Anal. Chem. 1999, 71, 5131-5136
Luminol/H2O2 Chemiluminescence Detector for the Analysis of Nitric Oxide in Exhaled Breath Jill K. Robinson,†,‡ Mark J. Bollinger,‡ and John W. Birks*,†,‡
Department of Chemistry and Biochemistry and Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, Colorado 80309-0215, and 2B Technologies, Inc., P.O. Box 288, Golden, Colorado 80402
A new instrument for the detection of nitric oxide has been developed and applied to the analysis of exhaled breath. The instrument is based on conversion of NO to NO2, using the oxidant chromium trioxide, followed by detection of chemiluminescence in the reaction of NO2 with an alkaline luminol/H2O2 solution. The presence of H2O2 is found to enhance the sensitivity of NO2 detection by a factor of ∼20. A bundle of porous polypropylene hollow fiber membranes is used to bring the gaseous sample into contact with the luminol solution. Chemiluminescence occurring within the translucent hollow fibers is detected using a miniature photomultiplier tube. The limit of detection for NO is 0.3 ppbv for S/N ) 3, and the 1/e response time is 2 s. A large interference resulting from the 4-6% CO2 concentration in exhaled breath is removed by use of an ascarite scrubber in the air stream. Breath measurements of NO were made using a sampling technique developed by Sensor Medics (Yorba Linda, CA) with simultaneous detection using the luminol/H2O2 and NO + O3 chemiluminescence techniques. The two instruments were found to be in excellent agreement. Nitric oxide levels were in the range 6.0-22.0 ppbv for healthy individuals and 40.0-80.0 ppbv for individuals with asthma or a respiratory infection. This new detector offers the advantages of compact size, low cost, and a simple configuration compared to NO detectors based on NO + O3 chemiluminescence. Nitric oxide has most often been studied as an atmospheric pollutant formed in combustion processes. In recent years, it was discovered that NO is produced by living organisms and plays a major role in many biological processes.1-4 NO is a messenger molecule and is involved in neurotransmission, blood pressure control, and immune system defense mechanisms.5 Endogenous NO is exhaled from the lungs of humans and animals, and NO levels can be used in the diagnosis and monitoring of asthma and other pulmonary diseases.6 Asthmatic patients exhale markedly higher levels of nitric oxide than normal individuals.7-12 Treatment †
University of Colorado. 2B Technologies, Inc. (1) Beckman, J. S.; Koppenol, W. H. Am. J. Physiol. 1996, 271, c1424-cc1434. (2) Barnes, P. J.; Liew, F. Y. Immunol. Today 1995, 16, 128-130. (3) Curran, A. D. Int. Arch. Allergy Immunol. 1996, 111, 1-4. (4) Gustafsson, L. E. Eur. Respir. J. 1998, 11, 49s-52s. (5) Feldman, P. L.; Griffith, O. W.; Stuehr, D. J. Chem. Eng. News 1993, 71 (Dec 20), 26-38. ‡
10.1021/ac990646d CCC: $18.00 Published on Web 10/08/1999
© 1999 American Chemical Society
of asthma with inhaled steroids has been shown to lower exhaled nitric oxide levels.10 It is expected that further research will lead to a greater understanding of the role of nitric oxide in the airways and aid in developing strategies for the treatment of asthma. A simple, inexpensive, and portable nitric oxide detector is needed to expand research on exhaled nitric oxide levels and asthma and for clinical diagnosis and treatment. Several methods have been used to detect nitric oxide in aqueous biological tissues and in the gas phase. The techniques used to detect aqueous nitric oxide include bioassays,13 electron magnetic resonance spectroscopy,14 electrochemical detection,15 and luminol/H2O2 chemiluminescence.16 Techniques commonly used for gas-phase nitric oxide analysis include laser-induced fluorescence,17 oxidation of NO to NO2, followed by detection of NO2 with luminol chemiluminescence (Luminox, Unisearch Associates, Inc., Ontario, Canada), and the gas-phase chemiluminescent reaction of NO with ozone.18 To date, the only instrument reported to be used for the analysis of NO in exhaled breath is the NO + O3 chemiluminescence instrument.7-12 Although this instrument exhibits excellent sensitivity and selectivity, it has several disadvantages with respect to use in a clinical setting. The NO + O3 instrument is relatively large and expensive. It requires a vacuum pump, generation and destruction of the toxic gas ozone, high voltages, and a cooled red-sensitive photomultiplier tube. (6) Silkoff, P. E.; McClean, P. A.; Slutsky, A. S.; Furlott, H. G.; Hoffstein, E.; Wakita, S.; Chapman, K. R.; Szalai, J. P.; Zamel, N. Am. J. Respir. Crit. Care Med. 1997, 155, 260-267. (7) Dupont, L. J.; Rochette, F.; Demedts, M. G.; Verleden, G. M. Am. J. Respir. Crit. Care Med. 1998, 157, 894-898. (8) Howarth, P. H.; Redington, A. E.; Springall, D. R.; Martin, U.; Bloom, S. R.; Polak, J. M.; Holgate, S. T. Int. Arch. Allergy Immunol. 1995, 107, 228230. (9) Kharitonov, S. A.; Yates, D.; Logan-Sinclair, R.; Shineborne, E. A.; Barnes, P. J. Lancet 1994, 343, 133-135. (10) Massaro, A. F.; Gaston, B.; Kita, D.; Fanta, C.; Stamler, J. S.; Drazen, J. M. Am. J. Respir. Crit. Care Med. 1995, 152, 800-803. (11) Perrson, M. G.; Zetterstrom, O.; Agrenius, V.; Ihre, E.; Gustafsson, L. E. Lancet 1994, 343, 146-147. (12) Sato, K.; Sakamaki, T.; Sumino, H.; Sakamoto, H.; Hoshino, J.; Masuda, H.; Sawada, Y.; Mochida, M.; Ohyama, Y.; Kurashina, T.; Nakamura, T.; Ono, Z. Am. J. Physiol. 1996, 270, L914-L919. (13) Wallace, J. L.; Woodman, R. C. Methods 1995, 7, 55-58. (14) Wilcox, D. E.; Smith, R. P. Methods 1995, 7, 1995. (15) Wink, D. A.; Christadoulou, D.; Ho, M.; Krishna, M. C.; Cook, J. A.; Haut, H.; Randolph, J. K.; Sullivan, M.; Coia, G.; Marray, R.; Meyer, T. Methods 1995, 7, 71-77. (16) Kikuchi, K.; Nagano, T.; Hiroshi, H.; Hirata, Y.; Hirobe, M. Anal. Chem. 1993, 65, 1794-1799. (17) Schulz, C.; Volker, S.; Heinze, J.; Stricker, W. Appl. Opt. 1997, 36, 32273232. (18) Dickerson, R. R.; Delany, A. C.; Wartburg, A. F. Rev. Sci. Instrum. 1984, 55, 1995-1998.
Analytical Chemistry, Vol. 71, No. 22, November 15, 1999 5131
Figure 1. Schematic diagram of the hollow fiber gas-liquid exchange module. The luminol/H2O2 solution flows through the interior of the fibers, and the gas flows around the exterior. The gas diffuses through the pores in the fiber membrane and into solution, resulting in the chemiluminescence reaction.
We have developed a new instrument for breath analysis based on oxidation of NO to NO2 followed by its chemiluminescent reaction with an alkaline luminol/H2O2 solution. The oxidation is accomplished by passing NO over glass beads coated with chromium trioxide in the humidity range 10-30%. A porous membrane is used to contact the gas-phase NO2 with the solution. The membrane consists of a bundle of ∼50 hollow polypropylene fibers encased in a polymer housing. The luminol/H2O2 solution flows through the interior of the fibers, while the gas flows around the outside of the fibers. Gas-phase molecules diffuse through the pores of the membrane and into solution, resulting in the chemiluminescence reaction. The fibers are translucent, allowing the transmitted light to be detected by a photomultiplier tube. Figure 1 is a schematic diagram of the hollow fiber gas-liquid exchange module. EXPERIMENTAL SECTION Reagents. Luminol (disodium salt of 5-amino-2,3-dihydro-1,4phthalazinedione), 3 wt % hydrogen peroxide, sodium bicarbonate (ACS reagent), sodium carbonate (anhydrous ACS reagent), chromium trioxide, ascarite, and glass beads (0.7-1.0 mm) were obtained from Sigma (St. Louis, MO). Acetonitrile, acetone, and toluene were from Fisher Scientific (Pittsburgh, PA). Isoprene, acetaldehyde, ethanol, and pentane were obtained from Aldrich (Milwaukee, WI). Nafion moisture exchange tubing was purchased from Perma Pure Inc. (Toms River, NJ). Two reagent solutions were prepared for the chemiluminescence detector and stored separately since H2O2 slowly oxidizes luminol in a reaction catalyzed by trace concentrations of metal ions. The first reagent solution is 4 mM luminol and 50 mM bicarbonate/carbonate buffer (pH 10.25). The second solution is 5 mM hydrogen peroxide. All gases purchased were of the highest purity. Nitrogen, zero air, and carbon dioxide were purchased from US Welding (Denver, CO). Nitric oxide (10 ppmv in nitrogen), nitrogen dioxide (110 ppbv in nitrogen), sulfur dioxide (410 ppbv in nitrogen), and ammonia (59 ppmv in nitrogen) were purchased from Scott Specialty Gases (Longmont, CO). For maximum stability and accuracy, the gas standards were prepared by dynamic dilution, and the flows of all gases were regulated with mass flow controllers from MKS Instruments (Andover, MA). 5132 Analytical Chemistry, Vol. 71, No. 22, November 15, 1999
Figure 2. Schematic diagram of the luminol/hydrogen peroxide instrument for NO detection. Breath samples or NO standards pass through an ascarite trap, Nafion moisture exchange tubing, and a CrO3 converter before entering the hollow fiber module. Flow controllers are used to regulate the pressure and flow of the exhaled breath and generate NO calibration standards.
CrO3 Converter. The chromium trioxide converter for oxidizing NO to NO2 consisted of ∼2 g of CrO3-coated glass beads packed in 4 in. of 3/8-in. glass tubing. The beads were held in place with glass wool. The coated beads were prepared by soaking glass beads (0.7-1.0 mm) in a 20 wt % aqueous solution of CrO3 for 10 min. The beads were then filtered from the solution and dried in an oven at 60 °C. The amount of CrO3 coated on the glass beads was found by weighing the beads before and after coating and found to be ∼30 mg. The conversion efficiency was measured as a function of relative humidity. Nitric oxide standards with relative humidity in the range 0-80% were generated by mixing a dry air stream with a humidified air stream from a bubbler. The relative humidity was measured using a capacitance thin-film hygrometer from Vaisala (Finland). Preparation of Volatile Organic Standards. The responses from potentially interfering organic compounds found in breath were measured by injecting 10 µL of pure or diluted analyte into a heated gas flow chamber at 120 °C. The vaporized compound was carried into the detector with a stream of zero air, and if the compound produced a signal, a peak was detected. The concentration of the organic analyte was calculated from the amount injected, the gas flow rate, and the Gaussian peak shape. Instrument. Figure 2 is a schematic diagram of the luminol/ H2O2 chemiluminescence detector. The hollow fiber gas-liquid exchange module, as designed by us, is manufactured by Rampro (Lakewood, CO). The polypropylene fibers have an outer diameter of 380 µm, a wall thickness of 50 µm, and a pore size of 0.2 µm (Akzo Nobel). A peristaltic pump, model 0798 (Alitea, Holland), is used to deliver the two reagents at a total flow rate of 1.25 mL/ min, split equally between the two solutions. The solutions are mixed in a tee prior to entering the gas-liquid exchange module.
A liquid valve, model E-980107B (Pneutronics, Hollis, NH), is used to switch to a deionized water flush prior to shutting down the instrument. A rotary vane pump, model 50311 (ASF Thomas, Norcross, GA), is used to draw the main air stream through a 10 slpm flow controller (Unit Instruments, Yorba Linda, CA) and into the instrument. Nitric oxide standards in the range 10-100 ppbv are generated by addition of NO (10 ppmv in nitrogen) with a 30 sccm flow controller (Unit Instruments) into the main air stream. The rotary vane pump and the 10 slpm flow controller also are used to regulate the flow rate of the exhaled breath. A valve (model P8053, Humphrey, Kalamazoo, MI) is used to switch between zero air and the breath sample. The same rotary valve pump, in combination with a flow restrictor, is used to pull a side stream off the main flow at 200 mL/min. A side stream with a lower flow rate is needed for a longer residence time in the ascarite scrubber, Nafion moisture exchange tubing, and the CrO3 converter. The side stream is passed through an ascarite trap to remove CO2 in exhaled breath and NO2 impurity in the NO standard, a Nafion moisture exchanger (Perma Pure) to equilibrate the stream to ambient humidity, and a CrO3 converter for NO-to-NO2 oxidation. The air stream then enters the hollow fiber gas-liquid exchange module, and light from the NO2 reaction with luminol/H2O2 is detected by a Hamamatsu model 5784 photomultiplier tube (Bridgewater, NJ). The voltage signal from the photomultiplier tube is filtered with a 0.2-s RC time constant, and the data are digitized and stored in a computer. Breath Sampling Technique and Intercomparison with an NO + O3 Instrument. A breath sampling technique developed by Sensor Medics (Yorba Linda, CA) was used in the analysis of NO in exhaled breath. The patient inhales NO-free medical air to eliminate possible contamination from high levels of atmospheric NO in polluted areas. Medical grade air flows into the patented mouthpiece and the subject inhales and exhales the air normally four times. After four normal breaths, referred to as “resting breathing”, the subject inhales to the full capacity of the lungs and then exhales for ∼30 s. The exhale is controlled at a flow rate of 6 L/min with the 10 slpm flow controller. The pressure produced when exhaling against the flow controller is sufficient to close the soft palette, thus excluding nasal NO. Three breath measurements were taken for each individual. The flow of the exhaled breath was split between the luminol/H2O2 instrument and the NO + O3 analyzer from Ionics-Sievers (Boulder, CO). The NO concentration in exhaled breath is reported as the average value reached during the plateau of the 30-s exhale. RESULTS AND DISCUSSION NO Detection. The current clinical requirements for measurements of NO in breath are detection in the concentration range 1-200 ppbv with a precision of 1 ppbv or better and a response time of ∼3 s. The luminol/H2O2 detector in combination with a CrO3 converter was able to meet these requirements. The luminol/H2O2 instrument was originally explored for direct detection of NO. Direct detection of NO in biological tissues was reported by a reaction with a luminol/H2O2 solution.16 NO reacted with H2O2 to form the potent oxidizer peroxynitrite (ONOO-).16 Peroxynitrite is capable of oxidizing luminol to an excited electronic state of 3-aminophthalate, which relaxes by emitting light.19 The NO signal resulting from peroxynitrite formation is detected on top of the background signal resulting from the
oxidation of luminol by hydrogen peroxide,16,20
NO + H2O2 + 2OH- f ONOO- + 2H2O
(1)
ONOO- + luminol f 3-aminophthalate + N2 + products + hυ (2) However, in our instrument, NO did not produce a chemiluminescent signal when put in contact with the luminol/H2O2 solution provided that any NO2 impurities were first removed from the NO source by using an ascarite trap. Several reaction conditions were varied in an attempt to increase the sensitivity to NO, including pH and concentrations of each of the reactants. The reaction of NO bubbled through a luminol/H2O2 solution also was monitored but did not produce a signal above background. NO2 is known to oxidize luminol in the absence of H2O2 and thus was expected to produce a signal in the instrument.21,22 However, the NO signal was predicted to be much greater than NO2 on the basis of work by other investigators.16 We are uncertain as to why we were unable to detect NO directly by luminol/H2O2 chemiluminescence; it could be that peroxynitrite does not form on the time scale of a few seconds. In the course of these experiments, however, we discovered that the presence of 5 mM H2O2 in the luminol solution enhanced the NO2 response by a factor of ∼20. This is possibly due to the formation of a stronger oxidant, possibly peroxynitrate (OONO2-), in a reaction of NO2 with H2O2. Because the direct reaction of NO with luminol/H2O2 solutions did not provide a detectable signal, an alternate approach to NO detection based on oxidation of NO to NO2 prior to reaction with a luminol/ H2O2 solution was pursued. Oxidation of NO to NO2 with CrO3. Chromium trioxide coated on an inert support has been used to oxidize NO to NO2 in luminol detectors used for atmospheric measurements.23 Our detector differs from those detectors in the method of putting the gas and liquid in contact and by the addition of H2O2 as a reagent solution. The oxidation efficiency of CrO3 was reported to vary with the relative humidity of the sample.24 Figure 3 shows the efficiency of oxidation of NO to NO2 as a function of relative humidity using CrO3-coated glass beads. The maximum conversion efficiency (99%) occurs at a relative humidity of ∼13%. The dependence of conversion efficiency on relative humidity presents a problem for breath analysis, since exhaled air is saturated with water vapor while the NO standards used for calibration are prepared with dry air. In order for the calibration to be accurate, the standards and the breath must be at the same relative humidity. This was achieved by adding a 24-in. segment of 1/8in.-o.d. Nafion moisture exchange (ME) tubing to the air stream prior to the CrO3 converter. The ME tubing equilibrates the humidity of the interior air stream with the humidity of the (19) Radi, R.; Cosgrove, T. P.; Beckman, J. S.; Freeman, B. A. Biochem. J. 1993, 290, 51-57. (20) Kikuchi, K.; Nagano, T.; Hayakawa, H.; Hirata, Y.; Hirobe, M. J. Biol. Chem. 1993, 268, 23106-23110. (21) Maeda, T.; Aoki, K.; Munemori, M. Anal. Chem. 1980, 52, 307-311. (22) Wendel, G. J.; Stedman, D. H.; Cantrell, C. A. Anal. Chem. 1983, 55, 937940. (23) Levaggi, D.; Kothny, E.; Belsky, T.; de Vera, E.; Mueller, P. Environ. Sci. Technol. 1974, 8, 348-350. (24) Hutchinson, G. L.; Yang, W. X.; Andre, C. E. Atmos. Environ. 1999, 33, 141-145.
Analytical Chemistry, Vol. 71, No. 22, November 15, 1999
5133
Figure 3. NO-to-NO2 oxidation efficiency as a function of percent relative humidity in the CrO3 converter. The line is a smooth curve through the data.
Figure 5. Calibration curve for nitric oxide standards in the range of breath analysis (1-100 ppbv). The curve was fit to polynomial represented by the equation, signal (mV) ) 0.1229[NO]2 + 6.039[NO] - 0.0097, with R2 ) 1.0000. The error bars represent the standard deviation in three measurements. Table 1. Summary of the Concentrations of Various Compounds in Exhaled Breath and Their Molar Selectivity Ratios compd
Figure 4. Signals from 20, 40, and 60 ppbv NO standards in the luminol/H2O2 peroxide instrument at 20% relative humidity.
ambient air surrounding the tubing. Therefore, the breath was dried and the standards were humidified to the ambient humidity of the laboratory (10-22%). This humidity range resulted in adequate oxidation efficiency for NO detection. However, if the ambient humidity is greater than 30%, additional humidity control may be required. This can be accomplished by enclosing the ME tubing in a humidity-controlled chamber. The effect of NO on the conversion efficiency of the CrO3 converter was measured by continuously passing 0.5 ppmv NO dilute in air equilibrated to ambient humidity (∼20% RH) through the converter. The conversion efficiency was found to drop to 60 and 5% of its original value after passage of 8 × 10-5 and 1.2 × 10-4 mol of NO, respectively. Thus, assuming that 50 ppbv NO passes through the converter for 10 min at a flow rate 200 mL/ min for each breath analysis, the converter could be used for ∼20 000 analyses before the conversion efficiency decreases to 60% of its original value. After subsequent exposure of the CrO3 catalyst to ambient air for one week, the conversion efficiency returned to 100% of its original value. On the basis of these results, CrO3 appears to behave as a true catalyst and could be used almost indefinitely if exposed to clean air between samples. Calibration and Limit of Detection. Responses of the detector to nitric oxide standards in the range 20-60 ppbv are shown in Figure 4. A working curve was generated by making three replicate measurements of the NO standard at several concentrations in the range expected in exhaled breath (1-100 ppbv), as shown in Figure 5. The response was fit to a secondorder polynomial with a correlation coefficient of r2 ) 1.0000. The precision of the method was 5% for 10 ppbv NO. The limit of detection was 0.3 ppbv for a signal-to-noise ratio of 3. The 1/e response time of the instrument was measured to be 2 s. 5134
Analytical Chemistry, Vol. 71, No. 22, November 15, 1999
CO2 CO NH3 acetaldehyde ethanol acetone isoprene acetonitrile pentane toluene
mixing ratio 4-6%12 0.5-15 ppmv25 0.2-2.0 ppmv26 50 ppbv27 100 ppbv27 50 ppbv27 30 ppbv27 30 ppbv27 10 ppbv27 5 ppbv27
selectivity (NO/cmpd) 3.5 × 105 >7 × 104 >1 × 105 750 >1 × 105 >1 × 105 >1 × 105 >1 × 105 >1 × 105 >1 × 105
Instrument Response to Potential Interferences. A wide variety of compounds have been measured in exhaled breath, including carbon dioxide,12 carbon monoxide,25 ammonia,26 and as many as 100 organic compounds.27 Many of these compounds were explored as possible interferences in the detection of NO. The compounds tested were CO2, CO, NH3, ethanol, acetone, isoprene, acetonitrile, pentane, toluene, and acetaldehyde. The volatile organic compounds tested are representative of various organic functional groups and are the most abundant trace species in breath.27 The concentrations of gases tested were at or above the highest level reported in exhaled breath. Table 1 summarizes the concentrations of these compounds found in exhaled breath and the selectivity ratios of the detector for those compounds. The only compound found to produce a significant interference was carbon dioxide. Although the response to NO is much greater than CO2, the concentration of CO2 is much higher (4-6%).12 Carbon Dioxide Interference. Although the luminol/H2O2 method has the sensitivity to measure nitric oxide at the levels in exhaled breath, the presence of percent levels of carbon dioxide creates a large interference. Carbon dioxide can interfere with the NO signal in two ways. The first is that carbon dioxide (25) Lee, P. S.; Majkowski, R. F.; Perry, T. A. IEEE Trans. Biomed. Eng. 1991, 38, 966-972. (26) Spanel, P.; Davies, S.; Smith, D. Rapid Commun. Mass Spectrosc. 1998, 12, 763-766. (27) Krotoszynski, B.; Gabriel, G.; O’Neill, H. J. Chromatogr. Sci. 1977, 15, 239244.
Figure 6. Signal from exhaled breath levels of CO2 (4-6%) in the luminol/H2O2 instrument.
hydrolyzes in water to form carbonic acid, which lowers the pH of the solution. Reducing the solution pH decreases both the background and the nitric oxide signal because the luminol oxidation is more efficient at high pH. This interference can be eliminated by buffering the solution. The second interference produces a positive signal, as shown in Figure 6. This results from the reaction of carbon dioxide with hydrogen peroxide to form peroxycarbonates, which decompose to highly reactive radical anions capable of oxidizing luminol.28
CO2 + H2O2 f -O(CdO)OOH + H+
(3)
O(CdO)OOH f CO2- + HO2
(4)
-
Figure 7. Analysis of exhaled breath from (a) a healthy individual and (b) an untreated asthmatic. Note differences in the scales for the y axes.
2-O(CdO)OOH f -O(CdO)OO(CdO)O- + HO2- + H+ (5) -
O(CdO)OO(CdO)O- f 2CO3-
(6)
Figure 6 shows the signal from 4, 5, and 6% CO2 in the luminol/ H2O2 instrument. A 5% CO2 signal is equivalent to ∼100 ppbv NO. The NO signal can be detected on top of the CO2 signal; however, since the CO2 signal is large and varies over the course of a breath, subtraction of the CO2 response to obtain the NO component of the signal would not be very accurate. Therefore, the interference must be eliminated in order to accurately measure NO in exhaled breath. A trap consisting of ascarite was placed in the air stream to remove CO2 by the reaction
CO2 + 2NaOH f Na2CO3 + H2O
(7)
The trap was tested for the removal of both CO2 and NO from the air stream. The signal from breath concentrations of CO2 was eliminated by the ascarite, while no detectable amount (
