Progress in SIFTMS: Breath analysis and other applicationsdownload.xuebalib.com/xuebalib.com.31345.pdf · Ligor et al., 2009) and membrane extraction (Lord et al., 2002). Multiple

PROGRESS IN SIFT-MS: BREATH ANALYSIS ANDOTHER APPLICATIONS

Patrik Španěl1* and David Smith21J. Heyrovský Institute of Physical Chemistry, Academy of Sciencesof the Czech Republic, Dolejškova 3, 182 23, Prague 8, Czech Republic2Institute for Science and Technology in Medicine, School of Medicine,Keele University, Thornburrow Drive, Hartshill, Stoke-on-Trent ST4 7QB, UK

Received 23 July 2009; received (revised) 12 September 2009; accepted 12 September 2009

Published online 20 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.20303

The development of selected ion flow tube mass spectrometry,SIFT-MS, is described from its inception as the modified verylarge SIFT instruments used to demonstrate the feasibility ofSIFT-MS as an analytical technique, towards the smaller butbulky transportable instruments and finally to the currentsmallest Profile 3 instruments that have been located in variousplaces, including hospitals and schools to obtain on-line breathanalyses. The essential physics and engineering principles arediscussed, which must be appreciated to design and construct aSIFT-MS instrument. The versatility and sensitivity of theProfile 3 instrument is illustrated by typical mass spectraobtained using the three precursor ions H3O

þ, NOþ and Oþ�2 ,and the need to account for differential ionic diffusion and massdiscrimination in the analytical algorithms is emphasized toobtain accurate trace gas analyses. The performance of theProfile 3 instrument is illustrated by the results of several pilotstudies, including (i) on-line real time quantification of severalbreath metabolites for cohorts of healthy adults and children,which have provided representative concentration/populationdistributions, and the comparative analyses of breath exhaledvia the mouth and nose that identify systemic and orally-generated compounds, (ii) the enhancement of breath metab-olites by drug ingestion, (iii) the identification of HCN as amarker of Pseudomonas colonization of the airways and (iv)emission of volatile compounds from urine, especially ketonebodies, and from skin. Some very recent developments arediscussed, including the quantification of carbon dioxide inbreath and the combination of SIFT-MS with GC and ATD, andtheir significance. Finally, prospects for future SIFT-MSdevelopments are alluded to. # 2010 Wiley Periodicals, Inc.,Mass Spec Rev 30:236–267, 2011Keywords: selected ion flow tube; SIFT-MS; trace gasanalysis; breath analysis; volatile organic compounds

I. INTRODUCTION AND HISTORICALPERSPECTIVE

This review is largely concerned with the advancement ofselected ion flow tube mass spectrometry, SIFT-MS, as ananalytical tool of increasing sensitivity and wider applicationand with instruments of smaller size. At the onset, the attentionof the reader is drawn to a major review that was published inMass Spectrometry Reviews several years ago, which describedthe essential physics and ion chemistry that are the basis ofSIFT-MS and which demonstrated the potential and the widescope and utility of this analytical technique in several fields ofresearch (Smith & Španěl, 2005). The focus of the presentreview is on the latest down-sized SIFT-MS instruments thatwere designed and constructed in response to the need for atruly transportable instrument that can be readily located indifferent environs to achieve real time analyses of ambient air,exhaled breath and other gaseous media. A major motivation forthese developments has been to realize instruments that can beused by health professionals in the clinical setting—hospitalwards, intensive care units and even general practitioner’ssurgeries—for exhaled breath analysis in support of clinicaldiagnosis and therapeutic monitoring. The results of severalpilot studies will be summarized to show that this importantobjective has been achieved. But the value of these SIFT-MSinstruments is by no means restricted to breath analysis and theyare finding much wider application, including the analyses ofurine and cell cultures headspace, volatile compounds emittedby bacterial cultures, monitoring of food freshness, andanalyzing combustion products, to name just a few. Yet themajor challenge has been the quantitative analysis of tracegases present in humid exhaled breath. When this hasbeen successfully achieved then the analysis of most otherless humid gaseous media offer relatively few problems, soin this spirit we outline the challenging aspects of breathanalysis.

The main thrust of clinical diagnosis focuses on blood andurine analysis. Clearly, such requires that samples of the bodyfluids be taken, which some consider invasive, especially bloodsampling which can be uncomfortable for the donor. Bloodanalysis is usually concerned with the large molecular weightnon-volatile compounds such as proteins and ions. However, itis now known that volatile compounds are present in blood; theycan cross the alveolar interface and appear in exhaled breath,being measured at trace concentrations in the parts-per-million

Mass Spectrometry Reviews, 2011, 30, 236– 267# 2010 by Wiley Periodicals, Inc.

————Contract grant sponsor: Keele University School of Medicine;

Contract grant sponsor: North Staffordshire Medical Institute; Contract

grant sponsor: Glaxo-Smith-Kline; Contract grant sponsor: Ministry of

Industry and Trade of the Czech Republic; Contract grant number: FT-

TA4/124; Contract grant sponsor: Grant Agency of the Czech

Republic; Contract grant numbers: 202/09/0800, 203/09/0256.

*Correspondence to: Patrik Španěl, J. Heyrovský Institute of Physical

Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 3,

182 23, Prague 8, Czech Republic.

E-mail: [email protected]

(ppm) and parts-per-billion (ppb) levels and lower (Gelmont,Stein, & Mead, 1981; Manolis, 1983; Phillips & Greenberg,1991; Španěl & Smith, 1996b; Phillips et al., 2003). Thesevolatile compounds can be valuable indicators of metabolicstatus, allowing distinction between the healthy and diseasedstate if their levels can be measured to an acceptable accuracy(Manolis, 1983; Kneepkens, Leparge, & Roy, 1994; Musa-Veloso, Likhodii, & Cunnane, 2002; Miekisch, Schubert, &Noeldge-Schomburg, 2004; Amann & Smith, 2005; Amann,Španěl, & Smith, 2007). Breath analysis as a clinical diagnosticis very attractive, because it is non-invasive and can generally becarried out for children, elderly persons and sick patients. It hasbeen used for some years in support of asthma diagnosis by thedetection of exhaled nitric oxide (Kharitonov & Barnes, 2001).The wider challenge is to identify and quantify a range ofvolatile compounds in exhaled breath to sufficient accuracy andprecision so that they may usefully support clinical diagnosis. Inresponse to this, remarkable developments in gas analyticaltechniques and in sampling methodology have been madeamongst which the exploitation of gas chromatography withmass spectrometry, GC-MS, has been very rewarding, espe-cially when combined with thermal desorption (Phillips &Greenberg, 1991; Jones, Lagesson, & Tagesson, 1995; Ligor,2009), solid-phase microextraction (Grote & Pawliszyn, 1997;Ligor et al., 2009) and membrane extraction (Lord et al., 2002).Multiple breath samples can be collected into bags or ontovarious forms of traps for release and subsequent analysis,but now on-line breath sampling can be achieved with theadvent of new analytical techniques (see Wenqing & Yixiang,2007). Of the newer techniques, SIFT-MS offers a uniqueapproach to the analysis of ambient air and exhaled breath, aswe will show.

What are the demanding analytical requirements thatwill allow breath analysis to become a reliable tool formedical diagnosis? Ideally, a wide range of diverse volatileorganic and inorganic compounds need be unambiguouslyidentified and accurately quantified in directly exhaled breathin real time, obviating sample collection into bags or ontotraps, which can compromise the sample by introducingexogenous impurities and selectively favor certain compounds,and delay analyses. These are serious demands made moreso by the fact that most of the breath metabolites are present atthe ppb level or lower, although it is now known that somedisease biomarkers are present in breath at the ppm leveland greater (Miekisch, Schubert, & Noeldge-Schomburg,2004; Amann & Smith, 2005). A serious point to note isthat the copious amount of water vapor present in exhaledbreath cannot be tolerated by most analytical methods andsteps need to be taken to remove water vapor from the sampleto be analyzed. However, this can seriously compromise thelevels of some trace metabolites, ammonia being a goodexample, and as such it is an undesirable practice if it can beavoided. This is a challenge that has been met successfully bySIFT-MS, as will be demonstrated in this paper. But it isimportant to stress, as mentioned above, that SIFT-MS has avaluable role to play in gas phase analyses in many other areasand the scope is widening as the smaller, more versatileinstruments become available. This review describes howdown-sizing has been achieved without diminishing instrumentperformance, but rather improving sensitivity and simplifyingoperation.

II. BRIEF OVERVIEW OF THE SIFT-MSANALYTICAL TECHNIQUE

The SIFT-MS analytical method is based on the selected ionflow tube (SIFT) technique that was conceived and developed,and exploited many years, for the study of the fundamentals ofion-molecule reactions at thermal energies (Adams & Smith,1976; Smith & Adams, 1987; Smith & Španěl, 2005). The SIFThas had a special role to play in the understanding of reactionsthat occur in the terrestrial atmosphere (Smith & Adams, 1980;Smith & Španěl, 1996b) and in interstellar clouds (Smith,1992). The use of SIFT for analytical purposes has beendescribed in some detail previously (Španěl & Smith, 1996a,b;Smith & Španěl, 1996a, 1999); the term SIFT-MS was thenquickly adopted and used in subsequent publications (Španěl &Smith, 1999d, 2001a; Smith & Španěl, 2005, 2007). A linediagram of a generic SIFT-MS instrument is shown in Figure 1,indicating the parts that are common to all such instruments. Achosen precursor ion (either H3O

þ, NOþ or Oþ�2 ) is selected by aquadrupole mass filter from a mixture of ions generated in amicrowave discharge and injected into fast-flowing heliumcarrier gas. The chosen precursor ion is used to ionize the tracegases in an air or breath sample that is introduced at a knownflow rate into the carrier gas downstream of the ion injectionpoint. The reactions between the precursor ions and the tracecompounds in the sample result in characteristic product ionsthat identify the compounds and their count rates allowquantification. The gas to be analyzed, which may be exhaledbreath sampled directly or collected and contained in a bag, orthe headspace of a liquid, is presented to the entry port of theinstrument and only a small fraction of it enters the carrier gasvia a heated, calibrated capillary. Single breath exhalations canbe sampled directly by simply exhaling through a disposablemouth piece coupled to the entry port of the instrument, asindicated in Figure 1. This displaces the ambient air withexhaled breath; inhaling via the mouth returns ambient air to thecapillary entrance for immediate analysis, as is desirable inbreath analysis studies.

There are two distinct analytical modes of operation ofSIFT-MS. Firstly, the full scan mode, FS, in which a conventionalmass spectrum is obtained over a chosen range of ion mass-to-charge ratio, m/z, to identify the precursor and product ions and todetermine their respective count rates. From these count rates,after appropriate corrections for mass discrimination and differ-ential diffusions (see Section IV.C), the on-line computerimmediately calculates the concentrations of those trace gascompounds present in the breath sample by exploiting the in-builtkinetics library, which comprises the rate coefficients and theproduct ions of the particular precursor ion/trace gas compoundreactions. This library has been constructed from numerousdetailed SIFT studies of the reactions of various classes ofcompounds (including alcohols, aldehydes, ketones, and hydro-carbons) with the three available SIFT-MS precursor ions (Španěl& Smith, 1995, 1996a, 1997, 1998a,b,c,d,e, 1999a,b,c; Španěl,Pavlik, & Smith, 1995; Španěl, Ji, & Smith, 1997; Španěl, VanDoren, & Smith, 2002; Španěl, Wang, & Smith, 2002; Smith,Wang, & Španěl, 2003; Diskin et al., 2002; Wang, Španěl, &Smith, 2003, 2004). It is worthy of note that all exothermic protontransfer reactions proceed at or close to the collisional rate and therate coefficients for these can be calculated, but such cannot beassumed for NOþ and Oþ�2 reactions and so their rate coefficients

PROGRESS IN SIFT-MS &

Mass Spectrometry Reviews DOI 10.1002/mas 237

are usually determined experimentally in relation to thecorresponding H3O

þ rate coefficients (Španěl & Smith, 1997).There are few compounds that cannot be detected by SIFT-MS;these include H2 and the smaller alkanes, principally because oftheir low proton affinities and relatively high ionization energies(Smith & Španěl, 2005).

Secondly, the multiple ion monitoring mode, MIM,in which the downstream analytical mass spectrometer israpidly switched between selected m/z values of both theprecursor ions and the product ions, to quantify both watervapor [used as an internal indicator of the sample integrity (seeŠpaněl & Smith, 2001c)] and the targeted trace compounds.This mode of operation provides more accurate quantificationof the targeted trace compounds than does the broad sweep fullscan mode and, because of the rapid time response of theinstrument, the time profiles of each metabolite in singlebreath exhalations are defined and the concentrations ofcompounds in alveolar (end tidal) breath can be obtained, asis well illustrated later.

The choice of the precursor ion in SIFT-MS depends on thetrace gas compounds in the air/breath sample to be analyzed,different characteristic product ions usually being produced inthe reactions of a given compound with the different precursorions. A simple example is all that is required in this paper, sincethe ion chemistry involved in the reactions of these precursor ionswith a wide variety of compounds has been reported fully inprevious papers and reviewed in Smith and Španěl (2005).Consider the analysis of acetone, a compound that is present inthe breath of all individuals. This compound reacts with all three

precursor ions used in SIFT-MS, but the characteristic productions differ, thus:

H3Oþ þ CH3COCH3 ! CH3COCH3Hþ þ H2O ð1Þ

NOþ þ CH3COCH3 þ He! NOþCH3COCH3 þ He ð2Þ

Oþ�2 þ CH3COCH3 ! CH3COCHþ�3 þ O2 ð60%Þ ð3aÞ

Oþ�2 þ CH3COCH3 ! CH3COþ þ CH�3 þ O2 ð40%Þ ð3bÞ

Hence, on a mass spectrum the product ions appear atdifferent m/z values, and this feature of SIFT-MS can be used topositively identify the acetone. Such can be used for othercompounds, especially isobaric compounds, although it must besaid that this is not possible for many compounds because of thecomplexity of the ion chemistry involved. The combination ofH3O

þ and NOþ precursor ions is especially useful, but in thisregard Oþ�2 ions are less useful because their reactions withpolyatomic molecules usually result in multiple ion fragments(Španěl & Smith, 1998e; Wang, Španěl, & Smith, 2004; Smith &Španěl, 2005). A downstream orifice (O2 in Fig. 1) samples theprecursor ions and the product ions, which then pass into adifferentially pumped quadrupole mass spectrometer and aredetected by an ion counting system. Hence, when acetone ispresent in the sample the product ions have m/z values of 59(using H3O

þ), 88 (using NOþ) and both 43 and 58 (using Oþ�2 ).The ratio of the total count rates of all the product ions (includingany hydrated product ions) to the count rates of all the precursor

FIGURE 1. A schematic diagram of a selected ion flow tube mass spectrometer, SIFT-MS, instrument. Thevarious ion transit and sampling orifices are labeled O0, O1, and O2, as discussed in the text. The ions

indicated in the flow tube are the precursor ions (upstream) and examples of the product ions (downstream).

Details of the ion chemistry involved are given in Smith and Španěl (2005). Some commonly used sampling

methods are indicated in the large circles; see also Figure 4.

& ŠPANĚL AND SMITH

238 Mass Spectrometry Reviews DOI 10.1002/mas

ions, the rate coefficients for the reactions of the precursor ionswith the particular trace gas compound and several critical para-meters are required, including the carrier gas and sample flowrates, for the proper quantification of trace gases, as explained indetail in a recent paper (Španěl, Dryahina, & Smith, 2006).

It is now important to stress the significance of the flow rateof the sample gas to be analyzed. In SIFT-MS analyses it mustbe appreciated that when water vapor is a component of thesample gas, and especially when using H3O

þ precursor ions toanalyze the sample, the water cluster ions H3O

þ(H2O)1,2,3 areformed in the carrier gas. If the sample gas is very humid, such asexhaled breath, then the sample flow rate must be small enoughto ensure that the fraction of the H3O

þ ions converted to thehydrates is not too large, because this complicates the ionchemistry on which the trace gas analyses rely (Španěl, Dryahina,& Smith, 2006). We have developed refined analyses to accountfor the partial conversion of H3O

þ to its hydrates (Španěl &Smith, 2000c), which have been shown to be reliable byexperiments on gases of varying humidity (Smith et al., 2001).However, much larger sample flow rates can be used to analyzedry samples for which water cluster ion formation is minimal.

A very important consequence of the formation of thehydrates of H3O

þ is that this phenomenon can be exploited todetermine the water vapor content of the sample to be analyzed.This is simply achieved by measuring the relative signal levels ofH3O

þ ions to the total signal level of the H3Oþ(H2O)0,1,2,3 ions

(which is equivalent to the H3Oþ signal level in the absence of

water vapor from the helium carrier gas). Then, knowing the ratecoefficient for the reactive conversion of the H3O

þ to H3OþH2O,

the water molecule number density in the carrier gas can bedetermined and knowing the sample gas flow rate its absolutehumidity can be obtained (Španěl & Smith, 2001c). This is aunique and valuable feature of SIFT-MS, since the absolutehumidity of exhaled breath is close to 6% (the saturation vaporpressure of water at body temperature) and so the measurement ofthe breath humidity acts as an internal check on the proper set upof the instrument. This is now a routine aspect of all SIFT-MSanalyses of breath. So, although validation studies have beencarried out (Španěl et al., 1997; Smith et al., 1998), calibration ofSIFT-MS instrument for each individual compounds usingprepared mixtures, which is difficult at low (ppb) concentrations,is not required when breath humidity can be measured.

On a related topic, we have developed a method by which thedeuterium content of water vapor can be measured. Initially, thiswas achieved using SIFT-MS (Španěl & Smith, 2000d), whichwas followed by the development of flowing afterglow massspectrometry, FA-MS (Smith & Španěl, 2001; Španěl & Smith,2001b, 2004). These methods are based on the determination ofthe relative signal levels of the cluster ions H3O

þ(H2O)3 orsimply H9O

þ4 (at m/z 73) and its isotopologues H8DO

þ4 and

H9O173 O

þ (at m/z 74) and H9O183 O

þ (at m/z 75) using accuratequantitative mass spectrometry. An important application of thisFA-MS technique is the measurement of total body water, TBW.Thus, a small but accurately known amount of D2O is given to aperson orally and the increase in the m/z 74 deuterated ion signalrelative to that for the m/z 75 ion (used as a reference level) istracked as the exhaled breath water vapor enters the instrument.The administered D2O molecules disperse throughout theTBW becoming HDO via isotope exchange with H2O, and byexploiting the simple idea of isotope dilution the TBW of theperson is obtained. This has been used to great effect to determine

TBW for healthy volunteers (Davies, Španěl, & Smith, 2001a;Smith et al., 2002; Engel et al., 2004) and, most recently, andmore importantly to determine TBW in haemodialysis patients(Chan et al., 2008) and to study water transport through theperitoneal membrane (Asghar et al., 2003, 2005).

Examples of the results of trace gas analysis studies inseveral areas of research, including breath analysis, using the firstgenerations of SIFT-MS instruments were presented a few yearsago in our previous mass spectrometry reviews paper (Smith &Španěl, 2005). The present paper largely reviews the new resultsobtained during the last few years using Profile 3 and otherSIFT-MS instruments.

III. DEVELOPMENT OF SIFT-MS INSTRUMENTS

All generations of SIFT-MS instrument have essentially the sameform, as depicted in Figure 1. They consist of an ion source andion selection quadrupole mass filter, a carrier gas flow tubereactor, a downstream analytical quadrupole mass spectrometer,a drive pump for the carrier gas and pumps to maintain thequadrupole chambers at suitably low pressures. The size, cost andperformance of a SIFT-MS instrument largely depends on thechoice of the following:

(i) Performance of the quadrupoles, which can be tracedback to their rod size and the frequency of the rf voltageused to energize them.

(ii) Length and diameter of the flow tube and the pumpingspeed of the carrier gas drive pump that is largelydetermined by its size and weight.

(iii) Speeds of the pumps required to maintain suitably lowpressures in the quadrupole chambers, which are largelydetermined by their sizes and weights.

(iv) Stability of the ion source and the currents of precursorions that it generates.

The ultimate sensitivity of the instrument as an analyticaldevice depends on the achievable precursor ion count rates(see (iv)) and the product ion count rates, as mentioned in theprevious section, but these are also dependent on the choice of theother three elements (i), (ii), and (iii) together with judiciouschoices of interdependent parameters such as the carrier gas andsample gas flow rates and the ion orifice aperture sizes, as will beseen later. It is self evident that a desirable goal is to producesmall, low cost instruments with superior performance that caneasily be moved and utilized in different locations. Remarkablestrides have been made towards these objectives, as reported inthis paper.

A. The Large Laboratory SIFT-MS Instrument

The first exploratory work on SIFT-MS in 1994 was initiatedusing a standard laboratory selected ion flow tube, SIFT,instrument that had been developed for the study of ion-moleculereactions (Španěl, Pavlik, & Smith, 1995; Smith & Španěl,1996a; Španěl & Smith, 1996a, 1999d, 2000b). In these earlystudies we demonstrated that proton transfer from H3O

þ reagentions (Španěl & Smith, 1995) and the reactions of NOþ and Oþ�2(Španěl & Smith, 1996a), as discussed above, could be used toanalyze the trace gases present in exhaled breath sampled directlyinto the helium carrier gas of the SIFT instrument. It is this early



observation of the value of H3Oþ precursor ions for trace gas

analysis that stimulated the development of proton transferreaction mass spectrometry, PTR-MS (Hansel et al., 1995;Lindinger, Hansel, & Jordan, 1998; Blake, Monks, & Ellis, 2009)that traditionally uses only H3O

þ precursor ions. But veryrecently, PTR-MS instruments have been modified to include theuse of both NOþ and Oþ�2 precursor ions (Jordan et al., 2009)following the precedence set by SIFT-MS. However, unlikeSIFT-MS where rapid switching between the precursor ions froman existing mixture in the ion source is achieved using aquadrupole mass filter, in the modified PTR-SRI-MS instrumentit is necessary to change the ion source gas mixture to favor eachparticular precursor ion species, which takes some time. Thus,the facility in SIFT-MS for rapid sequentially switching betweenthe H3O

þ, NOþ, and Oþ�2 precursor ions in times of order ofmilliseconds and to accumulate analytical data for each precursorion without delay, which is so valuable when analyzing limitedvolume and transient samples, is not achievable using the PTR-SRI-MS analytical method.

What are the characteristic dimensions of this largelaboratory instrument? They were typical of SIFT instrumentsof the day, being very large and heavy, following the precedenceof the early flowing afterglow instruments (Ferguson, Fehsen-feld, & Schmeltekopf, 1969; Dunkin et al., 1968, Smith et al.,1975; Adams & Smith, 1976), which surprisingly is stillfollowed today when new laboratory research flow tube instru-ments are built (Van Doren et al., 2008; Miller et al., 2009). Theflow tube was about a meter long and 8 cm diameter. The carriergas drive pump was a Roots-type kinematic pump weighingabout one metric ton with a pumping speed of about 500 L/sec.The flow speed of the helium carrier gas at a pressure of about0.5 Torr was such as to realize flow (reaction) times of order ofseveral milliseconds, which allowed the accurate study of ion-molecule reactions (Smith & Adams, 1987). Large quadrupoledevices were used in large vacuum housings with large oildiffusion pumps to evacuate them. Clearly, these instruments arevery heavy, in total weighing typically two metric tons, and areobviously strictly immobile, so gas samples to be analyzedusually had to be brought to the laboratory in bags. Using thisimmobile instrument at Keele University, UK, the basic principlesand the unique potential of SIFT-MS as an analytical tool weredemonstrated, and the earliest on-line, real time analyses ofexhaled breath of healthy volunteers and of patients with renalfailure were carried out (Smith & Španěl, 1996a,c; Španěl &Smith, 1996b; Španěl, Davies, & Smith, 1998; Smith, Španěl, &Davies, 1999) This instrument was also used for our initial studiesof urine headspace (Španěl et al., 1999; Smith et al., 1999).

The ion source that is common to all SIFT-MS instrumentsconstructed to date is a microwave cavity discharge throughhumid air at a pressure of about 0.5 Torr. This provides a source ofthe precursor (reagent) ions H3O

þ, NOþ and Oþ�2 , which arechosen because they do not react rapidly with the majorcomponents of air and exhaled breath (N2, O2, CO2, Ar, watervapor) but do react rapidly with most other permanent gases andvapors. The current of each of these three precursor ion that canbe extracted from these sources via the orifice O0 (see Fig. 1) is oforder of 10 nA, although there are ongoing programs to increasethese currents for reasons that will become apparent later. Acurrent of the mass selected precursor ion is then injected into thefast flowing helium carrier gas via a small orifice, O1. The heliumcarrier gas enters the flow tube via a Venturi inlet that is intended

to minimize the back flow of helium into the quadrupole massfilter chamber. The precursor ions are convected along the flowtube by the helium as a thermalized ion swarm where they reactwith the various trace gases present in the air/breath sample thatis introduced downstream. The precursor and product ions ofthe reactions are sampled by another small orifice, O2, into adifferentially pumped analytical quadrupole mass spectrometerand detected by an electron multiplier as ion counts per second,c/sec. Clearly, the ion current that passes into the carrier gas isdependent on the size of O1 and the ion current that entersthe analytical quadrupole is dependent on the size of O2. Thediameters of these orifices depend on the pumping speeds of thepumps evacuating the quadrupoles, and so they have to be chosenso that acceptable pressure in the quadrupole chambers can bemaintained. Typical diameters for O2 range from 0.3 to 0.5 mm.

It is neither appropriate nor necessary to present here adetailed description of the flow dynamics and kinetics involved inSIFT-MS analyses, since this has been given in detail previously(Španěl & Smith, 1996b; Španěl, Dryahina, & Smith, 2006).However, for that which follows, it is important to appreciate thebasic idea of SIFT-MS trace gas analysis. As the precursor ionsare convected along the flow tube by the carrier gas, their numberdensity, n1, near O1 (see Fig. 1) is continuously diminished bydiffusive loss to the flow tube walls resulting in a lower numberdensity, n2, near O2. After sampling via O2 and transmissionthrough the analytical quadrupole mass spectrometer, the countrate (c/sec) of the precursor ions at the ion detector is designatedI1. The ion diffusive loss is decreased by increasing the carrier gaspressure, p, but the limit to this pressure is determined by the gasflow that can be tolerated through O1 and O2 that ensuressufficiently low pressures in the quadrupole chambers. Clearly,the larger the diameters of O1 and O2 the greater are the ioncurrents that flow through them and hence the larger the I1 (and I2;see below). Reducing the length of the flow tube, l, and increasingthe flow velocity of the carrier gas and hence that of the ions, vi,will also reduce diffusive loss.

On introducing the sample gas to be analyzed, the precursorions react with the trace gases in the sample (rate coefficient k)producing characteristic product ions. These product ions arealso subject to diffusive loss along the flow tube; this complexsituation has been treated in detail previously (Španěl & Smith,2001a; Dryahina & Španěl, 2005). For the present purpose it issufficient to designate the count rate of a given product ionresulting from trace gas M as I2 and now the relation in itssimplest form that allows the number density of the trace gas,[M], in the carrier gas to be determined is:

I2 ¼ I1k½M�l

við4Þ

Clearly, the larger I1 the larger will be I2 (all otherparameters being equal) and the lower will be the detection limitof [M]. However, for a given [M], the larger the reaction time, l/vi,the greater will be I2. It is the proper balance between all thevariable parameters—O1, O2, p, l, vi—that has to be considered tooptimize the sensitivity of the instrument. Detailed discussions ofthese important principles are given for this laboratory instru-ment in our earliest SIFT-MS papers (Smith & Španěl, 1996a,c;Španěl & Smith, 1996b, 1999d). It is sufficient to state herethat the precursor ion count rates achieved were of order of5� 104/sec at best, and these combined with tolerable sampleflow rates and the relatively long reaction time of about 4 msec



gave a limiting sensitivity of this instrument of 10 ppb/sec of dataacquisition time for most trace compounds present in singlebreath exhalations (Španěl & Smith, 1996b).

The important factor that diminished the sensitivity of thismassive instrument for trace gas analysis was primarily the longflow tube and its large diameter, which could not be sufficientlycompensated even by the large kinematic pump. However, thisinstrument allowed much of the SIFT-MS exploratory workto be carried out and, in particular, showed that analysis ofsingle breath exhalations could be achieved in real time, withsufficient time resolution to allow the analysis of alveolar (end-tidal) breath, as described previously (Smith & Španěl, 1996a,c;Davies, Španěl, & Smith, 1996, 1997). This laboratory instrumentwas transported to Prague and the feasibility of scaling down itssize was tested by using a significantly smaller Roots pump (150 L/sec pumping speed) and a shorter flow tube of length 60 cm. Thisflow tube was used to investigate fundamental ion chemistry (e.g.,Španěl & Smith, 1998e; Španěl, Van Doren, & Smith, 2002;Dryahina, Polášek, & Španěl, 2004), to quantify anesthetic gasesin exhaled breath (Wang, Smith, & Španěl, 2002) and also toinvestigate the dependence of the diffusion losses of ions in heliumon their geometrical size (Dryahina & Španěl, 2005).

B. Transportable SIFT-MS Instruments,Mk.1 and Mk.2

The next obvious step in SIFT-MS development was to design aninstrument that could be transported to different locations toallow air and breath analysis on line and in real time obviatingsample collection. This required a leap into the unknown with adeparture from the massive SIFT instruments that have existed inseveral laboratories around the world for decades, some of whichwere beginning to be used for trace gas analysis (Kato et al., 2001;Wilson et al., 2001) following the precedence set in ourlaboratories.

The quantum leap in 1997 was the design and construction ofa transportable instrument with a shorter flow tube of length 40 cmand of diameter 4 cm using a smaller Roots pump (pumping speedabout 100 L/sec). This resulted in a compact instrument on wheelsof weight 350 kg. This combination, the Mk.1 instrument, resultedin a helium carrier gas flow velocity comparable to that forthe large laboratory instrument, the smaller pumping speed of thedrive pump being compensated for by the smaller diameter of theflow tube, with reaction times of order 3 msec. An additionaladvantage of this Mk.1 design was that the helium carrier gas flowrate was considerably smaller, typically 60 Torr L/sec (about5,000 sccm) as compared to some 250 Torr L/sec (20,000 sccm)for the large laboratory instrument. The oil diffusion pumps werereplaced by more efficient turbo molecular pumps to evacuate thequadrupoles, thus allowing some increase in the diameters ofthe O1 and O2 orifices and a higher carrier gas pressure. Thesefactors resulted in somewhat larger precursor ion count rates oftypically 105/sec. Using similar sample flow rates used for thelaboratory instrument, the analytical sensitivity of this Mk.1SIFT-MS instrument was marginally increased to about 5 ppb/secfor breath samples.

To obtain accurate trace gas quantification it has beenessential to gain an understanding of the differential diffusionphenomenon in which the product ions of the analytical reactionsdiffuse to the flow tube walls more slowly than the precursorions. This phenomenon is called ‘‘diffusion enhancement’’.

This, together with mass discrimination in the detection massspectrometer, has been parameterized as a function of m/z(Španěl & Smith, 2001a), which was then implemented into theSIFT-MS software. Thus, a truly quantitative SIFT-MS instru-ment was created. These physical phenomena are discussedfurther in relation to the development of the much smallerinstrument (see Section IV.C).

But the most significant point is that this instrument, whilstvery cumbersome, was mobile and it has been located in differentplaces to perform on-line analyses, including in the renal dialysisunit of the local hospital to analyze the breath of patients pre- andpost-haemodialysis (Davies, Španěl, & Smith, 2001b), and in adistant health and safety laboratory to analyze the exhaust gasesof a diesel engine (Smith et al., 2004). More importantly, theextensive use of this unique instrument put SIFT-MS analysesonto a firm foundation and established SIFT-MS as a veryvaluable addition to the methods available for trace gas analysis,especially demonstrating the value of real time analyses of breathfor physiological studies, clinical diagnosis, and therapeuticmonitoring. The numerous papers published on the methodologyand applications of Mk.1 are referenced in our previous reviews(Smith & Španěl, 2005; Španěl & Smith, 2007) and in a recentbook (Amann & Smith, 2005).

The first commercial SIFT-MS instrument, Mk.2, built in2001, was essentially designed with the same parameters asMk.1, but using more modern components (pumps and quadru-poles) with improved specifications, allowing some furtherrelaxation of the critical parameters (e.g., orifice diameters), asdiscussed above. This resulted in another improvement in thedetection limit for trace gas analysis of breath samples to about2 ppb for 1 sec integration time. This Mk.2 instrument has beenused to great effect by Turner, Španěl, and Smith (2006a,b,c,d),especially for promoting breath analysis by detailed studies of theconcentration distributions of several breath metabolites usinglarger cohorts of volunteers (Smith, Turner, & Španěl, 2007). It ispertinent to show a typical full scan (FS) spectrum obtained forthe analysis of the breath of a healthy volunteer using this Mk.2instrument. This is shown in Figure 2 for the analysis of a breathsample collected into a Nalophan bag (contrast this with the

10 20 30 40 50 60 70 80 90 100 110 12010 0

10 1

10 2

10 3

10 4

10 5

10 6 c/s

18

19

30

32

36

37

51

55

59 65

69

73

77

79

81

83

85

87

91

93 95 97

99 103 107

117

ammonia acetone

ethanol

methanol

H3O+(H2O)0,1,2,3

47

m/z

33

FIGURE 2. A typical full scan (FS) mode mass spectrum (countsper second, c/sec, against m/z) ranging from m/z values of 10–120

obtained using H3Oþ precursor ions when a sample of breath from a bag

is introduced into the Mk.2 SIFT-MS instrument. The major trace

compounds in the breath sample are indicated, as recognized by their

characteristic product ions.



improved spectral data obtained using the Profile 3 instrumentshown later in Fig. 5). The major breath metabolites are indicatedby their characteristic product ion peaks. As mentionedpreviously, to improve the accuracy of quantification of specificmetabolites in single breath exhalations the multiple ionmonitoring mode (MIM) is used. Thus, longitudinal studies of

the concentration distributions of several breath metabolites havebeen obtained for significant populations of healthy adultvolunteers by monitoring mouth-exhaled breath (see laterdiscussion in Section V.D). The concentration distributions soobtained for a cohort of 30 adult volunteers over a period of6 months are shown in Figure 3. These data demonstrate the great

Ammonia (median 833 ppb)

Methanol (461 ppb)

Ethanol (112 ppb)

ppb1 10 100 1000200 30020 30

Isoprene (106 ppb)

ppb1 10 100 1000200 30020 30

ppb1 10 100 1000200 30020 30

Acetone (477 ppb)

ppb1 10 100 1000200 30020 30

ppb1 10 100 1000200 30020

GSD = 1.62

GSD = 1.58

GSD = 1.62

GSD = 3.24

GSD = 1.65

FIGURE 3. Concentration distributions for the five most prominent trace metabolites present in the mouth-exhaled breath of the healthy population. The concentrations on the horizontal axes are shown by

logarithmic scales in parts-per-billion, ppb. The median values are given in parentheses for each metabolite

together with their associated geometrical standard deviation, GSD. The curves through the ammonia,

acetone and methanol data describe logarithmic distributions. The wider distribution for ethanol is partially

explained by oral production of this compound (Španěl et al., 2006; Turner, Španěl, & Smith, 2006c). The

collected data are taken from Turner, Španěl, and Smith (2006a,b,c,d). Reproduced from Smith and Španěl

(2007) by permission of The Royal Society of Chemistry (on-line http://dx.doi.org/10.1039/b700542n).



potential of SIFT-MS for on-line non-invasive breath analysis;more examples are shown later in Section V. Further to thesestudies, Turner’s group have carried out a study of the metabolitespresent in the headspace of the serum extracted from wildbadgers suffering from tuberculosis, TB (Spooner et al., 2009).Multivariate classification algorithms were then employed toextract a simple TB diagnosis from the complex multivariateresponse provided by the SIFT-MS analytical mass spectra. Thisis the first publication that describes the application of multi-variate analysis to SIFT-MS data. ATSIFT Mk 2-type instrumentwas also built by Canterbury University using documentationand know-how provided by us. This was later followed bythe production of smaller instruments by Syft Technologies Ltd,New Zealand (Francis et al., 2007). Their latest instrument,Voice 200, has a weight of 212 kg, which is a significantreduction on the weight of Mk.1 and Mk.2 instruments and theirVoice 100.

C. Portable SIFT-MS Instruments, Profile 3

Notwithstanding the great success of the Mk.1 and Mk.2instruments there remained an obvious need to produce a SIFT-MS instrument of smaller and more manageable size, lower cost,lower running costs and quieter and simpler to operate thatcould be used by non-specialists in hospital wards, intensivecare units and GP surgeries. This has required considerablethought around the physics (especially flow dynamics andVenturi design), the engineering (allowed pumping systemsand vacuum housings) and the ion chemistry underpinningSIFT-MS. This resulted in the production in the UK of the Profile3 SIFT-MS instrument1 in 2006 that has many of the desiredfeatures. With this major development, improvements havebeen made in sampling methodology, especially for on linebreath analysis.

In designing SIFT-MS instruments it is necessary to beguided by the scientific principles expressed by Equation (4),especially the requirements of a sufficiently high I1 and areaction time, l/vi, long enough to allow ion-molecule reactionslike reactions (1) to (3) to result in I2 count rates that can bemeasured to acceptable precision. For high I1 a short flow tube, l,is favored, but this diminishes l/vi unless vi is reduced. However, asmall vi can result in a greater loss of ions via diffusion unless thehelium carrier gas pressure, p, is increased. But a higher value of prequires smaller orifices O1 and O2. So, as always, a compromiseis required and some exploratory experimentation was needed.However, the design challenge was alleviated because of theunderstanding gained over the years of the essential physics andion chemistry of SIFT-MS, as learned from the development ofthe Mk.1 and Mk.2 models.

It turns out that in terms of the physics and ion chemistrythere is no practical limit to the size and weight of a SIFT-MSinstrument, although it is certain that some loss of sensitivity willhave to be expected for very small instruments; rather, it is theavailability and performance of the engineering components thatcurrently are the limiting factors. To reduce the size and weight

significantly, the large pumps utilized in Mk.1 and Mk.2, inparticular the Roots pumps, have to be dispensed with. Yet a drivepump is required to flow the carrier gas rapidly enough tomaintain the rapid time response of the instrument. Currently,rotary backing pumps are an available alternative with pumpingspeeds of order 10 L/sec at the allowed carrier gas operatingpressure of about 1 Torr, and this is the starting point for thedesign of the current Profile 3 instrument. This lower pumpingspeed now requires downscaling of the flow tube dimensions inaccordance with the principles expounded above. The turbomolecular pumps chosen to evacuate the upstream and down-stream mass spectrometer housings have speeds within the range70–550 L/sec, depending on the sizes of the sampling orifices O0,O1, and O2 that are chosen.

The actual dimension of the Profile 3 flow tube is 5 cm longand 1 cm diameter (see Fig. 4). The operating helium carriergas pressure, p, is typically 1.0 Torr and the reaction timeis about 0.5 msec. Because of the shorter flow tube andthe somewhat higher p than in the larger instruments,the diffusive loss of precursor ions to the walls of the flowtube is much reduced resulting in I1 values greater than 10

6/sec(using the improved microwave discharge ion source con-ditions, as discussed later in Section IV.A; also see Fig. 4). Notethat these greater I1 values do not reflect directly in an increasedsensitivity vis-à-vis the larger instruments because of thesmaller reaction time in this instrument. These developmentshave realized a SIFT-MS instrument of total weight only 120 kgmounted on wheels, which is readily moveable by oneindividual around hospital environments. It is very quiet whenoperating and so it can be used without serious disturbance topatients and healthcare workers alike. The consumption ofhelium carrier gas is also very much reduced when compared tothe larger SIFT-MS instruments, such that it is only a smalladdition to the running costs.

So what is the current sensitivity or limit of detection of thisProfile 3 instrument and what is the accuracy and precision thatcan be realized? As for the previous generations of SIFT-MSinstruments, these are dependent on the I1 values, the allowablesample flow rates (sample humidity) and the proper set up of theanalytical software to account for diffusion and mass discrim-ination effects. These are discussed individually in the nextsection, but it can be stated here that the detection limit of theProfile 3 instrument can typically approach 0.1 ppb for 1 sec ofintegration.

IV. PERFORMANCE OF THE PROFILE 3INSTRUMENT

The performance of Profile 3 SIFT-MS instruments has beenillustrated by the data presented in several research papersproduced by the teams at Keele (Pysanenko, Španěl, & Smith,2008, 2009; Smith et al., 2008; Wang et al., 2008a; Wang, Španěl,& Smith, 2008b; Pysanenko et al., 2009), at Prague (Španěl,Dryahina, & Smith, 2007a,b,c) and at Thunder Bay (Ross &Vermeulen, 2007; Ross, 2008; Hryniuk & Ross, 2009; Ross,Babay, & Ladouceur, 2009; Ross et al., 2009). Scrutiny of thesepapers reveals the value and the quality of trace gas analysesobtained using current Profile 3 instruments. However, in thisreview it is pertinent to outline the special features of theseinstruments and some new developments and improvements intheir sensitivity and in the analysis procedure, which are a direct

————1The Profile 3 SIFT-MS instrument is a joint development by Trans

Spectra Limited, Newcastle-under-Lyme, UK (TSL) and and Instru-

ment Science Limited, Crewe, UK (ISL) and is now commercially

available from ISL.



result of their greater sensitivity. Following this, some recentexciting results that have been obtained using Profile 3 instru-ments for breath and urine headspace analysis and in other areasof trace gas analysis will be reviewed.

A. Improvement in Sensitivity: Quality ofFull Scan Spectra

The performance of the ion sources used in SIFT-MS instrumentsis paramount in determining their sensitivities and detectionlimits for trace gas analysis. We have carried out a careful study ofthe ion chemistry occurring within the microwave dischargeplasma ion sources that are currently used for the production ofcurrents of the precursor ions H3O

þ, NOþ and Oþ�2 ions andshown that the most suitable ion source gas composition is amixture comprising maximal water vapor and minimal air at thelowest total pressure at which the discharge is sustained andstable (Španěl, Dryahina, & Smith, 2007c). Under theseconditions, count rates, I1, greater than 10

6/sec of all threeprecursor ions with less than 1% of impurity ions have beenachieved, even approaching 107/sec with careful tuning. This hasresulted in a massive improvement in the quality of the massspectra obtained by these SIFT-MS instruments.

‘‘Quality’’ in this context refers to the signal levels, that is,the achievable count rates of the precursor ions and thecharacteristic analytical product ions, which have a first orderinfluence on the sensitivity of the SIFT-MS instrument fortrace gas analysis. The gradual improvements in the perform-ance of the microwave discharge ion sources now allows countrates for the precursor ions of several million per second to berealized, which are so high that the ability of most electronmultipliers to record accurately is challenged. This is not initself a limiting factor, because then the minor isotopologues ofthe precursor ions can be used and an appropriate scaling factorapplied to obtain the count rates of the major isotopologues,although to date this has not yet been implemented. Such anapproach, however, must to some extent diminish the accuracyof trace gas quantification, because the isotopic ratios of theisotopologues is not always well defined when complex gasmixtures containing large number densities of reactive com-pounds, especially water vapor, are being analyzed, because ofkinetic isotope effects (Španěl & Smith, 2000d). Examplesof the spectra obtained for the current Profile 3 instrumentsare shown in Figure 5 for H3O

þ, NOþ and Oþ�2 precursorions. These spectra were obtained as samples of exhaledbreath entered the instrument at the entry port (see Fig. 4).

FIGURE 4. A schematic diagram of the Profile 3 SIFT-MS instrument showing the microwave dischargeion source, injection mass filter, the detection quadrupole mass spectrometer and the three metal discs to

which ion current can be measured and which support the orifices O0, O1, and O2 through which,

respectively, ions pass from the ion source into the injection mass filter, mass selected ions enter the flow

tube, and via which ions are sampled into the analytical quadrupole mass spectrometer. Both direct breath

sampling into the instrument and sampling from bags are illustrated. Reprinted from Smith, Pysanenko, and

Španěl (2009b) with permission from Elsevier.



As well as the peaks due to the precursor ion signals andtheir hydrates, some of the peaks due to product ionsresulting from the presence of some major breath metabolitesare indicated.

From such quality spectra the major breath compoundspresent in single breath exhalations at levels of typically 50–1,000 ppb, such as ammonia, acetone, and isoprene (Čáp et al.,2008; Wang et al., 2008a) can be quantified to a precision of a few

FIGURE 5. Profile 3 SIFT-MS spectra (ion counts per second, c/sec, against m/z) obtained using (a) H3Oþ,

(b) NOþ and (c) Oþ2 precursor ions for the analysis of exhaled breath. The precursor ions, their isotopologuesand their hydrates are indicated by the open bars. The major trace compounds in the breath sample are

indicated, as recognized by their characteristic product ions. Reprinted from Španěl and Smith (2009) with

permission from Elsevier.



percent. Equally significant is that other compounds present atlevels of just a few ppb can be identified and quantified toclinically acceptable precision, say 20%, for example, hydrogensulfide (Španěl & Smith, 2000b; Pysanenko, Španěl, & Smith,2008), hydrogen cyanide (Španěl, Wang, & Smith, 2004; Carrollet al., 2005), and acetic acid (Pysanenko, Španěl, & Smith, 2009).Thus, the limit of detection, LOD, by SIFT-MS for the volatilemetabolites present in air and even in single breath exhalationscan be moved into the 0.1–1 ppb regime using longer dataacquisition times (e.g., see Milligan et al., 2007) and samplecollection techniques such as solid phase microextraction andthermal desorption in combination with SIFT-MS (Ross, 2008).The actual LOD can be evaluated for any trace gas in terms of theprecursor ion count rate, the sample flow rate, the reaction ratecoefficient and the data acquisition time (Španěl, Dryahina, &Smith, 2006).

It is worthy of note here that accurate analyses of theheadspace of very small volumes of liquid can be made over awide dynamic range, which is especially useful for blood plasmasamples. To demonstrate this, experiments have been carried outto assay methanol, ethanol and isopropanol in the headspace ofaqueous solutions of volume ranging from, 50 to 1,000 mLcontained in air-tight tubes held at 378C (Rowbottom, Workman,& Roberts, 2009). The vapor above the sample was aspirateddirectly into the Profile 3 instrument. The headspace concen-trations of the alcohols were measured using the MIM mode ofanalysis and were seen to be linearly dependent on the respectiveliquid phase concentrations over a wide concentration range,independent of the sample volume and relatively independent ofthe osmolar concentration. This novel application of SIFT-MS iseasy to follow, requires no sample preparation and the widedynamic range will facilitate measurement of alcohols present inurine and blood due to normal metabolism as well as when takenin excess or in accidental poisoning.

B. Important Additional Ion Chemistry Revealed

Before the advent of this more sensitive generation of SIFT-MSinstruments it was tacitly assumed that the presence of the majorcompounds in air and breath samples, viz. N2, O2, Ar, CO2, hadlittle influence on the ion chemistry occurring in the carrier gasreactor. This was simply because it was known from many ionchemistry studies, notably of the terrestrial ionosphere (Smith &Adams, 1980; Smith & Španěl, 1996b), that reactions of theSIFT-MS precursor ions with these neutral compounds were veryslow. Indeed, this is why the precursor ion that are used for SIFT-MS analyses are chosen! Also, any product ions formed in anysuch reactions were not visible in the ion mass spectra obtainedat the relatively low sensitivities of the earlier instruments.However, this is no longer the situation, because adduct ions suchas H3O

þN2, H3OþCO2, NO

þCO2, and Oþ2 CO2 are clearly seen in

Profile 3 analytical spectra. Such are apparent in the spectrashown in Figure 6, which were obtained as a mixture of dry aircontaining 5% carbon dioxide is introduced into the heliumcarrier gas. These adduct ions are formed in the relatively slowthree-body association reactions of the precursor ions with N2and CO2, as has been discussed in detail in a recent paper (Španěl& Smith, 2009). Such association reactions are promoted by highcarrier gas pressures and so they are more likely to be formed atthe higher helium pressures that are required for the Profile 3instruments as compared to those used in the first generation of

SIFT-MS instruments. It must also be said that these looselybound adduct ions react rapidly with water molecules by the well-known process of ligand switching (Adams et al., 1970; Feng &Lifshitz, 1995; Španěl & Smith, 1995) producing hydrates of theprecursor ions, for example, H3O

þH2O, and so they are largelydestroyed when humid samples like exhaled breath are beinganalyzed.

Now it is important to understand that these ions caninterfere with the analyses of certain compounds, for example,the H3O

þN2 adduct ion at m/z 47 overlaps with that of protonatedethanol, C2H5OH

þ2 , and if not accounted for they can result in

erroneous quantification of ethanol present at low levels in asample of air or breath. But if the ion chemistry that leads to theformation and reactive loss of these adduct ions is understoodthen their interference in the analysis of trace level compoundscan essentially be eliminated. This is well illustrated by ouranalysis of acetaldehyde in exhaled breath that contains CO2typically at the 3–5% level, which inevitably results in theformation of H3O

þCO2 adduct ions at m/z 63 when using H3Oþ

precursor ions. This ion overlaps with the monohydrate ofprotonated acetaldehyde, CH3CHOH

þH2O, also at m/z 63, and soit would seem that acetaldehyde in breath could not be properlyquantified. However, we have carried out a study of the complexion chemistry that occurs when humid air containing CO2 andtrace amounts of acetaldehyde (to simulate exhaled breath) isintroduced into the helium carrier gas of the Profile 3 instrument.The complex scheme of the reactions that occur is shown inFigure 7. From this work it was shown to be possible to analyzeacetaldehyde in breath by excluding the m/z 63 ion from theanalysis of this compound (Španěl & Smith, 2008) and anabsolute quantification equation accounting for these interfer-ences was formulated and appropriate entries into the SIFT-MSkinetics library were constructed. This represents yet anotherextension of the detailed ion chemistry work that is required tocontinuously build the kinetics library that is needed for SIFT-MS(Španěl, Dryahina, & Smith, 2006). This almost hidden danger ofadduct ion formation is the price paid for increased sensitivityand so greater vigilance is needed in the exploitation of thesemore sensitive instruments. But clearly, enhanced sensitivity is adesirable development and one very beneficial aspect is that thedetection of the H3O

þCO2 adduct ion has provided a method forthe quantification of CO2 in air and exhaled breath, as is discussedin Section V on breath analysis.

C. Understanding Diffusion and Mass Discriminationto Obtain Accurate Analyses

The physics and chemistry of ion production and loss in the flowtubes of SIFT-MS instruments together with ion sampling into theanalytical mass spectrometer and mass discrimination in thelatter have been discussed in detail in previous papers (Španěl &Smith, 1996b, 2001a). The essential point to note is that if bothdiffusion enhancement of the (heavier) product ion currents at thedownstream sampling orifice, O2 (see Fig. 4), above that of the(lighter) precursor ions and mass discrimination against heavierions in the analytical mass spectrometer are not accounted forthen inaccurate analyses will result. These phenomena areinstrument dependent and so a separate study must be carried outto quantify them under the specific operating conditions of eachinstrument and their influences must be implemented in theanalytical software.



FIGURE 6. Profile 3 SIFT-MS spectra (ion counts per second, c/sec, against m/z) obtained when analyzingdry cylinder air containing about 5% CO2. The precursor ions, their isotopologues and their hydrates are

indicated by the open bars. a: H3Oþ precursor ions; note the clear adduct ion peaks of H3O

þN2 andH3O

þCO2 at m/z, 47 and 63 and that at m/z 88, which is COþ2 CO2. b: NO

þ precursor ions; note the adduct ionpeak of NOþCO2 at m/z 74. c: O

þ2 precursor ions; note the adduct ion peaks, O

þ2 H2O, O

þ2 O2, and O

þ2 CO2 at

m/z 50, 64, and 76 respectively and that of H2OþH2O at m/z 36. Reprinted from Španěl and Smith (2009)

with permission from Elsevier.



The required experiments involve the total conversionof the precursor ions to product ions at several m/z values andthe measurement of the increased ion current, usually in pA,to the disc supporting orifice O2 resulting from the decreaseddiffusive loss of the heavier ions; a typical plot of the relativecurrent increase with increasing m/z of the ions is shown inFigure 8a. Simultaneously, the count rates of the ions collected bythe mass spectrometer counting system in terms of the count rateper pA are obtained for ions at the different m/z values and a massdiscrimination factor is obtained for each ion, as exemplified inFigure 8b. A detailed study of this kind for the Keele Profile 3instruments has been carried out (Smith, Pysanenko, & Španěl,2009b) and it was shown that consistent and accurate analyses ofacetone and ammonia in exhaled breath can be obtained using thedifferent precursor ions available in SIFT-MS. On the basis of thisexperimental work, parameterized functions accounting for bothdiffusion enhancement and mass discrimination have beenimplemented in the SIFT-MS analytical software. Thus, it isnow a straightforward procedure to quickly characterize anySIFT-MS instrument during software configuration and thenaccurate analyses of compounds that result in a wide range of m/zvalues of characteristic product ions can routinely be achieved.

Accurate quantitative analyses are based on a completelydocumented numerical method that allows the calculation, inreal time, of absolute concentrations of trace gases, includingvolatile organic compounds, and water vapor in exhaled breath(Španěl, Dryahina, & Smith, 2006). In this calculation noassumptions are made concerning the SIFT-MS instrument sizeor its configuration and thus the calculation can be applied toboth the large SIFT-MS instruments and the smaller Profile 3instruments. But this numerical method must clearly recognizethose parameters that are specific to a particular instrument,including flow tube geometry, reaction time, differential

diffusion and mass discrimination factors and the essentialreaction kinetics that describe the formation and loss of theproduct ions formed in the chemical ionization of the trace gasesby the precursor ions. A generalized calculation of the requiredionic diffusion coefficients is introduced with options either fortheir accurate determination from the molecular geometry of ions(Dryahina & Španěl, 2005) or for less accurate but simplerestimates obtained using just the ionic mass (Španěl, Dryahina, &Smith, 2006). A numerical example of the calculation of theacetone concentration in exhaled breath from the precursor andproduct ion count rates has been presented (Španěl, Dryahina, &Smith, 2006) as an illustration of the calculation sequence that isnormally performed automatically in real time by the SIFT-MSinstrument software. Recently a thorough study of the repeat-ability of the measurement of exhaled volatile metabolites usingSIFT-MS has been carried out under clinical conditions, whichshows the quality of the data obtainable (Boshier, Marczin, &Hanna, 2010).

D. Recent Extensions to SIFT-MS

The major contribution that gas chromatography (GC) has madeand continues to make to the analysis of multicomponent mixtures,especially when combined with mass spectrometry (GC-MS), iswell known (Karasek & Clement, 1988). It has been exploited forthe analysis of numerous media, both gases and liquids, includingexhaled breath (Phillips & Greenberg, 1991; Grote & Pawliszyn,1997; Lord et al., 2002; Phillips et al., 2003; Miekisch, Schubert,&Noeldge-Schomburg, 2004; Ligor, 2009). A unique feature of GCis the clear temporal separation of the individual componentseluting from the column, which essentially allows each componentof the mixture to be identified and quantified separately. However,in one respect GC techniques are somewhat less than perfect

H3O+ H3O

+(H2O)H2O

H3O+(H2O)2

H2O H2OH3O

+(H2O)3

C2H5O+ C2H5O

+(H2O)(m/z 63)

H2O

CH3CHO CH3CHO

CH3CHO

CH3CHO

CH3CHO

O2+

C2H5OH

(C2H5OH+, CH3O

+)

C2H5O+(H2O)2

(m/z 81)

H2O

CO2

H3O+(CO2)

(m/z 63)

H2O

FIGURE 7. The ion chemistry of H3Oþ precursor ions reacting with acetaldehyde in the presence of water

vapor, including the ‘‘interfering’’ reactions of ‘‘impurity’’ Oþ2 ions with ethanol and the association ofH3O

þ with CO2 (for discussion of the latter reaction see Section V.B and Smith, Pysanenko, & Španěl,2009a). The dotted arrows indicate three-body association reactions, the one thick solid arrow indicates a

two-body proton transfer reaction; the one reverse reaction indicates collisional dissociation and the thin

solid arrows indicate other two-body reactions (ligand switching, charge transfer and dissociative charge

transfer). Adopted with permission from IOP Publishing from the scheme given in Španěl and Smith (2008).



because real time (rapid) analysis is not readily achievable.Additionally, for every compound to be quantified in a sample it isnecessary to define the retention time and to calibrate the detectorsensitivity using standard mixtures. So, if reliable quantification isto be achieved, a regular check on the sensitivity of the associateddetector (the MS ion source) must be carried out. To alleviate thiswe have explored the use of SIFT-MS as a mass spectrometer toquantify the compounds eluting from a GC column (Kubista et al.,2006). Hence, we have shown that the GC/SIFT-MS combinationallows accurate trace gas quantification obviating the regulartime consuming calibrations that are usually required for the morecommonly used detectors in GC systems. Additionally, positiveidentification of isomers in mixtures can be accomplished, which isoften challenging using SIFT-MS alone. Thus, the GC/SIFT-MScombination paves the way to more confident analyses of complexmixtures such as exhaled breath.

A more recent development by Ross and Vermeulen (2007)has involved the combination of SIFT-MS with sample collection

techniques, specifically automated thermal desorption (ATD)that is more commonly used in combination with GC systems(Phillips & Greenberg, 1991; Jones, Lagesson, & Tagesson,1995). ATD involves the adsorption of trace gases from air orbreath samples onto the surface of a suitable material and theirrelease (by heating) into a neutral gas stream and thence into thedetector. When a GC system is used then the final analysis isachieved in times of many minutes, but the release into a SIFT-MS instrument provides more rapid analyses, which obviously isadvantageous when many samples are to be analyzed. This ATD/SIFT-MS combination not only provides rapid analysis, but theeffective preconcentration of trace gases from the sampleultimately lowers their limit of detection to sub-ppb levels.Using standard samples the linearity of this system has beendemonstrated within the concentration range from 1 to 10 ppb(Ross & Vermeulen, 2007). Hryniuk and Ross (2009) have nowsuccessfully used this ATD/SIFT-MS combination for breathanalysis and found that there was a 1 to 1 concordance betweenthe levels of isoprene and acetone in the breath of 12 healthyvolunteers as measured using this combination technique anddirect real-time SIFT-MS measurements.

A common problem in mass spectral analysis is the presenceof isobaric compounds and structural isomers. The classicexample in breath analysis is the simultaneous presence ofaldehydes and ketones (Ligor et al., 2009). These can beseparated and analyzed using SIFT-MS by exploiting thediffering ion chemistries of these compounds with H3O

þ andNOþ precursor ions; for example, whereas both the isomersacetone and propanal react with H3O

þ ions to form theprotonated compounds both at m/z values of 59, acetone reactswith NOþ to form the adduct ion NOþCH3COCH3 at m/z 88whereas it reacts with the isomeric propanal to form C2H5CO

þ atm/z 57 (Španěl, Ji, & Smith, 1997).

A particular problem has been that the analyses of exhaledbreath using SIFT-MS over the last few years has revealed thepresence of a volatile compound or compounds with molecularweight 60 and the concern has been to identify which of thepossible volatile isobaric compounds 1-propanol, 2-propanol,acetic acid and methyl formate is present. This problem iscompounded by the formation of hydrates of the characteristicproduct analytical ions (formed in the reactions of thesecompounds with the precursor ions used for their analysis, viz.H3O

þ and NOþ), which is particularly efficient when humidsamples such as exhaled breath are to be analyzed. Thus, theproduct ion spectra formed are complex and it is not obviouswhich of the characteristic product ions can best be used for theanalyses. To investigate this, a study has been made (Pysanenko,Španěl, & Smith, 2009) of the ion chemistry of H3O

þ and NOþ

with the propanol isomers, acetic acid and methyl formate undervarying conditions of sample humidity up to that of exhaledbreath (6% by volume). The problems involved in the separateanalysis of propanol had been met and solved by previous SIFT-MS studies (Španěl & Smith, 1997; Wang et al., 2006) and nowthe study by Pysanenko, Španěl, and Smith (2009) revealed howacetic acid and methyl formate can be separately identified in ahumid mixture using NOþ precursor ions. Following this work,the kinetics library for the SIFT-MS analyses of these compoundsin breath was constructed and the analysis of the exhaled breathof five healthy volunteers showed that acetic acid was present atlevels typically within the range from 30 to 50 ppb and thatmethyl formate was not present above the detection limit. This is

FIGURE 8. a: The variation of r, the ratio of the current collected to theO2 orifice disc in the Profile 3 instrument (see Fig. 4) when only H3O

þ

ions are present in the helium/sample air flowing gas (r¼ 1) to thecurrents measured when the H3O

þ ions are totally converted to otherterminating ions at several specific m/z values. The error bars are

estimated from the fractional conversion of the precursor ions into the

terminating ions using a full kinetic model. b: The dependence of theanalytical mass spectrometer discrimination factor, Mr, on m/z

normalized to the Oþ2 m/z 32 value. The inset shows the measureddependence of the ratios of the ion count rates to the disc currents,

R (c/sec per pA), on m/z. Reprinted from Smith, Pysanenko, and Španěl

(2009b) with permission from Elsevier.



an excellent example of how careful studies of the reactions of theprecursor ions with candidate compounds present in gas mixturessuch as breath can lead to proper identification and analysis bySIFT-MS.

V. ANALYSES OF EXHALED BREATH OFHEALTHY VOLUNTEERS

Breath analysis is a new and rapidly evolving research area thatwill ultimately lead to an additional diagnostic technique to thosealready available to the clinician. For this to become a reality,continual improvements in instrument sensitivity (as exemplifiedfor SIFT-MS in previous sections of this review) and in samplingmethodology are necessary. Base line levels of metabolitespresent in the exhaled breath of healthy volunteers are required sothat abnormal levels can be recognized. It is also crucial that thecontribution of orally produced compounds to the content ofmouth exhaled breath is known and, as is emphasized below, thesampling of alveolar breath via the nose needs to be adopted.The sections below summarize the progress made in some ofthese areas.

A. Wide Dynamic Range of SimultaneousAnalyses in Real Time

A special feature of SIFT-MS is the very wide concentrationrange of compounds that can be analyzed simultaneously insingle breath exhalations. These range from water vapor and CO2at the few percent levels to trace compounds present at the ppblevel. Figure 9a shows the data obtained for the detection andquantification of three compounds in six sequential single breathexhalations/inhalations—water vapor, carbon dioxide and ace-tone—obtained using a Profile 3 instrument operated in the MIMmode. The method of detection and quantification of CO2 bySIFT-MS is given in the next section. Note that the concentrationrange covered in these data exceeds 105 times. Note also theconsistency of the concentrations determined in the alveolarportions of the three exhalations as identified by the time profiles.In the similar data in Figure 9b, which additionally shows theanalysis of ammonia and hydrogen cyanide in exhaled breath, theconcentration range span is 107! For trace compounds alone (notwater vapor and CO2) the concentration span can be as much as105. This remarkable dynamic range is unsurpassed by othertechniques. The rapid time response allows the simultaneoustracking of the concentrations of several trace compounds thatmay vary on short time scales. A good example is the in vivometabolic conversion of ethanol to acetaldehyde and the accuratedescription of the kinetics of this process via breath analysiswhen the acetaldehyde is present in the exhaled breath at a level103 times lower than the ethanol (Smith, Wang, & Španěl,2002b). Recently, the latter study has been complemented by acoordinated FA-MS and SIFT-MS analysis of single breathexhalations following ingestion of both D2O and ethanol whenthe dispersal kinetics of isotopically labeled water and ethanolwere directly compared (Španěl, Wang, & Smith, 2005). It is ourcontention that analysis of exhaled breath using direct exhala-tions are simpler and more accurate than bag sampling (see, e.g.,O’Hara et al., 2008), especially now that carbon dioxide can bemeasured in single exhalations.

B. Quantification of CO2 in Air and Exhaled Breath

As discussed in Section IV.B, the recent improvement in thesensitivity of SIFT-MS instruments has revealed the presenceof the adduct ions H3O

þCO2 in the analytical spectra obtainedwhen analyzing exhaled breath. Whilst this has complicatedthe analysis of acetaldehyde, it has provided a desired route to theanalysis of CO2 in breath, which some utilize to identifying thealveolar portion of the exhalation (Dolch et al., 2008; Risby,2008), although we consider that the water vapor exhalationprofile is equally good if it can be obtained simultaneously withbreath metabolites of interest (Španěl & Smith, 2001c). Thedetails of the ion chemistry involved in the SIFT-MS quantifi-cation of CO2 are given in a very recent paper (Smith, Pysanenko,& Španěl, 2009a). It is sufficient to say here that the formationrate of the H3O

þCO2 adduct ion obviously depends on the CO2levels present in the helium carrier gas and this is the basis of itsquantification. However, this adduct ion reacts rapidly with watermolecules, as discussed in Section IV.B, so a simultaneousmeasurement of the water vapor level is required, which isroutinely achieved and is one of the unique features of SIFT-MS,as discussed in Section II. We have carried out a detailed study ofthe formation H3O

þCO2 adduct ion and its loss in reaction withwater molecules and constructed appropriate SIFT-MS kineticslibrary entries that allow the CO2 concentration to be determinedover the range of humidity from 1% to 2% (ambient air; CO2 atabout 0.04%) to 6% (exhaled breath; CO2 varying between 3%and 6%). This remarkable advance in the SIFT-MS analyticaltechnique is demonstrated by the MIM mode data in Figure 9a,which shows the wide range of quantification for breath gases bySIFT-MS, now including CO2. We have yet to exploit this veryrecent advance, but it will surely assist in obtaining a betterunderstanding of lung function and the coupling between theexpiration of CO2 and the trace gases released from the blood atthe alveolar interface.

C. Distributions of Metabolites in the Breath ofHealthy Adults and Children

We described in Section III.B how the distributions of the severaltrace gas metabolites in mouth-exhaled breath of significantcohort of healthy adults have been determined by longitudinalmeasurements over periods of a few weeks to a few months usingSIFT-MS Mk.1 and Mk.2 instruments (Diskin, Španěl, & Smith,2003a; Turner, Španěl, & Smith, 2006a,b,c,d). This work wascarried out primarily to establish the typical levels of metabolitesand their variations in the exhaled breath of the healthypopulation in preparation for studies of patients with knowndiseases. These surveys have recently been extended by usingProfile 3 instruments to encompass cohorts of younger people ofschool age both in Prague (Španěl, Dryahina, & Smith, 2007a,b)and in Keele (Enderby et al., 2009a) and to include more breathmetabolite compounds. The results of these studies have beenreported in detail in the cited papers, but a flavor of the results isgiven here. In general, the mouth-exhaled breath levels of mostmetabolites are similar in children to those for adults, but thereare exceptions, one of which is ammonia, which is clearly lowerin the exhaled breath of the young and increases with age. Thedistribution obtained for a particular cohort of Prague schoolchildren is apparently different for the 17- and 18-year olds,although the number in each age group is small (see caption to



FIGURE 9. a: Plots of the derived concentrations of water vapor and CO2 (in % and also in parts-per-billion, ppb) and acetone (in ppb) obtained using the Profile 3 SIFT-MS instrument in the multi ion

monitoring, MIM, mode for six sequential breath exhalations by one volunteer over the time indicated

in seconds. These data show the remarkable consistency in the derived levels of these compounds in the

alveolar regions of the exhalations, as indicated by the shaded portions. The mean values (with standard

deviations) are water vapor, 6.1 (0.1)%, CO2 3.8 (0.1)% and acetone, 428 (14) ppb. Also indicated are the

laboratory air levels of these compounds. Reproduced, with permission from Wiley, from Smith,

Pysanenko, and Španěl (2009a). b: Simultaneous absolute quantification of water vapor, ammonia,acetone and hydrogen cyanide in three sequential breath exhalations. Note that the water vapor is again

about 6% of the exhaled breath and that the concentration of hydrogen cyanide obtained in the same breath

exhalations is at a mean value of 10 ppb, which is some seven orders-of-magnitude lower. Reproduced

from Smith and Španěl (2007) by permission of The Royal Society of Chemistry (on-line http://

dx.doi.org/10.1039/b700542n).



Fig. 10a). Most recently, in collaboration with local paediatri-cians at the University Hospital of North Staffordshire, UK, wecarried out a detailed study of several metabolites in the breath of200 children and young adults in the age range 7–18 years bylocating a Profile 3 instrument in a local school (Enderby et al.,2009a). The most notable result of this comprehensive study isthat the mean level of the trace compound isoprene (a systemiccompound; see the next section), which is present in the exhaledbreath of all individuals, is about a factor of three lower inchildren than in adults and a clear increase with age occurs. Thedistributions obtained for this young cohort and that obtainedpreviously for adults are shown in Figure 10b. There is a clearincrease in breath isoprene with age around puberty. The detailsof this study and the possible reasons for this very obvious agevariation are discussed in a recent paper (Smith et al., 2009).Clearly, such studies are necessary if the most desirable goal ofnon-invasive breath diagnosis of disease is to be extended toinclude children. It should be emphasized that all these analyseswere made of mouth-exhaled breath, as has been used almostuniversally to date (Amann & Smith, 2005), but now we

understand that systemic blood concentrations of many meta-bolites are best monitored by analyzing nose-exhaled breath, asdiscussed below.

D. Composition of Mouth-Exhaled and Nose-ExhaledBreath and Air in the Oral Cavity: Halitosis

For obvious reasons, there has been on-going interest in thenature of and origin of the compounds that are responsible fororal malodour (halitosis), but the fact that compounds generatedin the oral cavity can add to those released at the alveolarinterface (systemic compounds) in mouth exhaled breath has notbeen adequately addressed by those interested in using breathanalysis as a clinical tool to detect systemic disease. It is certainlytrue that compounds can be generated in the oral cavity whencertain foods or substances are introduced. For example, we haveshown that ammonia is released following a mouth wash ofurea solution and that ethanol is similarly released when sugaris introduced into the mouth (Španěl et al., 2006b). Studiescomparing mouth-exhaled and nose-exhaled breath are surelyoverdue and so we have developed a protocol to measuresequentially the various trace compounds present in mouth-exhaled and nose-exhaled breath and the static gas in the oralcavity. This was achieved by including a small pump in the SIFT-MS sampling line to draw the sample across the always openheated sampling capillary, as shown in Figure 11. Clearly, nose-exhaled breath should have less contamination from mouthgenerated compounds and although some contamination couldoriginate from the airways, the nose-exhaled breath should

FIGURE 10. a: The distributions of mouth-exhaled breath concen-trations of ammonia in parts-per-billion (ppb), in a cohort of young adult

volunteers age 17 (n¼ 12) and 18 (n¼ 14) years (y). Also shown is thedistribution obtained previously for a cohort of older adults (Turner,

Španěl, & Smith, 2006a). The medianvalues are indicated for both young

cohorts and for the adult cohort. Reproduced, with permission from IOP

Publishing, from Španěl, Dryahina, and Smith (2007a). b: Histogram ofmouth-exhaled breath isoprene levels in parts-per-billion (ppb), obtained

for 200 children and young adults in the age range 7–18 years in this case

shown as a percentage, %, within chosen ppb windows. Both the mean

level and the standard deviation (SD) and the median level and the

geometric standard deviation (GSD) are given. Note the few outliers at

low and zero isoprene levels. The data are taken from Enderby et al.

(2009a). For comparison, the distribution for adults, reproduced from

Figure 1, is shown as a shaded/dotted distribution.

FIGURE 11. A representation of the sampling of (a) breath exhaled viathe mouth, (b) breath exhaled via the nose and (c) the air in the oral cavity.The air/breath samples are drawn along the individual (interchangeable)

plastic sampling tube and across the sampling calibrated capillary

(shown in the enlargement) by the action of a small pump. The in-line

needle valve is used to regulate the flow rate of the air/breath past the

sampling capillary. Reproduced, with permission from IOP Publishing,

from Wang et al. (2008a).



largely contains trace systemic compounds. In two recent pilotstudies the level distributions of ammonia (Smith et al., 2008) andseveral trace compounds (Wang et al., 2008a) have been obtainedin mouth-exhaled and nose-exhaled breath and in the oral cavity.These distributions are exemplified by the data obtained forammonia and acetone shown in Figure 12, where it can be seenthat ammonia is largely generated in the mouth whereas acetoneis essentially systemic. In this way it is also seen that bothmethanol and isoprene are systemic, both ethanol and hydrogencyanide are largely of oral origin and both acetaldehyde andpropanol are apparently produced partly in the mouth and arepartly systemic. The recommendation must be that for breathanalysis to be of value in the detection of systemic disease itshould be made for nose-exhaled breath. It should be stressed thatthese level distributions are for individuals whereas the datagiven in Figure 3 are the distributions obtained from populationstudies.

The above approach to breath analysis can be turned toadvantage for the study of halitosis, which is generally attributedto the presence in mouth-exhaled breath of odorous sulfurcompounds like hydrogen sulfide, methanthiol and dimethylsul-fide and ammonia and diamines such as putrescine andcadavarine (Goldberg et al., 1994; van den Velde et al., 2007).These compounds, especially the diamines, are extremely‘‘sticky’’ at surfaces (Ross, Babay, & Ladouceur, 2009) and soare very difficult to collect and then release from containers.

Remembering that sample collection is unnecessary using SIFT-MS methodology, the study of these orally generated compoundsis relatively easy and to demonstrate this we have carried out apilot study of several sulfur-bearing compounds in mouth-exhaled and nose-exhaled breath and in the oral cavity of twohealthy volunteers (Pysanenko, Španěl, & Smith, 2008). Thelevel distributions for hydrogen sulfide and methanthiol for onevolunteer are shown in Figure 13, where it can clearly be seen thatthese compounds are almost entirely generated in the oral cavity.However, the actual levels of these compounds in the breathof these volunteers are well below those that are known to besocially unacceptable (Hirsch, 2008) and they illustrate thesensitivity of the Profile 3 instrument.

In addition to compounds generated in the oral cavity,exogenous compounds can be inadvertently ingested that mayinterfere with breath analysis. One example is the use of inhalersby asthmatics that results in the presence of chlorofluorocarbonsin exhaled breath as was observed using SIFT-MS in NewZealand (Epton et al., 2009).

VI. PHYSIOLOGICAL AND CLINICAL STUDIES BYBREATH ANALYSIS

The studies described in the previous section are largelyconcerned with the nature, origin and level distributions of

FIGURE 12. The intra-individual distributions of (a) acetone and (b) ammonia obtained from a healthyvolunteer measured in mouth-exhaled breath, nose-exhaled breath and in the air in the closed oral cavity. The

median ammonia levels, estimated as geometric mean, m*, and geometric standard deviation, s*, values areindicated for each distribution and are also indicated by the vertical lines. The mean ambient air level of

acetone is 11� 5 ppb, presented as m� s, where m is the mean value and s is the standard deviation, thesevalues being too low to show on the figure. Similarly, the mean ambient air level of ammonia is 80� 10 ppbas shown by the vertical shaded region. Reproduced, with permission from IOP Publishing, from Wang et al.

(2008a).



common trace gases present in the exhaled breath of healthyindividuals. The results obtained are an essential prelude tostudies of breath compounds in disease such as in renal failure(Davies, Španěl, & Smith, 1996, 1997, 2001b) and cancer(Španěl et al., 1999; Španěl & Smith, 2008), areas that we haveonly touched on so far using SIFT-MS, but which will be a focusfor future work. However, since breath analysis is a window to thenormal metabolic processes occurring in the body, there is a verywide area of application of breath analysis, and hence SIFT-MS,in physiology. Much can be learned about pharmokinetics by‘‘intervention’’ studies, as we demonstrated by studying themetabolic removal of ethanol from the body following theingestion of alcohol and the conversion of the ethanol toacetaldehyde (Smith, Wang, & Španěl, 2002b; Španěl, Wang, &Smith, 2005). This study demonstrated the simplicity of on-linebreath analysis and the rapid sampling rate achievable (see decaycurves in Smith, Wang, & Španěl, 2002b; Smith, Pysanenko, &Španěl, 2010), which can be every few seconds for severalcompounds simultaneously, which has great advantage inaccurately defining formation and decay rates of breath tracegases and the biochemical coupling between various compounds.Our specific studies in this area have been partly directed by theinterests of local cl

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