Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin 9

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Breath ammonia analysis: Clinical application and measurement

Troy Hibbard and Anthony J. Killard

Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin 9


This review covers in detail the complexity of human breath, how the body metabolises ammonia, clinical conditions which are directly related to regulation of ammonia concentration, and analysis of current techniques that are capable of detecting breath ammonia. Focusing on these areas provides the information needed to develop a breath ammonia sensor for monitoring dysfunction of the human body. Human breath has been broken down into its key components which are necessary for proper understanding of what to look for when attempting to isolate volatile organic compounds. A pathway has been shown which explains the origin of ammonia in the body and how it is processed within a healthy system. Following this, the hazards of several dysfunctions related to the broken ammonia pathway have been discussed. It is essential that technicians have knowledge of these inner workings of the human body along with current technology. Thus, the advantages and disadvantages of techniques from chemical ionisation, gas chromatography, laser spectroscopy, and chemical sensing have been discussed.


Breath ammonia analysis, ammonia metabolism, clinical applications, analytical techniques

1. Introduction

The diagnostic potential of clinical breath analysis has been recognised for centuries. It is said that the original research can be found within the writings of Hippocrates [1]. However, the first published quantitative analysis was not until 1784 when Lavoisier examined carbon dioxide in breath [2]. By the 1950s, separation of individual gas molecules became possible with gas chromatography [3]. Since then, more and more compounds found in human breath have been linked to physiological conditions. For example, acetone has been linked to diabetes, whereas ammonia is indicative of liver and/or kidney dysfunction. Human breath is a highly complex substance with numerous variables that can interfere with one another. Each human breath contains over 1,000 trace volatile organic compounds (VOCs) [4]. On average, exhaled human breath is a mixture of 78.6% (w/v) nitrogen, 16% (w/v) oxygen, 4.5% (w/v) carbon dioxide, and 0.9% (w/v) inert gases and VOCs [5]. This mixture is exhaled at temperatures between 34oC [6] and 37oC [7] while relative humidity may range from 91% to 96% in oral exhalations, and from 82% to 85% in nasal exhalations [8]. Human breath cannot have a relative humidity above 99% since 100% implies that the water has gone from the vapour to the condensed phase [9]. Additional respiratory variables such as flow rate and lung volume must also be considered when making measurements of trace gases in breath, and these can vary according to an individual’s height, weight, age, and body surface area [10]. Essentially, larger volumes have the potential for a greater mass of gas. Flow rates are required in order to calculate the concentration of gas present. Several parameters are important for exhaled flow rate and volume analysis:

  • Forced vital capacity is a volume measurement where the full volume of inhaled air is added to the full volume of forced exhaled breath [5].

  • Minute volume (or maximum voluntary ventilation - MVV) is a volume-to-rate measurement of the litres of breath exhaled over a period of one minute [5].

  • Peak expiratory flow is a rate measurement performed by calculating how fast the breath volume can be forced out of the lungs [5].

Calculations and values can be found in any number of spirometry related articles such as those published by the American Thoracic Society [11], Bass [12], Tomlinson [13], and Quanjer [14]. Within the 0.9% (w/v) of breath which constitutes inert gases and VOCs, the individual gas

Breath gas


range (ppb)



2 - 5



6 - 33



293 - 870



200 - 2,000



50 - 2,000



559 - 639



425 - 1,800



422 - 2,389



200 - 2,000



1,500 - 2,000



200 - 300


Carbon dioxide










27 - 153



100 - 3,358


Hydrogen cyanide




55 - 121











Nitric oxide










0 - 135

Table 1. Concentrations of Gases in Human Breath
oncentrations can range between parts-per-million (ppm) and parts-per-trillion (ppt). Some of the gases that have been detected so far down to parts-per-billion (ppb) levels are shown in Table 1. Of these gases, ammonia has attracted increasing interest for clinical diagnostics such as in haemodialysis monitoring [20], asthma assessment [25], diagnosis of hepatic encephalopathy [26], detection of Helicobacter pylori [27], and analysis of halitosis [28]. The normal physiological range for human breath ammonia is in the region of 50 to 2,000 ppb [17]. To be effective, analytical techniques for breath ammonia quantification must be capable of a limit of detection of some 50 ppb. While there are several analytical technologies capable of this, they also possess many limitations for application in clinical settings. While it is true that these techniques are moving from invasive to non-invasive, most detection methods are still extremely complex instrumental systems and require special training to use. Aside from breath analysis, other non-invasive techniques based on the analysis of urine, saliva, hair, and nails may also offer potential solutions [1]. With regard to breath analysis, however, development of breath monitors that are simple and portable for point-of-care use is a critical next step. Furthermore, the possibility of performing real-time analysis of breath has recently become a reality. Originally, breath analysis depended on collection of exhaled breath condensate (EBC) which was placed within the detection region of a device [18]. However, by collecting breath samples into containers such as balloons, samples undergo significant losses and contamination [29]. Typically, collection of EBC is only recommended if pH analysis of breath compounds is necessary [30].

2. Ammonia metabolism and the urea cycle

When food is ingested, a fine balance of nutritional absorption and toxin removal takes place. The body must be specific about how amino acids are processed, or nitrogenous compound concentrations could prove fatal. Initially, the stomach, lumen and intestines break down food into amino acids, nucleotide bases, and other nitrogenous compounds which diffuse into the blood [31]. These excess nitrogenous compounds are then absorbed from the blood into the liver. The liver converts them into less toxic soluble forms which can be safely removed in relatively low volumes of water. In mammals, this less toxic form is urea. The urea cycle, as it applies to humans, is the pathway upon which amino acids are effectively broken down (Fig. 1). Ammonia is first absorbed into the liver and combined with carbon dioxide to form carbamoyl phosphate. This enters the urea cycle and combines with ornithine to form citrulline. Amino acids are fed into the cycle via their transamination by aspartate which combines with citrulline to form argininosuccinate [32]. Aspartate also acts to drive the availability of free ammonia which is used in the initial steps with carbon dioxide [33]. Argininosuccinate is then split into fumarate (which is fed into the citric acid cycle) and arginine. Arginine then reacts with arginase and water to produce urea and regenerated ornithine [31]. As the liver finishes processing, the urea is excreted into the bloodstream among excess ammonia and is absorbed by the kidneys via the glomerulus. The typical glomerular filtration rate (GFR) is about 0.125 L/min creating 1 to 2 Litres of urine a day [33]. However, this rate decreases if the concentration of materials is high enough to impede absorption. Kidneys serve the purpose of filtering the blood urea and excess ammonia out of the body in the form of urine [32]. Normal concentrations of ammonia in blood range between 1.2 ppb and 6.6 ppb [34]. However, if the liver loses the ability to enzymatically break down nitrogenous compounds, or the kidneys can no longer remove them from the blood, then complications such as hyperammonaemia [31], hepatic encephalopathy [35], and / or uraemia [33] can arise. In order to monitor these levels, current methods depend on invasively measuring the nitrogen concentration found within the urea in the blood (i.e. blood urea nitrogen, BUN) [20].

Figure 1. The urea cycle. Taking place in the liver (yellow box), the urea cycle breaks down nitrogenous compounds such as ammonia into the less toxic form of urea. The processes of transamination and oxidative deamination also allow for the conversion of aspartate into free ammonia [33].

3. Current and potential clinical applications for breath ammonia monitoring

Clinically, several conditions are related to changes of blood nitrogen levels and consequently ammonia levels. These are impairments in relation to the liver, brain, kidneys, stomach, duodenum, oral cavity, and lungs. In all cases, if ammonia levels in the blood are of a higher concentration than those found in the air, then ammonia can diffuse out of the blood and into the lungs [36]. Doing so allows for potential clinical measurements of blood ammonia from a non-invasive perspective.

3.1 Hepatic encephalopathy

With reference to the organs involved in nitrogen metabolism, the liver and kidneys are central to the proper removal of ammonia from the body. If there is a problem associated with either of these, ammonia levels in the blood may escalate to toxic levels. With liver dysfunction, the result is hyperammonaemia (i.e. increased ammonia in blood) which has further consequences including damage to brain tissue (i.e. hepatic encephalopathy) [31]. Studies have shown a 0.61 correlation between arterial ammonia levels and severity of hepatic encephalopathy [37]. Normally, the brain is protected by a blood-brain barrier that prevents toxins from entering. However, if there is an obstruction in the synthesis of the urea cycle, components are created that can modify the permeability of the blood-brain barrier. An example of a compound that can do this is glutamine. During the transamination process of the urea cycle, glutamate is capable of joining with excess ammonia via glutamine synthetase to create glutamine. Glutamine in elevated levels is then able to change the osmotic tendencies around brain tissue resulting in swelling of the brain [31]. This swelling is due to higher concentrations of toxins outside the barrier flowing into the lower concentrated area of the brain. Included in this flow would be ammonia if levels in the blood were high. By entering the brain, ammonia is capable of modifying the gene expression and signal transmission of astrocytes and neurons. Such modifications primarily induce type II Alzheimer’s disease. Though glutamine production can cause damage to the brain, its production may also be able to prevent cell damage. Astrocytes can generate glutamine synthetase which catalyses the reaction of ammonia with glutamate so as to reduce the ammonia levels. However, this would not reduce the swelling nor assist much with the already effected neurons [35]. Methods for analysis involve taking blood measurements for ammonia levels and correlating the data against known neuropsychiatric standards such as the Trail Making Test (TMT) [38], the West Haven Criteria (WHC), and the Glasgow Coma Scale (GCS) [26]. The potential for measuring breath ammonia could replace the need for such invasive methods.

3.2 Haemodialysis

Assuming the liver is functioning properly, kidney failure can also result in harmful conditions such as uraemia (i.e. increased urea in the blood) [39], acidosis (i.e. elevated H+ levels), and edema (i.e. extreme water retention) [33]. Furthermore, hormones become imbalanced, bones lose strength, blood pressure increases, and fewer red blood cells are produced [40]. In the case where the filtration rate from the blood into the renal tubules is hindered or blocked, solutes that are normally filtered out of the body begin to build up in the blood. Urea reaches toxic levels and hydrogen compounds turn the blood acidic. This increase in solute concentration forces the body to retain as much water as possible to maintain equilibrium [33]. In time, the same consequences arise that result from liver dysfunction. Currently, the primary method for assisting renal failure is haemodialysis (Fig. 2). This begins by removing aliquots of blood from the body which then go through a dialyser to filter the toxins. Dialysers are reusable pieces of equipment that must be sterilised between uses. Within the dialyser, the blood is filtered by way of thousands of small fibre membranes. Blood passes through the fibres leaving the toxins trapped behind [40]. The rate at which toxins are removed from the blood is dependent upon blood flow rate due to solute concentrations, mass, and the area of diffusion [41]. To determine the individual filtration requirements, toxins are isolated according to Fick’s Law:

J = - DA (dc/dx) = - DA (c/x) (1)

where the flux of toxins J flowing over a distance of dx is proportional to the difference in concentration dc and the area of diffusion A. Diffusivity D is a constant value with units of cm2/sec that results from balancing the rest of the equation at a given temperature [41]. Once the toxins are isolated by diffusion, a cleaning solution known as dialysate flushes the waste material away from the dialyser fibres [40]. Dialysate is a solution made specific to individual needs, and hence the concentrations of solute vary. However, the general composition consists of sodium, potassium, calcium, magnesium, chloride, acetate, bicarbonate, and glucose [42]. Once the waste material is removed, the dialyser returns the clean blood to the body [40]. This is a time-consuming technique that requires most patients to visit a clinic about three times a week for six or more hours at a time [39]. While a patient is undergoing haemodialysis, a calculation is performed that shows how well urea is being filtered from the body. This is known as the urea reduction ratio (URR):

URR = ((BUN before treatment – BUN after treatment) / BUN before treatment) X 100% (2)

A URR of at least 65% is necessary for effective haemodialysis [20]. By focusing on blood urea nitrogen (BUN) and creatinine levels, a standard of excess nitrogen in the blood can be compared. The correlation between breath ammonia and BUN has been found to be 0.95, and 0.83 between breath ammonia and creatinine [20] and suggests that breath ammonia analysis has the potential to be an effective surrogate for BUN for monitoring haemodialysis efficacy.

Figure 2. Haemodialysis. Nitrogenous waste products of the blood are diffusing across the dialysis membrane and into the dialyser. From there, the dialysis fluid carries the waste away allowing filtered blood to return to the body [40].

3.3 Peptic ulcers

Aside from liver and kidney dysfunction, ammonia concentrations in breath can also be used to diagnose peptic ulcers affecting either the stomach or duodenum. The causal link between these ulcers and breath ammonia is a bacterium known as Helicobacter pylori. Currently, about 40% of adults are infected with H. pylori [43]. It is assumed that the infection is contracted through food or water, but the origin is still unclear. Stomach acids have little effect on the bacteria since H. pylori secrete urease enzymes that neutralise acids and weaken the surrounding tissue. By weakening the lining of the stomach and/or duodenum, H. pylori allows biological acids to deteriorate the tissue and form ulcers [44]. The current method of diagnosis is the urea breath test (UBT) and involves measuring the urease activity of the organism via the ingestion of 13C or 14C labelled urea [45]. If H.pylori is present in the stomach the high levels of urease excreted by H. pylori are detected by monitoring the breakdown of the labelled urea into radioactive carbon dioxide and ammonia as follows [46]:

(NH2)2CO + H2O CO2 + 2NH3 (3)

The compounds then pass through the blood, diffuse into the lungs, and can be measured in the breath which is detected using a scintillation counter [47]. Since initial ammonia baseline levels can vary from individual to individual, the rate of ammonia increase upon urea absorption is monitored. It has been shown graphically that the ammonia concentration of H. pylori negative individuals increases by approximately 0.12 ppm while H. pylori positive individuals increases by approximately 0.40 ppm [27]. Ammonia breath monitoring has the potential to play a role in measuring urease breakdown without the need for radioactive labels if the low detection limits of breath ammonia generated can be reached.

3.4 Halitosis

H. pylori is a urease positive bacteria which is specific to the stomach and duodenum. However, there are bacteria which can generate ammonia in the oral cavity. In the mouth, anaerobic bacteria metabolise food debris and create numerous byproducts which are the cause of the smells associated with halitosis [28]. About 90% of breath odour originates from the oral cavity as a result of orolaryngeal and/or gastrointestinal disorders [48]. Studies show that halitosis is primarily due to volatile sulphur compounds (VSC) in the oral cavity causing tissue damage and malodour. VSCs can be measured using a gas chromatograph in conjunction with a flame photometric detector system. Of the VSCs that develop, methyl mercaptan has been shown to have a correlation with ammonia [28]. Furthermore, the relationship between VSCs and ammonia has been shown to have a 0.39 correlation [49]. Normal concentrations of oral ammonia are usually too low to measure. However, as methyl mercaptan levels change, ammonia levels show proportional change as well. Furthermore, an increase in the bacterial load of the oral region correlates with increase in ammonia. Hence, by measuring the ammonia concentration from the bacteria that grow within the tongue coating and dental plague, and relating it to the methyl mercaptan levels associated with VSC measurements, ammonia in the oral cavity has the potential for assessing halitosis and oral hygiene [28].

3.5 Pulmonary dysfunction

Lung dysfunction or impairment such as asthma also has potential links with breath ammonia. It has been shown that individuals with asthma have lower levels of ammonia in their breath than healthy individuals. It is speculated that concentrations of ammonia produced by glutaminase may be directly affected by the levels of corticosteroids and cytokines produced by asthma patients. The methods used for analysis rely on collecting exhaled breath condensate in a lamellar condenser followed by measurement with a solid state ion selective electrode [50]. However, examining breath in gaseous form may simplify this technique.

4. Techniques for quantifying ammonia gas in breath

When it comes to quantifying ammonia gas, being able to isolate them from complex gaseous mixtures is important. Since the 1950’s, techniques such as chemical ionisation, gas chromatography, laser spectroscopy, and chemical detection have emerged as the key methods. Their sensitivity, precision and accuracy have proven suitable for selectively detecting and identifying low molecular weight species in gaseous form. Furthermore, combining these methods with each other in various ways (e.g. with mass spectrometry) shows potential for strengthening their detection capabilities. The techniques that follow and their detection limits for ammonia are summarised in Table 2.

4.1 Techniques based on chemical ionisation

Chemical ionisation uses the charge of a molecule to control how it reacts with other molecules. Under various pressures, the molecular reaction rate can also be controlled [51]. By combining atmospheric pressure chemical ionisation with mass spectrometry (APCI-MS), a spectral image displaying the mass-to-charge ratio can be obtained. APCI-MS was first developed as a method for analyzing trace gases in breath. However, due to interference problems with breath ammonia, an improvement was devised using protonated water clusters formed from ion-molecule reactions (Fig. 3) [8]. It was later confirmed that the proton transfer reactions of APCI were

Figure 3. Atmospheric Pressure Chemical Ionisation–Mass Spectroscopy (APCI-MS). By reacting protonated water clusters H+(H20)n with analyte T, a proton-transfer reaction takes place forming protonated analyte-water clusters TH+(H20)n of varying sizes. From here, unreacted nitrogen N2 molecules are forced to collide with the weakly bound water molecules of the cluster, thereby separating the water molecules (H20)n from the cluster and leaving only the protonated analyte TH+. The structure of the analyte is then analysed by mass spectrometry [52].

capable of increasing the sensitivity of the detection of compounds by several orders of magnitude [53]. Furthermore, data indicated that higher humidity levels decreased fragmentation of ionization gases and, therefore, increased overall sensitivity. Since normal human breath has an average relative humidity of around 84% from the nose and 94% from the mouth, this is a variable that must be considered [8]. This technique shows strong potential for analysis of ammonia in human breath since protonated analyte-water clusters of (H2O)nNH3+ have shown stability with other techniques [54]. Two chemical ionisation techniques that are capable of ammonia detection are proton transfer reaction – mass spectrometry (PTR-MS) and selected ion flow tube – mass spectrometry (SIFT-MS). PTR-MS (Fig. 4) has been used mostly for air analysis and environmental studies where atmospheric ammonia gas can be detected between 90 and 270 ppt [55]. However, PTR-MS has shown potential for analysing VOCs from exhaled breath as well [56]. Primarily, the precursor ion H3O+ is used to initiate proton transfer

Figure 4. Proton Transfer Reaction – Mass Spectrometry (PTR-MS). The initial section known as the “ion production section” collides electrons with carrier gas molecules so as to assert an ionic charge upon them. This ionised carrier gas, known as the precursor ion H3O+, reacts with trace gases which are added downstream. These reactants are carried to the “ion separation section” by gas flow, typically in the form of a buffer gas such as helium, to maintain a neutral atmosphere within the tube. Since the “ion shutter” only interacts with one ion charge at a time, specificity is high. Depending on what factors are being examined in the “drift-reaction section”, the shutter can be set to open at pulse intervals or simply remain open continuously. If set to pulse, then drift velocities can be analysed. In continuous flow, the reaction rates can be focused on [57].

with trace gases (T) such as VOCs, because H3O+ will react with most VOCs found in air. The majority of VOCs have a proton affinity higher than that of water, and will therefore transfer protons with almost every interaction [56]. Furthermore, only one ion species (TH+) will emerge from each collision along with the dissociation of water [58]. Using a flow-drift tube to control ion movement prevents cluster ions from forming which, in turn, results in a clear spectrum for analysis [16]. Cluster ions do not form since the ion separation section uses an electric field to send negative and positive ions in different directions [57]. However, the disadvantage of using an electric field is that ionic-molecular reaction times are unpredictable. PTR-MS has the advantage of being able to select for specific molecules via precursor ions. However, there is still a problem with separating compounds of similar pressure, mass, or density due to the use of H3O+ precursor ions alone [16]. With SIFT-MS, the initial concept was used to investigate the kinetic behaviour of gas phase ion-neutral reactions. Since then, thousands of gas reactions ranging from environmental to clinical have been studied using the SIFT-MS technique. It can be seen from Figure 5 that SIFT-MS is similar to PTR-MS in that it uses helium as the buffer gas at a given flow rate in the flow tube. However, unlike PTR-MS, three precursor ions (H3O+, NO+ and O2+) are available allowing for additional proton transfer reactions [59]. Having more

Figure 5. Selected Ion Flow Tube – Mass Spectrometry (SIFT-MS). The ions are introduced into the helium flow from an ion source at the beginning of the tube. Upon which, a gas sample (e.g. breath) is introduced directly and reactions take place. Adding breath samples to the system directly reduces the uncertainties that tend to arrive with collection methods [15]. Once the protonated analyte flows to the end of the tube, the ions are selected by a sampling orifice and directed into the mass spectrometer for analysis [60].

precursor ions allows for multiple ion spectra and greater quantification in mixed samples [61]. With SIFT-MS, analysis of breath VOCs (including ammonia) is achieved in about 10 ms with a sensitivity as low as 10 ppb [19]. Furthermore, water vapour and metabolite condensation are prevented by heating the tubing [16]. With real-time measurements and high sensitivities possible for clinical measurement of human breath, the SIFT-MS is being modified into a more portable version known as the Profile 3. The key benefits of this system are that it is smaller and more sensitive than previous versions [60].

4.2 Gas Chromatography

Another technique which has been widely used in quantifying breath gases is gas chromatography. This has been combined with sampling methods such as solid phase micro extraction (SPME) to collect the breath prior to their addition to the chromatographic column [4]. As the breath sample flows through the column, the individual gas molecules separate by their affinity for either the carrier gas or the liquid coating on the column [62]. However, since gas chromatography is reliant on first collecting a sample, quantification is not performed in real-time. Furthermore, SPME provides greater sensitivity, but concentration accuracy decreases due to possible loss during the collection process [16]. Chromatographic columns can also be damaged by moisture such as that found in humid breath. Hence, an additional drying method must be used to remove any water vapour that is present or the accuracy of readings could be effected [63]. The rate at which a species flows through the column is dependent upon the nature, amount, and surface area of the individual molecules being examined at a specific temperature [64]. In conjunction with gas chromatography, ion mobility spectrometry (GC-IMS) is capable of analysing the concentrations of ammonia found in human breath as low as 14 ppt [65]. Initially termed plasma chromatography (PC), the adaptation of IMS to GC first took place in the 1970s [54] and is capable of identifying breath gases by their mobility and drift velocity in an electric field at atmospheric pressure [4]. To calculate the mobility (K) of ions under the conditions of the electric field (E), the following equation can be used:

K = (Vd)(E-1) (4)

where Vd is the velocity of the drift ion [54]. The difference in mobility and drift velocity are dependent upon the differences in the unique mass and geometry of the specific gas particles [16]. However, due to IMS originally being designed for detection of chemical warfare agents, explosives and drugs, it relies heavily on known ion-molecular reactions [66]. This means GC-IMS spectrometers have limited sensitivity and cannot directly analyse breath samples. Hence, the trace gases from breath must be separated by way of column chromatography before the ion mobility can be deduced in the drift tube (Fig. 6). In some cases, the use of multi-capillary columns (MCC) have been recommended over a single narrow column because MCCs allow for a higher flow rate and sample capacity [65]. Furthermore, results can be obtained from 20 ms to

Figure 6. Gas Chromatography – Ion Mobility Spectrometry (GC-IMS). While separation is taking place in the capillary column, reactant ions are being produced in the “reaction region”. Once separation is complete, the analytes from the column are introduced into the reaction region where protonation takes place. The protonated gases then enter the electric field of the drift tube and head towards the detector at a constant velocity. Since there is a counter-flow of drift gas, the opposing ions collide with each other causing separation based on the individual charges and masses. From here, a spectrum based upon unique ion mobility is generated from impact intervals with the Faraday plate [67].

a few minutes depending on the number of spectra being studied. Using IMS spectrum peaks to identify differences between the breath of patients and healthy people has already been found to provide fast and accurate ammonia concentrations [66].

4.3 Laser Spectroscopy

Laser spectroscopy utilises the characteristic absorption or emission of energy by matter (in this case gas particles) at specific wavelengths when excited by a laser energy source [68]. Use of laser spectroscopy has shown high selectivity, high sensitivity, and real time potential for clinical breath ammonia analysis [25]. Specific techniques used for ammonia quantification are laser induced fluorescence (LIF), cavity ring down spectroscopy (CRDS), tunable diode laser absorption spectroscopy (TDLAS), photoacoustic spectroscopy (PAS), and optical frequency comb cavity – enhanced absorption spectroscopy (OFC-CEAS). With LIF, detection of breath molecules as low as 10 ppt has been demonstrated with gases such as nitric oxide [69]. Recently, however, LIF has shown the capability for making qualitative measurements of ammonia [70]. Ammonia detection is currently qualitative because of the typical problem of collisional quenching which results in no radiation from the fluorescing state during transition [71]. Using fluorescence techniques, ammonia molecules were originally detected at an excited state between 220 and 115 nm by way of a Nd:YAG-based dye laser source [70]. However, it has since been discovered that ammonia has multiple excitation states and investigation is underway to define these. Using the LIF technique of double-photon excitation, it is possible to excite ammonia molecules between the regions of 300 and 310nm. Doing so enables ammonia gas to fluoresce at wavelengths as high as 565nm (Fig. 7). Furthermore, LIF provides the advantage of being able to

Figure 7. Laser Induced Fluorescence (LIF). Initially, the laser interacts with the gas molecules (e.g. ammonia) at a specific wavelength. The molecules increase in energy from their original ground state to an excited level. To get back to the ground state, the molecules release the energy (de-excitation). This energy is radiated in the form of fluorescence which is detected and analysed by spectroscopy [70].

detect more than one species of gas at a time using a single laser emission. This would enable multiple gases to be measured simultaneously [70]. LIF is also considered to be a “background-free” technique implying that spectral images are not impeded by overwhelming emission light [71]. In CRDS, the molecules in breath are quantified according to the absorption rate of pulsed light within an optical cavity (Fig. 8) [72]. The rate of absorption is directly related to the amount of time required for light to leave the cavity. It is expected that the decay rate will be shorter if the absorption is larger [71]. This information can then be used to calculate the decay rate. The decay rate indicates the amount of photons lost, which in turn, defines the species of gases in the cavity [73]. The following equation shows how the decay rate (-1) decreases according to the rate at which the breath sample absorbs light:

-1 = (c (1 – R) / d) + c 

where c is the speed of light, d is the length of the cavity, R is the reflectivity of the mirrors, and is the absorption coefficient of the medium between the mirrors [73]. With CRDS, breath ammonia can be detected as low as 25 ppb over a period of 20 seconds. Using a mid-IR

Figure 8. Cavity Ring Down Spectroscopy (CRDS). The collected breath sample is first placed into the cavity between two mirrors. Once the sample has been added, a laser pulse is transmitted into the cavity at which point it bounces back and forth between the two mirrors. With each reflected hit, a fraction of the laser leaks out of the cavity and is detected by a photodetector [73].

Quantum Cascade laser at 967.35 cm-1, ammonia coincides with its strongest spectral region [74]. This method is highly sensitive, but due to the noisy transmitted intensity required from the laser, the accuracy is limited [75]. In the case of TDLAS (Fig. 9), its typical use has been to provide high-resolution spectra for gases from industrial pollution. However, due to its ability to detect various trace gases at low levels with little interference, it has also shown potential for clinical breath analysis [76]. TDLAS is capable of detecting breath ammonia at concentrations as low as 1 ppm in approximately 10 s [77]. However, breath samples must be pre-collected using such techniques as exhaling into a container [78] which, in turn, removes the possibility for real-time analysis. The TDLAS gas measurement is based on the Beer-Lambert relationship using an infrared laser to measure the transmitted intensity, Iv:

Iv = Iv,0 exp[- S(T)g(V-Vo)NL] (6)

Figure 9. Tunable Diode Laser Absorption Spectroscopy (TDLAS). The process begins with an infrared laser being introduced to the first lens. The lens divides the laser light into two beams. One beam is sent through a reference cell that contains the gas species that is being searched for in the breath sample. By doing this, an absorbance reference is provided to detector one for analysis comparison. The second beam is focused into and out of a monochromator by two mirrors. Monochromators serve the purpose of characterizing spectrums and wavelength calibrations. This is the half point of the laser beams full distance. Beyond the monochromator, the laser passes through the breath sampling chamber and intersects with detector two. Using the reference of beam one, beam two is then able to lock on to specific sample gases according to their unique absorbances [77].

where Iv,0 is the initial laser intensity, S(T) is the temperature-dependent absorption line strength, g(V-Vo) is the frequency dependence of the line strength, N is the target gas number density, and L is the optical path length through the gas [79]. PAS (Fig. 10) differs from other laser-based techniques in that it uses acoustics to measure gas concentrations. After a CO2 laser excites a molecule in the photoacoustic chamber, the light energy converts to heat [20]. As the molecule de-excites, the heat is emitted as vibrational energy and is monitored by a microphone [25]. This photoacoustic measurement (P) can be calculated as follows:

P = Poe -Nl (Po-P) ≈ PoNl (7)

where (Po-P) is the absorbed laser intensity which is converted to acoustic energy, is the area of absorption per molecule, N is the number of absorbing molecules, and l is the absorption path length [80]. In the case of breath ammonia, the gas molecules can be detected at a wavelength of 1531.68 nm [81]. PAS is capable of detecting breath ammonia at concentrations as low as 10 ppb in approximately 13 s [82]. This technique has high sensitivity, but difficulty with specificity can arise where spectra overlap due to similar acoustic frequencies that form between similar molecules [81].

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