Real-Time Analysis of Exhaled Breath via Resonance-Enhanced Multiphoton Ionization-Mass Spectrometry with a Medium Pressure Laser Ionization Source: Observed Nitric Oxide Profile LUKE CHANDLER SHORT,* RU ¨ DIGER FREY, and THORSTEN BENTER Bergische Universita ¨t Wuppertal, Fachbereich C—Mathematik und Naturwissenschaften, Gaußstr. 20, D-42119 Wuppertal, Germany (L.C.S., T.B.); and Bruker Daltonic, GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany (R.F.) An elevated concentration of nitric oxide (NO) in alveolar ventilation is indicative of inflammatory stress within the lung. We present here the first description of time-resolved measurement of NO in breath using photoionization mass spectrometry, providing new capabilities for the medical investigator, such as isotopic tracing. Here we use resonance- enhanced multiphoton ionization (REMPI) with time-of-flight mass spectrometry (TOF-MS) coupled with a medium pressure laser ionization (MPLI) source for the selective detection of NO in breath. To demonstrate this technology, a single male subject breathes NO-free air for several minutes, and then the exhaled breath is monitored. The ability of REMPI to differentiate among three different isotopomers of NO is demonstrated, and then the concentration profile of NO in exhaled breath is measured. A similar time-dependence concentration is found as observed by previous techniques. The advantages of this approach compared to other techniques are: (1) parts-per-billion by volume (ppbV) mixing ratios of NO can be measured on a sub-second time scale, (2) since the technique operates optically as well as mass-resolved, isotopomers of NO are discernable, permitting the use of isotopic tracing, and (3) other biologically significant gas molecules can be measured via REMPI. Index Headings: Breath analysis; Inflammation; Resonance-enhanced multiphoton ionization; Mass spectrometry; REMPI-MS; NO; Nitric oxide. INTRODUCTION Analysis of breath for specific trace gases or profiles of multiple gas molecules is a promising new diagnostic technique for certain health conditions, including lung cancer, 1,2 breast cancer, 3 ulcerative colitis, 4 and oxidative stress in the lung. 5,6 In addition, breath analysis is minimally or noninvasive, ideally suited to patients sensitive to more invasive techniques, e.g., pediatric patients. Breath analysis can be broadly divided into techniques that first collect samples for an analysis at a later time, i.e., off-line, and techniques that monitor the breath in real time, i.e., on-line. The off-line analysis of volatile organic compounds in breath has been shown to be a promising tool for the investigation of lung cancer. 7 On-line techniques have been used to monitor breath with sub-second resolution, permitting the measurement of exhaled nitric oxide (NO), an indicator of pulmonary in- flammation. 8 These on-line techniques are of particular interest for the diagnosis and monitoring of lung inflammation, because the part of the breath originating from the lower lung can be monitored separate from the bulk of the air within a single breath. In the field of atmospheric chemistry, on-line monitoring of NO has seen considerable success with numerous techniques, and these techniques have been successfully applied to medical diagnosis. Due to its biological significance, NO remains an ideal molecule to demonstrate the application of new atmospheric techniques to the field of medicine. Within the human body, NO behaves as a secondary messenger molecule involved in the inflammation response. 9 One biosynthetic source for NO is from epithelial cells from L- arginine by the enzyme NO synthase. Once generated, NO travels to the surrounding tissues, triggering the relaxation of capillary muscles, resulting in an increase in blood flow to damaged or irritated tissues. 10 In the cellular environment, NO is oxidized to nitrite; however, in the presence of the enzymes oxyhemoglobin or oxymyoglobin, NO is oxidized to ni- trate. 11,12 These higher oxidized species have less of a vaso- dilatory effect, 12 resulting in a gradual decrease of vasodilation around the site of infection or inflammation. Production of excess NO from the human epithelial tissue is consequently used as an indication of inflammation and infection. 13 The use of 15 N 16 O as an isotopic tracer (dominant natural isotope is 14 N 16 O) from metabolized L-[guanidino- 15 N 2 ]argi- nine has been shown to be effective in monitoring certain physiological and pathophysiological processes. 14,15 Measure- ment of biosynthetic 15 N 16 O in breath has been measured using an off-line sampling technique, i.e., it lacks temporal resolution and thus cannot distinguish between the alveolar and dead- space portion of each breath. 16 Since a rapid equilibrium exists between the liquid-phase concentration of NO within the capillary bed of the lung and the gas-phase concentration in the air above the epithelial tissue, a sample of exhaled air is an accurate and timely indicator of NO production from the capillary bed. During the inhalation and rest phases of breathing, the concentration of NO accumulates within the airway dead space. As one exhales, first the gas from the mouth is removed, then the trachea, followed by that of the lung. This concentration profile, specifically the concentration of NO during the last phase of breathing, i.e., alveolar air, is used to determine the severity of inflammation in the lung. Not all parts within the respiratory system produce the same amount of NO. It is highest in the nasopharynx and decreases as one moves further into the lung. Several studies have suggested that the high concentration of NO in the upper respiratory system is due to both the antimicrobial properties of NO, 9 as well as the ability of NO to facilitate oxygen uptake. 17 Within the paranasal sinuses the mixing ratio of NO can be up to approximately 1 part per million by volume (ppmV), 17,18 while the mixing ratio of NO originating from the lung (orally sampled breath) ranges from a few parts-per-billion by volume (ppbV) to 50 ppbV. 13,19 Given that the concentration of NO resulting from production in the lung can be up to a thousand- Received 10 July 2005; accepted 8 December 2005. * Author to whom correspondence should be sent. E-mail: lcshort@ uni-wuppertal.de or lcshort@uci.edu. Volume 60, Number 2, 2006 APPLIED SPECTROSCOPY 217 0003-7028/06/6002-0217$2.00/0 Ó 2006 Society for Applied Spectroscopy