CHEMICAL ANALYSIS OF EXHALED HUMAN BREATH USING HIGH RESOLUTION MM-WAVE ROTATIONAL SPECTRA Tianle Guo, Jessica R. Thomas, Daniela R. Branco, Ivan R. Medvedev Department of Physics DAVID DOLSON, Department of Chemistry Wright State University, Dayton, OH HYUN-JOO NAM, Department of Bioengineering, TX,KENNETH O, Electrical Engineering, University of Texas at Dallas, Dallas, TX

Experimental Setup Continuous Wave THz Spectrometer Microwave SynthesizerCustom Built Diode MultipliersVirginia Diodes Heterodyne Reciever, 210~270 GHz Absorption Cell2 m long by 4 inches wide (14 L), Large; 2 m long by 1 inches wide (0.875 L), Small PreconcentratorENTECH 7100A; Markes Sorbent tubes Preconcentrator Absorption Cell(Large) Custom Built Microwave Synthesizer Absorption Cell(Small)

MM-WAVE ROTATIONALGas Chromatography– Mass Spectrometry Do no need Calibration. Only need to acquire a library once. Day to Day calibration High number of resolution elements 100,000 leads to high specificity Much lower number of resolution elements 200, leads to possible ‘false positive’ and ‘false negative’ Suitable for lighter polar moleculesmore accurate results for larger molecules YoungSophisticated Our System versus GC-MS

Blood glucose Assessment based in Breath analysis - Prior Work Breath acetone (1) and methyl nitrate (2) level were reported linear related to blood-sugar level. (1)C.N. Tassopoulos. (1969). BREATH-ACETONE AND BLOOD-SUGAR MEASUREMENTS IN DIABETES. the lancet. 293 (7609), p (2)Galassetti, P. R.. "Exhaled methyl nitrate as a noninvasive marker of hyperglycemia in type 1 diabetes." Proceedings of the National Academy of Sciences: The eventuality of metabolizing glucose and aspartame into Methanol is known from professional literature. A higher amount of breath CO in diabetic person was noticed, and there was a positive correlation between exhaled CO levels and the incidence of glycemia. (3)Paredi, P.. "Exhaled Carbon Monoxide Levels Elevated in Diabetes and Correlated With Glucose Concentration in Blood: A New Test for Monitoring the Disease?." Chest: Patients with diabetics may have a higher Breath Isoprene (4)Barker M, Hengst M, Schmid J, et al. Volatile organic compounds in the exhaled breath of young patients with cystic fibrosis. Eur Respir J 2006;27:929e36. Toluene is one of the potential markers used for the diagnosis of diabetes (5)Shin, Jungwoo. "Thin-Wall Assembled SnO2 Fibers Functionalized by Catalytic Pt Nanoparticles and their Superior Exhaled-Breath-Sensing Properties for the Diagnosis of Diabetes." Advanced Functional Materials 23: p

Analytical Chemical Detection Algorithm 1. Create the spectral libraries Collect overview spectra of the pure samples at well defined pressures (1 mTorr, 5 mTorr, 10 mTorr) These overview spectra are then overlaid in order to choose 5 of the strongest lines of each chemical that do not overlap with other chemicals. The amount of pressure for each chemical used in our library is determined by matching pressure broadening of the library spectra to the breath spectra. 2. Record spectra of the chemicals in breath Breath/Air samples were then collected into a Tedlar bag/Sorbent tubes Use preconcentrator/Sorbent tubes to remove major air constituents (O 2, N 2, H 2 O, and CO 2 ) Inject preconcentrated breath into the absorption cell Record the snippet spectra 3. Perform spectral analysis Calculate partial pressures of every chemical present in the absorption cell by performing the Least Squares Fitting (LSF) of the mixture spectrum to the library spectrum. Can get the volumetric dilution of each chemical in the original breath sample based on the volume of the absorption cell and the preconcentration efficiency

Spectra – Example

Breath Collection Method Tenax TA 35/60, Carborgraph 1TD 40/60, Carboxen /60 Bio-VOC Breath sampler 127CC Exhaled Breath Tedlar Bag Vs Sorbent tubes 1.By using Tedlar Bags, we collect all the exhaled breath. However, most valuable chemicals from metabolism are in the alveolar volume (last portion of exhalation). Using Tedlar Bag will lower the sensitivity and bring contamination from environment. 2.Entech 7100A use cold trap, and high boiling temperature chemical will be taken away. 3.Tenax TA is a weak sorbent, cannot catch Carbon- Monoxide. Entech 7100A With Tenax TA

Contamination of the cell (Old cell – Large volume) Black—Baseline(empty) Red—--Breath

Contamination of the cell (New Cell – Small Volume) Blue—Baseline(empty) Red—--Breath

Comparison of Two Method Black – Old system Red – New system

Comparison of Two Method Black – Old system Red – New system

Comparison of Two Method

ChemicalsNormal BreathSensitivity(Old)Sensitivity(New) Ethanol ppb24 ppb2.4ppb Methanol ppb8.1 ppb1.6ppb Acetone ppb68 ppb11ppb Acetaldehyde ppb10 ppb1.5ppb Chloromethane-33 ppb0.12ppb HCN4.4 ppb0.5 ppb10ppt Methyl Cyanide4.4 ppb12 ppb80ppt CO0~100ppm16ppb60ppt Formalradehyde40ppb(breath), 100ppb(environment) 14.7ppb2.6ppb Methyl Nitrate* ppt3 ppb- Toluene0-0.1ppb450 ppb- Isoprene50~1000ppb1200ppb- Chemicals Currently Studied

Breath Chemicals & Blood Sugar Experimental Process Subject I, female, healthy. Subject II, Diabetic type 1 breath (500cc) was collected Randomly, (no condition controlled), over several days, Breath samples consisted of approximately 2 exhalations. Blood Sugar level was collected right after the breath by OneTouch® UltraMini® blood glucose meter. Breath spectra was recorded for each sample.

Breath Results – Subject I (non diabetic)

Breath Results – Subject II (Type 1)

Breath Results – Subject II at high levels at the high blood glucose level, we found a possible negative linear relationship in Acetone, Acetaldehyde, and Methanol.

Type 2 Diabetic with New Cell, Strong Sorbent tube

NAMEAMOUNT IN BREATH/ppb Methyl Cynaide9.86±0.08 Chloromethane0.83±0.01 Acetaldehyde33.6±1.5 Acetone46.7±8.2 HCN6.99±0.02 Ethanol33.8±2.4 Methanol61.5±2.6 CO13.5±0.1 Formaldehyde71.8±0.3

Conclusion & Path Forward We now can see 9 chemicals which are potentially related to diabetes and blood glucose level. We successfully detected CO and Formaldehyde and improved the sensitivity of our system by a factor of 10. The blood glucose experiment is still in progress. Our preliminary results show that there may exist a relationship between blood glucose level and concentrations of acetone, acetaldehyde and methanol. Conclusion Path forward Improve Sensitivity and extend the current chemical list. Collect more data with the new system Conduct a more controlled experiment on blood glucose level. (overnight fasting, food)