixing mixing ratio (10(10 averaged scans in 30 s)30 calculated acetone concentrations mixing ratiowith withnitrogen nitrogen (10 averaged scans 30s)s) including the linear fit. including the linear fit. including the linear fit.


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4. Ethanol Influence on Detection of Exhaled Acetone

4. Ethanol Detectionabsorption of Exhaledprofile Acetone Figure 3Influence shows a on simulated of 500 ppbv acetone and 200 ppbv ethanol in ´ 1 the region of 1150–1300 cm for a path length of 1 kmppbv at a acetone pressure of200 1 atm. inset Figure 3 shows a simulated absorption profile of 500 and ppbv The ethanol in in thethe ´ 1 ´ 1 −1 top rightofcorner shows 1216.5 1220 cm thatThe wasinset proposed forright breath region 1150–1300 cm the forregion a path of length of 1cm km atand a pressure of 1 atm. in the top acetone earlier Although this 1220 wavelength was chosen because of the minimal corneranalysis shows the region[18]. of 1216.5 cm−1 and cm−1 thatregion was proposed for breath acetone analysis earlier [18]. Although thisfrom wavelength region becauseofofethanol the minimal spectroscopic interference water and CO2was , thechosen contribution resultsspectroscopic in an elevated interference from water and CO 2 , the contribution of ethanol results in an elevated absorption linethe absorption line without any significant lineshape change. The relative error for this region and without any significant lineshape change. The the relative error for region and the chosen chosen concentrations is about 5%. Furthermore, contribution of this ethanol is visible for the entire concentrations is aboutresulting 5%. Furthermore, the contribution ofconcentration. ethanol is visible the entire broadband scan region, in an overestimated acetone Thisfor overestimation broadband scan region,since resulting an overestimated concentration. This can be quite significant interinperson variabilityacetone of ethanol and acetone inoverestimation exhaled breathcan may be quite significant since inter person variability of ethanol and acetone in exhaled breath may vary vary greatly. greatly. Therefore, for accurate determination of acetone in exhaled breath, ethanol contribution must be Therefore, for accurate determination of acetone in exhaled breath, ethanol contribution must be considered for both broadband as well as narrowband scans and corrections should be applied. considered for both broadband as well as narrowband scans and corrections should be applied. 16

500 ppbv Acetone 200 ppbv Ethanol Sum Acetone + Ethanol

Absorption (%)

12

1216

1217

1218

1219

1220

1221

Wavenumbers (cm -1)

8

4

0 1150

1200

1250

1300

Wavenumbers (cm-1) Figure Simulationofofabsorption absorption of of 500 500 ppbv ppbv acetone at at a pressure of of 1 atm Figure 3. 3. Simulation acetoneand and200 200ppbv ppbvethanol ethanol a pressure 1 atm and a path length 1 km from PNNL (Pacific Northwest National Laboratory) database. The and a path length of of 1 km from thethe PNNL (Pacific Northwest National Laboratory) database. The inset −1 region, showing the contribution of ethanol in this region, which inset a shows the 1216 shows zooma zoom of theof1216 cm´1cmregion, showing the contribution of ethanol in this region, which results elevatedabsorption absorptionsignal signal without without significant forfor both contributions results inin ananelevated significantlineshape lineshapevariation variation both contributions (upper curve, blue). (upper curve, blue).

5. Interference Free Acetone Determination 5. Interference Free Acetone Determination A typical absorption spectrum of the breath from a healthy human subject is given in Figure 4. A typical absorption spectrum of the breath from a healthy human subject is given in Figure 4. In this spectrum, the acetone contribution is clearly visible; however, several other features are In this spectrum, the acetone contribution is clearly visible; however, several other features are present present and can be identified as ethanol and water. The modulation present in the background is due and be identified ethanol and water. The present in the background is due to the to can the residual opticalasfringes due to the ICOS gasmodulation cell. The recorded spectrum is a linear combination residual optical fringes due to the ICOS gas cell. The recorded spectrum is a linear combination of spectra from the different molecules present in the breath and absorbing in this wavelength region. of spectra from the different present the breath absorbing wavelength region. Considering that acetone molecules and ethanol are thein main species and contributing to in thethis recorded absorption Considering that acetone and ethanol are the main species contributing to the recorded absorption spectrum, the individual concentrations of the both gases can be retrieved. Prior the measurements, spectrum, the individual concentrations of the gasesand canethanol be retrieved. Priormixtures the measurements, the responses of the sensor are recorded forboth acetone calibrated separately,the responses of two the sensor aresignals. recorded acetone and ethanol separately, providing providing reference Forfor each wavelength scan ofcalibrated the system,mixtures the recorded signal is fitted two reference For each wavelength of the system, the is fitted with a linear with a linearsignals. combination of both referencescan signals minimizing therecorded residual, signal using the General Linear Fit model. In way, the relative acetone andusing ethanol can be retrieved. the combination ofsuch botha reference signalscontribution minimizingof the residual, the General LinearWith Fit model. therelative absolute concentration of the references gases, the of both gases in In knowledge such a way,ofthe contribution of acetone and ethanol can beconcentration retrieved. With the knowledge is known. of the thesample absolute concentration of the references gases, the concentration of both gases in the sample is known.



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This approach can be successfully implemented in both broadband as well as narrowband This approach can be successfully implemented in both broadband as well as narrowband scanning mode. However, for the narrowband region, the targeted molecules should display sufficient scanning mode. However, for the narrowband region, the targeted molecules should display spectroscopic differences in their absorption spectra. sufficient spectroscopic differences in their absorption spectra.

Figure 4. Typical measured absorption spectrum of a breath sample consisting of a combined spectrum of acetone, ethanol and water. The recorded spectrum is fittedsample with a consisting linear combination of two Figure 4. Typical measured absorption spectrum of a breath of a combined referenceof spectra of acetone ethanol. After minimizing theisresidual, the arelative contributionof of spectrum acetone, ethanol and water. The recorded spectrum fitted with linear combination bothreference gases contained inacetone the sample known.After The minimizing concentration both gases the sample two spectra of and is ethanol. theofresidual, the present relative in contribution can be gases then calculated reference gases (acetone and ethanolofinboth N2 ) gases with known of both contained as in the sample is known. The concentration presentconcentrations in the sample have been measured prior the experiment. can be then calculated as the reference gases (acetone and ethanol in N2) with known concentrations

have been measured prior the experiment.

6. Validation of Ethanol-Corrected Breath Acetone Measurements with PTR-MS 6. Validation of Ethanol-Corrected Breath Acetone Measurements with PTR-MS Samples of end-tidal breath from ten healthy persons who did not consume any alcohol for 48 h ofoff-line end-tidal healthy persons whoPA, didUSA) not consume alcohol for 48 h wereSamples collected in breath Tedlar from bags ten (SKC Inc., Eighty Four, using a any standard procedure were collected off-line in Tedlar bags (SKC Inc., Eighty Four, PA, USA) using a standard procedure described in [29]. Breath samples were introduced in the EC-QCL-based OA-ICOS setup at a flow described in [29]. Breath samples were introduced in the EC-QCL-based OA-ICOS at a flow rate of 1 L/h. Each sample was measured with the EC-QCL-based OA-ICOS setup insetup broadband and rate of 1 L/h. Each samplerespectively, was measured with EC-QCL-based OA-ICOS setupPTR-MS in broadband narrowband scan mode, using thethe correction for ethanol. In parallel, [30,31]and was narrowband scan mode, used for comparison andrespectively, validation. using the correction for ethanol. In parallel, PTR-MS [30,31] was used comparison and validation. The for bags were measured on the same day as the sampling. The Bland-Altman plot in Figure 5 The bags were measured on day as the sampling. The Bland-Altman plot in Figure 5 displays the comparison betweenthe thesame two systems. displays the comparison between the two systems.

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Figure 5. Bland-Altman plot withthe theEC-QCL-based EC-QCL-based Figure 5. Bland-Altman plotofofbreath breathacetone acetonefrom fromten ten persons persons measured measured with system compared proton-transferreaction reactionmass mass spectrometer spectrometer (PTR-MS). system andand compared to to proton-transfer (PTR-MS).(a) (a)broadband broadbandscan scan mode narrowband scanmode. mode.Solid Solid line line represents represents the mean mode andand (b)(b) narrowband scan mean difference differencebetween betweenthe thetwo two systems. Dashed lines represent limits agreement:(a) (a)´3.2 −3.2 ppbv and 9.6 systems. Dashed lines represent thethe limits ofof agreement: 9.6 ppbv ppbvand and(b) (b)−4.7 ´4.7ppbv ppbv ppbv; is standard deviation. andand 13.113.1 ppbv; SDSD is standard deviation.

In general, the determination of gas concentrations is more accurate when the absorption profile In general, the determination of gas concentrations is more accurate when the absorption profile over a wide range is measured, as the fit of the broadband spectrum to a database reference spectrum over a wide range is measured, as the fit of the broadband spectrum to a database reference spectrum will become more reliable, due to the distinction of multiple components in the spectrum. Using the willethanol become more reliable, due to the distinction of multiple in thescan spectrum. the correction, the accuracy of acetone detection is 1.3% components for the broadband (FigureUsing 5a) and ethanol the accuracy acetone is 1.3% for standard the broadband scan 5a)The and 2.8% correction, for the narrowband scan of (Figure 5b)detection as compared to the reference of(Figure PTR-MS. 2.8% for the narrowband scan (Figure 5b) as compared to the standard reference of PTR-MS. The accuracy values are relatively low, possibly within the gas calibration error, indicating that breath accuracy values are relatively low, possibly within the gas calibration error, indicating that breath acetone can be sufficiently accurate detected also within narrowband region, if the scanning domain acetone cansufficient be sufficiently accurateinformation detected also within region, if the scanning domain contains spectroscopic from H2O, narrowband CO2 and ethanol. contains sufficient spectroscopic information from H2 O, CO2 and ethanol. 7. Narrowband Detection of Exhaled Acetone 7. Narrowband Detection of Exhaled Acetone To verify the effect of ethanol on narrowband breath acetone measurements, a wavelength at To verify the used effectwith of ethanol on range narrowband a wavelength 1216.5 cm−1 was a scanning of 2 cm−1breath . Figureacetone 6 showsmeasurements, the acetone concentrations withat ´1 was used with a scanning range of 2 cm´1 . Figure 6 shows the acetone concentrations 1216.5 andcm without ethanol correction. In each pair of bars, the left (black) bar represents the acetone concentration without considering theof absorption ethanol (no correction) and theacetone right with and withoutdetermined ethanol correction. In each pair bars, the of left (black) bar represents the (red) bar after retrievingwithout the individual contribution of acetone and ethanol. The errorand bars were concentration determined considering the absorption of ethanol (no correction) the right derived fromretrieving the 1σ standard deviation of five consecutive measurements and theThe numbers topwere of (red) bar after the individual contribution of acetone and ethanol. error on bars the bars represent the relative error in the determined acetone concentration. The error in acetone derived from the 1σ standard deviation of five consecutive measurements and the numbers on top concentration fromthe all 10 samples ranges from 0% to 39%.acetone This significant contribution to theinamount of the bars represent relative error in the determined concentration. The error acetone of acetone found in breath can have a profound impact on the determination of the health status of a concentration from all 10 samples ranges from 0% to 39%. This significant contribution to the amount person. of acetone found in breath can have a profound impact on the determination of the health status of

a person.


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−1 from 10 breath 6. Comparison acetone concentration concentration at at 1216.5 1216.5 cm cm´1 Figure 6. Comparison of measured acetone from samples without correction for ethanol (left bars, black) and with (right bars, red). Numbers on top give the the presence presence of of ethanol ethanol is is not not considered. considered. relative error of acetone concentration if the

8. Conclusions 8. Conclusions In this this work, work, we we demonstrated demonstrated the of ethanol ethanol on on exhaled exhaled In the potentially potentially strong strong spectroscopic spectroscopic effect effect of breath acetone measurements (up to 39% error) using an EC-QCL in the wavelength region of breath acetone measurements (up to 39% error) using an EC-QCL in the wavelength region of −1 ´ 1 1150–1300 cm observed good agreement between measured spectra of acetone withwith the EC1150–1300 cm . .WeWe observed good agreement between measured spectra of acetone the QCL-based OA-ICOS in comparison to measurements from a PTR-MS. By including a correction EC-QCL-based OA-ICOS in comparison to measurements from a PTR-MS. By including a correction for for ethanol, both bothbroadband broadband and narrowband approaches accurately the acetone ethanol, and narrowband approaches determinedetermine accurately the acetone concentration concentration in human breath. The importancespectroscopic of considering spectroscopic in human breath. The importance of considering ethanol influencesethanol for the influences assessmentfor of ´ 1 the assessment of acetone in human breath is essential, especially for the narrowband scans, (spectral acetone in human breath is essential, especially for the narrowband scans, (spectral region 1216 cm ), −1), for which the error can go up to 39% if it is not taken into account. Furthermore, region 1216 cm for which the error can go up to 39% if it is not taken into account. Furthermore, other molecules of other molecules of interest features in this wavelength region ofhere. the EC-QCL used interest exhibit broadband exhibit featuresbroadband in this wavelength region of the EC-QCL used here. Acknowledgments: This work was financially supported by the Foundation for Fundamental Research on Matter under contract N0304M thewas European Regional Development Fund, province of GelderlandResearch (GO-EFRO Acknowledgments: Thisand work financially supported by the Foundation for Fundamental on project (No. 2009-010034)) and MPNS COST Action “MP-1204 TERA-MIR Radiation: Materials, Generation, Matter under contract N0304M and the European Regional Development Fund, province of Gelderland (GODetection and Applications”. EFRO project (No. 2009-010034)) and MPNS COST Action “MP-1204 TERA-MIR Radiation: Materials, Author Contributions: R.C., F.J.M.H. and S.M.C proposed the concept and initiated the study, F.J.M.H., S.M.C. and Generation, Detection and and Applications”. R.C. guided the study, R.C. J.M. carried out the measurements, and R.C., J.M. and S.M.C wrote the manuscript. Conflicts of Interest: The authors declare conflict of interest. Author Contributions: R.C., F.J.M.H. andno S.M.C proposed the concept and initiated the study, F.J.M.H., S.M.C.

and R.C. guided the study, R.C. and J.M. carried out the measurements, and R.C., J.M. and S.M.C wrote the

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