Analytica Chimica Acta 535 (2005) 189–199

Determination of acetone in breath

Norio Teshima1 , Jianzhong Li, Kei Toda2 , Purnendu K. Dasgupta∗
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA

Received 30 July 2004; received in revised form 9 December 2004; accepted 9 December 2004
Available online 12 January 2005

Abstract

A light emitting diode (LED)-based photometric method for the measurement of gaseous acetone in human breath is presented. The
detection chemistry is based on the reaction of acetone with alkaline salicylaldehyde to form a colored product, which absorbs in the blue and
can be monitored with GaN-based LEDs with emission centered at 465 nm. Gaseous acetone in breath is sampled with a porous membrane
based diffusion scrubber (DS). The collected sample in the continuously flowing water carrier reacts with the reagent solution. We have used
two approaches to collect breath acetone: the use of a face mask and a Mylar balloon as a collection bag. With the face mask approach, the
expired air can be measured over long periods without major subject discomfort, balloon collection (5 l) permits four measurements from a
single fill. The LED-based liquid core waveguide (LCW) absorbance detector utilized sapphire ball lenses to prevent exposure of other optical
components to a hot alkaline reagent solution. The high refractive index of the final mixture permitted the use of an inexpensive fluorinated
ethylene copolymer (FEP Teflon® ) tube as a 10 cm long LCW. The limit of detection (S/N = 3) is 14 ppbv gaseous acetone, and the linear
range extends to 1.21 ppmv. The concentration range in 11 volunteer subjects ranged from 176 to 518 ppbv.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Breath acetone; Diffusion scrubber; Liquid core waveguide; Absorbance detector

1. Introduction useful for toxicology and especially, disease diagnosis, in

Noninvasive breath analysis has the potential of replacing
blood and urine analyses for many compounds; in particular
volatile organic compounds (VOCs) in exhaled breath reflect
products of metabolism in our body in the expired air. As early
as the early 1900s, a method of obtaining alveolar air was
reported, and a study on the relationship between atmospheric
pressure and the partial pressure of exhaled CO2 was carried
out in detail [1]. However, as most VOCs in breath are present
at levels of ppmv or less, their measurement have become
more practical only since the mid-1900s. Since that time it
has been recognized that the analysis of trace VOCs can be
∗ Corresponding author. Tel.: +1 806 742 3067; fax: +1 806 742 1289.
E-mail address: sandy.dasgupta@ttu.edu (P.K. Dasgupta).
1 Permanent address: Department of Applied Chemistry, Aichi Institute
of Technology, 1247 Yachigusa, Yakusa-cho, Toyota 470-0392, Japan
2 Permanent address: Department of Environmental Science, Faculty of
Science, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555,
Japan
seemingly healthy to critically ill patients [2–5].
Gas chromatography (GC) followed by flame ionization
detection (FID) [6–9], ion mobility spectrometry [7] and
mass spectrometric detection [10–12] has been widely used
to separate, identify and quantify a number of VOCs in
human breath. These methods generally involve a collec-
tion/preconcentration step using solid sorbents [6,9] or a solid
phase microextraction device [7,10] prior to the introduc-
tion of the collected sample into a gas chromatographic col-
umn. For example, charcoal-based adsorbent or silica gel was
packed in a cartridge prepared from a syringe needle as the
collector and the collected analytes then thermally desorbed
into the carrier gas [6]. Schubert et al. [13] have in partic-
ular extensively used activated carbon preconcentration fol-
lowed by GC–MS; even in mechanically ventilated patients
the method was applicable due to small sample requirement
[14]. Sorption/desorption techniques have also been coupled
with a dimethylsilicone membrane extraction method, such
membranes are believed to be similar in nature to that of

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2004.12.018
190 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199
the nonpolar lipid bilayer cell membrane of the alveoli [7].
Phillips et al. [12] reported that on the average at least 204
VOCs are detectable in the breath from an individual subject
(n = 50) and more than 3000 different VOCs were detected
in at least one individual’s breath.
Selected ion flow tube mass spectrometry (SIFT/MS) was
conceived and developed by Adams and Smith [15] and is
applicable to many fields from interstellar chemistry [16] to
breath analysis [17–19]. The SIFT technique is based on soft
chemical ionization of trace gases to the exclusion of the ma-
jor breath gases, exploiting the ion-molecule reactions that
occur between the trace gases and preselected reactant ions
(H3 O+ , NO+ and O2 + ). A SIFT/MS study of the concen-
trations of the common breath constituents in the breath of
five subjects over 30 days was carried out [19]; for acetone
the intrasubject variation over the period had a relative stan-
dard deviation of 21–29%; this bore no correlation with the
average level of acetone measured for the individual, which
ranged from 293 to 870 ppbv. This group showed that acetone
levels increase both in urine headspace and breath in women
during ovulation [20]. Acetone levels have also been reported
to increase after alcohol ingestion [21] but this may be due
to lack of food consumption rather than an effect of alcohol
intake. Water vapor can cause significant errors in the quan-
titation of SIFT–MS results and appropriate measures must
be taken [22]. While breath acetone has been widely used as
a disease marker, there are many occasions where it is a poor
indicator of the particular problem. Studer et al. [23] found
breath acetone to be a poor predictor of organ rejection in
lung transplant patients.
Proton transfer reaction mass spectrometry (PTRMS) has
also been used for breath acetone measurement and at least
in some cases, acetone levels have been shown to increase
with exercise [24]. Formation of acetone is related to the
metabolism of triglycerides and lipids (vide infra). Taucher
et al. [25] tested the claim about health benefits of garlic: they
did find that following garlic ingestion, breath acetone levels
do rise slowly and remain elevated for as long as 30 h.
Mass spectrometry and chromatography–mass spectrom-
etry are versatile techniques capable of simultaneous deter-
mination of a number of breath constituents. Nevertheless,
capital cost needs and operator skill requirements for these
techniques are high and a more affordable dedicated tech-
nique for acetone will be a welcome development. Xie et al.
[26] custom-designed an ion mobility spectrometer with a
UV ionization source and a high-speed capillary column as
an analytical device for the sensitive detection of selected ke-
tones (acetone, 2-butanone and diethyl ketone). For acetone,
the limit of detection (LOD) was 2.7 ␮g l−1 which translates
to ∼1140 ppbv, much too high for breath analysis applica-
tions.
Flow injection (FI) based methods may be less generally
applicable than some of the above techniques, but there are
many devotees of FI who can benefit from simple and af-
fordable methods for the measurement of some of the more
abundant VOCs in human breath.
Some of more common VOCs in the breath of healthy
human subjects are isoprene (12–580 ppbv), acetone (1.2–
1880 ppbv), ethanol (13–1000 ppbv), methanol (160–2000
ppbv) and other alcohols [27]. Elevated levels of some com-
mon metabolites are known to be indicative of disease con-
ditions; renal impairment raises breath isoprene [28] and/or
ammonia [29], and acetone is closely related to diabetes mel-
litus [30]; acetone levels can rise as high as 12 ppmv [9] in
acute diabetes.
Monitoring breath acetone can be useful to follow patients
on a prescribed diet regimen as well as to monitor diabetic
patients. For instance, the efficacy of a ketogenic diet for
epileptic children was assessed using breath acetone analysis
by GC/FID [31]. Acetone and hydrogen emanating from hu-
man skin were first measured by GC/FID [32]. The emission
rate of acetone from skin correlated with the concentration of
acetone in breath (r2 = 0.807, n = 22). The measurement ap-
proach required a cryotrapping preconcentration procedure.
Reports on simple and affordable approaches to breath
acetone measurement are few. Henderson et al. [30] reacted
acetone with 2,4-dinitrophenylhydrazine to form the corre-
sponding hydrazone for the determination of acetone in hu-
man breath. The derivatization mixture was extracted with
CCl4 to separate the hydrazone. Even with a cryotrap cooled
with liquid air, the breath sample had to be collected for
∼1 h. In general, there are few reported methods for solution
phase measurement of acetone that are selective. We looked
at a number of methods reported for acetone measurement
[33–42]. Based both on ease of automation considerations
and experimentally attainable sensitivity in our exploratory
studies, we concentrated on a colorimetric method due to
Berntsson [42] that relies on the base-catalyzed aldol con-
densation of acetone with salicylaldehyde. This early work
indicated adherence to Beer’s law in the range of 3–60 ␮M
acetone. However, the original method recommended an op-
timum reaction time of 2 h. This would be difficult or im-
possible to implement in any near continuous measurement
approach.
In the present work, we have modified the salicylaldehyde
chemistry for use in a automated flow system with a rea-
sonable reaction time in conjunction with a long path light
emitting diode (LED)-based liquid core waveguide (LCW)
absorbance detector [43]. The analytical system includes an
in-line porous polypropylene membrane diffusion scrubber.
We also report on breath acetone levels of a number of healthy
subjects.
2. Experimental
2.1. Reagent
To prepare an acetone standard solution, a 50 ml volumet-
ric flask with ∼25 ml of water was pre-weighed. Acetone
(HPLC grade, EM Science), ∼130 ␮l in volume (∼0.1 g),
was added to the water in the flask and the flask was capped,
 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199 191

Fig. 1. Diffusion scrubber: TM, porous tubular membrane; PT, polymer tube (poly(methylmethacrylate)), ST, L-shaped stainless steel tube.

reweighed and then the solution was diluted with water
to the mark. The acetone stock standard thus produced is
∼40–45 mM in concentration.
Salicylaldehyde (SA, Acros, 99%) and NaOH (EM Sci-
ence) used were reagent grade. A mixed reagent solution
containing SA and NaOH: to 3 ml of 10 M NaOH in a 50 ml
volumetric flask, about 22 ml of water and 0.6 ml (∼0.7 g)
of SA were added, and the solution was mixed vigorously to
disperse SA (ultrasonication is helpful; if macro droplets of
SA remain in solution, a white precipitate is produced upon
the addition of concentrated NaOH in the following step).
Next, more NaOH (10 M, 24 ml) was added to the solution
followed by dilution with water to the mark (the final NaOH
concentration is 5.4 M). At the end of the NaOH addition step,
a crystalline white precipitate may be formed. This is redis-
solved by putting the flask in a 95 ◦ C bath for 30 s and agita-
tion. Any bubbles entrained in the relatively viscous solution
is removed by ultrasonication in a room temperature bath for
5 min. During this time, the temperature of the solution also
returns to ambient. This constitutes reagent R referred to in
the section describing the analytical system.
2.2. Diffusion scrubber
A cylindrical diffusion scrubber of annular geome-
try is shown in Fig. 1. Two small holes, 22 cm apart,
were made through the wall of a polymer tubing (PT,
poly(methylmethacrylate), 6.4 mm o.d., 3.1 mm i.d., http://
www.boedeker.com) for insertion of L-shaped stainless steel
(SS) tubing elbows (ST, 1.6 mm o.d., 1.2 mm i.d., Small Parts
Inc., Miami Lakes, FL). To bend these tubes without shutting
down the aperture, an SS wire (1.0 mm o.d., Small Parts) was
first inserted into the tube segments before they were bent; the
wire was removed after bending. A long segment (ca. >40 cm)
tubular porous capillary membrane (TM, Accurel® PP Q3/2,
0.9 mm o.d., 0.6 mm i.d., Membrana, Wuppertal, Germany)
was passed through one of the SS L-pieces ST and through
the hole in the tube PT, through the opposite axial end of tube
PT and then the terminus reinserted through the other hole
made in PT, from the inside. This end of tube TM was then in-
serted through the other SS L-segment and both L-segments
were cemented to PT with fast acting epoxy adhesive, one at
a time, in a manner that TM becomes located along the axial
centerline of PT. One end of TM was then epoxied to ST
(TM is sufficiently smaller than ST that the adhesive can go
between the exterior of TM and the ST wall). After the adhe-
sive cured, excess membrane was cut off. The membrane was
pulled taut from the other side and epoxied to ST in that con-
dition. Again, excess protruding membrane was trimmed off
after curing. The effective length of the collection membrane
was 20 cm.
2.3. LED-source LCW-based absorbance detector
The design of a long-path (100 mm) LED-based LCW cell
is shown in Fig. 2. One arm each of two polypropylene tees
(T, 0416TEEP, Ark-Plas, Flippin, AR) was bored out to a
diameter of 2.75 mm. A sapphire ball lens (3.0 mm diame-
ter; http://www.edmundoptics.com) was forcibly inserted to
close to the middle of the tee where it is securely self-seals the
insertion arm but leaves the passage to the vertical arm still
at least partially open (heating the tee with a heat gun softens
the tee and eases the insertion; care must be taken not to mar
the optical quality of the ball during insertion). Large core
(1 mm) fused silica optical fibers (TF-1300H, 1.8 mm o.d.,
http://www.meshtel.com) were cut and end-polished and in-
serted into each bored out T-arm to be in contact with the

Fig. 2. LED-based LCW absorbance cell with sapphire ball lenses. FO, large core (1 mm) fused silica optical fibers; FEP, Teflon FEP tube (20 LW tube, 0.86 mm
i.d., 1.16 mm o.d.) jacketed inside a TFE tubing (14 SW tube, 1.67 mm i.d., 2.49 mm o.d.) and covered with a black opaque tube, not shown here. SA, sapphire
ball lens (3.0 mm diameter); T, polypropylene tees.
192 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199
sapphire ball and sealed in place with Teflon® (TFE) tubing
sleeves (1.93 mm i.d., 2.74 mm o.d.; all Teflon tubes were
from http://www.zeusinc.com). The sapphire balls prevent
direct contact between the optics and the hot caustic reagent
mixture. One of the optical fibers led to a high brightness blue
LED (λpeak 465 nm, half bandwidth 25 nm, L200CWPB500,
http://www.ledtronics.com). The LED is provided on the bot-
tom with another optical fiber; this acts as the reference
light source. The light output from the reference and the
detector fiber are processed by previously described elec-
tronics [44], such detectors are also commercially available
(http://www.globalfia.com). The LCW itself consisted of a
0.86 mm i.d., 1.16 mm o.d., FEP Teflon tubing, 100 mm in
length, that was sleeved with 1.68 mm i.d., 2.49 mm o.d.,
TFE tubing for sealing. Liquid inlet/outlet connections were
made with 0.71 mm i.d., 1.32 mm o.d., TFE tubing through
the vertical arms of the tee as shown.
The detector output was acquired on a laptop PC with 12-
bit resolution at a rate of 1 Hz using custom software written
in HP-VEE. Detector baseline drift at high sensitivities was
corrected by software postprocessing. In some early opti-
mization experiments, a commercial HPLC-type absorbance
detector (Model 757, Kratos, 8 mm pathlength cell) was also
used as stated.
2.4. Breath collection/analysis system
The two different sampling/collection systems studied are
shown schematically in Fig. 3(a) and (b). In (a), a respirator-
type face mask (3M, Type 6200, http://www.fishersci.com)
that is provided with inlet/outlet one-way flap valves was
used for sampling. A tube was connected to the mask inte-
rior so that the air can be sampled. A miniature air pump
(AP) was used to aspirate the breath sample through the
DS at 0.4 standard liters per minute (SLPM). The en-
tire sampling line between the respirator mask and the DS
as well as the DS itself were wrapped with heating tape
and maintained at 40 ◦ C. When solenoid valve V (075T3,
http://www.biochemvalve.com) is turned off, the air flow in
the DS stops and the pump samples ambient air directly and
the instrument signal gradually returns to the baseline. The in-
strument operated on an automated 3 min sample–7 min zero
cycle, resulting in an overall measurement cycle of 10 min.
A trap is used before the flow controller to prevent water
condensation in the controller.
In arrangement (b), the subject was allowed to
inflate a Mylar balloon (Type CV212PB, Durafloat,
http://www.ctiindustries.com), which has been used previ-
ously by others for off-line breath collection [45] (full ca-
pacity 9.0 l), maintained in a heated enclosure (box capacity
allowed balloon to be inflated to <5.0 l) through a heated line
and solenoid valve V2. Valve V2 was then turned off and the
system sampled the air contained in the balloon in much the
same manner as in (a). When the balloon was filled with zero
gas and then sampled, no discernible signal was observed, in-
dicating negligible acetone offgassing from balloon material.
Similarly, when the balloon was ‘washed’ between samples
with zero gas and refilled with zero gas, there was no de-
tectable signal, indicating that memory effects were minimal.
The analytical system is shown in Fig. 3(c). Water is as-
pirated through the DS at 70 ␮l min−1 and normally passes
straight through six-port injection valve I and merges with
reagent (R) (165 ␮l min−1 ) and flows through a reaction coil
(RC) (0.46 mm × 1500 mm) maintained in a thermostated
bath (we have developed a more space and energy-efficient
inline heater that would be of utility in such applications
[46,47]) that provides a ∼1 min residence time for the mix-
ture. The mixture then flows through the absorbance detector
(D) provided with a backpressure restrictor (BPC) that con-
sists of a 10 mm length of a 100 ␮m i.d. capillary tubing. A
variable speed Rainin Dynamax peristaltic pump (10 rollers)
was used for liquid pumping.
2.5. Instrument calibration
Referring to Fig. 4, the calibration source consisted of
a syringe pump S (Model 341, containing a 10 ␮l syringe,
http://www.sageinst.com) that dispensed a standard acetone
solution via a 75 ␮m i.d. fused silica capillary tip through the
perpendicular arm of a T. The capillary was passed through
a 25-gauge needle and the end of capillary was placed at the
needle tip from which the acetone standard solution can be
volatilized; the capillary tip was affixed to the needle hous-
ing body by hot-melt adhesive. Typically, the acetone stan-
dard solution was delivered at a flow rate of 10 nl min−1
and completely evaporated by the airflow through the T-
arm. Ambient air, purified through a column serially packed
with Carulite 200 (activated manganese oxides, Carus Chem-
ical) and pelletized activated carbon (4–8 mm, Fluka), passed
through a humidifier containing water maintained at 37 ◦ C.
All downstream components up to and including the DS were
maintained at 40 ◦ C. The rest of the calibration system ar-
rangement was the same as the measurement arrangement; a
3/7 min sample/zero cycle and a sampling rate of 0.4 SLPM
was used. The acetone concentration generated is readily
computed from the solution concentration, syringe delivery
rate and air flow rate. For example, a commonly used calibra-
tion point was 3.53% (w/v) acetone delivered at 10 nl min−1
with 0.4 SLPM air flow, this corresponded to a gaseous ace-
tone concentration of 339 ppbv.
3. Results and discussion
3.1. Choice of chemistry
Among reported colorimetric methods for acetone, aro-
matic aldehydes such as SA [42], furfural [34], vanillin
[35] and o-nitrobenzaldehyde [40] are prominent. Our ini-
tial experiments with these showed that the SA method ex-
hibits the best sensitivity. In addition, the furfural method re-
quires the use of both concentrated H2 SO4 and base and the
 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199 193

Fig. 3. Schematic diagrams of the (a) collection system with face mask, (b) balloon and (c) the analytical system. Broken lines indicate regions maintained
at 40 ◦ C. M, face mask; DS, diffusion scrubber; C, carrier water; V, V2 three-way solenoid valves; Trap, water condensation trap; MFC, mass flow controller
(0.4 SLPM); AP, air pump; HB, heated box (43 ◦ C, volume ∼5 L); B, Balloon; R, reagent mixture (0.11 M salicylaldehyde + 5.4 M NaOH); S(l), acetone
solution for liquid-phase calibration; I, six-way injection valve with loop (50 ␮l); P, multichannel peristaltic pump; RC, PTFE-knotted reaction coil (0.46 mm
i.d., 1500 mm long); TB, thermostated bath (95 ◦ C); D, LED-based LCW absorbance detector (465 nm); BPC, back pressure capillary (10 mm, 100 ␮m i.d.);
W, waste.
194 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199
Fig. 4. Schematic diagram of instrument calibration setup. Broken lines, heating with heating tape at 40 ◦ C. Carulite 200 (Carus) and activated charcoal (Purum
p.a. 4–8 mm, Fluka) are used for zero gas. H, humidifier (37 ◦ C water in it); T, tee; S, 10 ␮l syringe pump (standard acetone solution in it); Y, y-type connector;
other symbols as in Fig. 3.
o-nitrobenzaldehyde method has a high blank absorbance.
Although diazotized o-aminobenzoic [37] and p-amino-
benzoic [38] acids have been used for acetone determina-
tion, the diazotized reagents are unstable. The SA method
was therefore further examined.
3.2. SA chemistry and optimization
In Berntsson’s SA method [42], the sample and reagent
solutions are placed in a 50 ml volumetric flask in this or-
der: 2 ml of 10.6 M NaOH, x (x < 23) ml of the acetone-
containing sample, 23 − x ml water, 0.6 ml of SA, 20 ml of
10.6 M NaOH and finally making up to 50 ml with water. The
addition of NaOH is divided into two steps to prevent imme-
diate precipitation of the sodium salt of SA. Without the ini-
tial presence of NaOH solution, water-insoluble SA formed
a separate organic phase that caused significant precipitation
when NaOH is added. When some NaOH is initially present
(∼0.8 M), the added SA can be dispersed in it, and the subse-
quent NaOH addition step did not lead to precipitation (final
[NaOH] 4.66 M). Liquid-phase acetone concentrations rang-
ing from 0.17 to 3.3 mg l−1 could be determined from the ab-
sorbance at 474 nm by the manual assay method. However,
the optimum reaction time (2 h) is too long to be applied to
flow analysis. We found that the reaction can be dramatically
accelerated by increasing the temperature. We also noted that
one must operate in a concentrated NaOH medium, a decrease
in the NaOH concentration remarkably decreased the sensi-
tivity either by altering the degree to which the condensation
was achieved or in ionization of the final product. The two-
channel flow system used in this study essentially dilutes a
reagent solution by the sample containing carrier. Too high
a reagent:carrier ratio will result in lowered sensitivity be-
cause too little sample will be injected. Conversely, too low a
reagent:carrier ratio will result in lowered sensitivity because
of decreased base concentration. The carrier solution could
not be made strongly basic because of the potential precipita-
tion of insoluble Na2 CO3 in the pores of the DS membrane.
We found the highest NaOH concentration that could be con-
veniently attained in the reagent solution was 5.4 M and then
we optimized the reagent:carrier mixing ratio.
These studies were carried out with a commercial flow-
through HPLC-type detector. The monitoring wavelength
was 474 nm as suggested by Berntsson [42] with a 50 ␮l ace-
tone standard injected into the water carrier.
Flow rate optimization was carried out in two steps. First,
the carrier flow rate was varied while holding the reagent flow
rate constant at 265 ␮l min−1 . Fig. 5(a) shows that the maxi-
mum signal was obtained at a carrier flow rate of 47 ␮l min−1 .
The reagent flow rate was then varied from 39 to 264 ␮l min−1
at a constant carrier flow rate of 47 ␮l min−1 (Fig. 5(b)). The
highest signal was obtained at a reagent:carrier flow rate ratio
of 2.3–2.4. For the final system, relative to the optima that
Fig. 5(b) represents, we wanted to reduce the absolute res-
idence time to improve sample throughput. We maintained
the reagent:carrier flow ratio but increased the absolute flow
rates to 165 and 70 ␮l min−1 .
The effect of SA concentration on the signal response was
studied for two different injected acetone concentrations. As
shown in Fig. 6, the response increased monotonically with
SA concentration. However, at SA concentrations higher than
0.11 M, there was more baseline drift, possibly from incipient
precipitation of the Na salt of SA in the system. We chose
therefore an SA concentration of 0.11 M.
3.3. Absorbance spectra
Fig. 7 shows the spectroscopic details relevant to the mea-
surement system. Although the net signal continues to in-
crease at wavelengths below 460 nm, the blank also rises
steeply and the actual attainable LODs deteriorate. Us-
ing a light source centered at 465 nm offers a reasonable
 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199
Fig. 5. Effect of flow rates of (a) carrier and (b) reagent streams on the net signal.
compromise between an optimum signal level and a manage-
able background absorbance level (the latter is of particular
concern with a long path cell used as a detector).
3.4. LCW detector
With 50 ␮l solution phase standards injected, the commer-
cial detector allowed an S/N = 3 LOD of 0.56 mg l−1 . The
linear r2 value up to an injected concentration of 317 mg l−1
was 0.9960. This attainable LOD was deemed insufficient to
measure acetone levels in exhaled breath of healthy subjects
Fig. 6. Effect of salicylaldehyde concentration on the net signal.
195
and hence improved sensitivity was sought with a detector
based on a longer path LCW cell. Under the optimized con-
ditions, the NaOH concentration in the final mixture is 3.8 M,
from standard sources of tabulated data, one obtains a refrac-
tive index of 1.3681. This is significantly above the refractive
index of FEP Teflon, 1.338. Indeed, the computed numerical
aperture (NA) of a Teflon FEP tube filled with such a solution
is 0.29, only slightly lower than the NA value of 0.32 for a
tube of Teflon AF 2400 (RI 1.29) filled with water (RI 1.33)
and is in fact better than the NA of a tube of Teflon AF 1600
(RI 1.31) filled with water. Light transmission of the reaction
mixture through a FEP Teflon tube was found to be satisfac-
tory and avoided the need for an expensive Teflon AF tube
and the necessity to execute prior agreements that are cur-
rently required by the manufacturer for the use of Teflon AF
for any application. This general approach may be useful to
many other applications where a significant amount of an in-
different solute, e.g., NaCl or CaCl2 can be added to raise the
RI of the solution. For reaction mixtures that must be heated
to accelerate the reaction, as in the present case, this has the
added benefit of increasing the boiling point and reducing gas
solubility, thus decreasing bubble formation problems. The
100 mm path LCW absorbance detector allowed an S/N = 3
LOD of 0.072 mg l−1 ∼8× better than the commercial detec-
tor. The improvement was less than what would be expected
on the basis of path length difference (12.5×) because of in-
creased baseline noise with the longer path due to the not
insubstantial background absorbance. For the same reason,
the temporal drift of the baseline was higher but could be ad-
equately compensated by software postprocessing. Detailed
calibration of the LCW cell was conducted directly with the
DS sampling gas phase acetone standards as described in the
next section.
196 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199
Fig. 7. Absorbance spectra in the SA method: (a) blank solution; (b) reaction product; (c) subtraction; (d) emission spectrum of blue LED (λpeak 465 nm, half
bandwidth 25 nm).
There was also some concern with adsorptive loss of ace-
tone on the acrylic walls of the DS housing. Experiments
with a Teflon lined DS showed that the loss was measurable
(∼18%) but not overwhelmingly large, likely because the
wall is heated. As calibration with all components in place
takes into account small system losses, we chose to use the
simpler unlined DS construction.
3.5. Gas phase calibration and analytical performance
The analytical system was calibrated with gas phase ace-
tone standards in the range of 0–1.2 ppmv and resulted in a
linear r2 value of 0.987:
Net signal (V ) = (5.58 ± 0.32) × 10−4 [acetone, ppbv]
+(1.2 ± 1.0) × 10−2
This linearity is worse than that obtained with the monochro-
mator equipped commercial detector due to the significant
bandwidth of the light source and appreciable change in the
absorption spectrum within this bandwidth. A typical cali-
bration sequence is shown in Fig. 8.
The S/N = 3 LOD for gaseous acetone was 14 ppbv. The
observed variance at levels higher than about 3× the LOD be-
come concentration independent and seem to be controlled
by system losses due to adsorption etc. The R.S.D. values
were 3.8, 6.8, 3.2, and 7.6%, respectively (n = 4 ea.) for the
responses at 159, 339, 670 and 1210 ppbv standards shown,
the line was not cleaned out between samples; so this re-
sults in an observable response at the end of each quartet. In
DS based vapor collection work, it is possible to put the DS
within the loop of a six-port valve and operate the DS in a
stopped liquid flow mode with periodic injections to obtain
better sensitivity [48]. Due to the potential complications of
keeping the receptor liquid stationary in an elevated temper-
ature enclosure, this approach was not pursued.
3.6. Interference study
Phillips et al. [12] reported that the highest concentration
of a VOC in human breath was that of 4,5-dimorpholino-2-
methoxy-6-phenylpyrimidine. However, this compound was
present in the breath of only 2 of the 50 subjects studied. In
contrast, acetone was detectable in their work in the breath of
46 out of 50 subjects. We studied the potential interference of
several compounds that are known to be commonly present
at human breath at significant levels and may represent po-
tential interferences. We studied the effect of added HCHO,
(1)
methanol, ethanol, methylisobutyl ketone (MIBK) and am-
monia in 5–100 mg l−1 concentration added to 1.15 mg l−1
acetone as a solution phase standard. The R.S.D. for so-
lution phase acetone response at this level was 2.3% and
this response is normalized to 100. The results are shown
in Table 1. Methanol, ethanol and ammonia did not interfere
at ∼100× the concentration. MIBK, a metabolite generated
from some fatty acids, produced a positive interference at
∼20× the acetone concentration but at ∼10× concentration
the interference was marginal. In actual gas phase analysis,
since the collection step is based on the diffusive transport of
gas molecules, there will be added discrimination against the
bulkier and slower diffusing MIBK molecule. Formaldehyde
showed a small negative interference at ∼10× concentration
but there was no significant effect at ∼5× the acetone con-
centration. In addition, HCHO itself is not very water soluble,
it needs to be hydrated to methylene glycol, CH2 (OH)2 , to
 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199 197

Fig. 8. Typical system outputs for gas phase acetone standards and for exhaled human breath from two volunteer subjects.

dissolve. This hydration kinetics is acid or base catalyzed and repeated over several exhalations. For each exhalation, the
is slow in water. In order to use water as the DS liquid for first part of the exhaled air was not collected), so that the bal-
the efficient collection of HCHO, one needs to use either an loon contents will more closely represent alveolar air. For one
acidic hydrophilic membrane like Nafion® [49] or a porous volunteer, the breath sample was balloon-collected and the
membrane with an acidic or basic solution as the DS liquid acetone concentration was determined to be 331 ± 10 ppbv
[50]. With a porous membrane DS and a water absorber, the (n = 3). Immediately afterwards the same subject was allowed
collection of HCHO will be quite inefficient [50]. to breath normally in the face mask based collection/analysis
system for 1 h, exhaling in normal fashion through the
nose for the initial 30 min, and then exhaling through the
3.7. Breath analysis
mouth for 30 min. The breath acetone concentrations for
nose and mouth exhalations were 217 ± 7 and 230 ± 9 ppbv,
Alveolar air (end-expired air) reflects metabolic products
respectively, not markedly different from each other.
in the bloodstream that equilibrate with the gas phase through
More importantly, these values were approximately two-
the alveoli. Usual expired air consists of alveolar air diluted
thirds of value measured by the balloon collection/analysis
by ambient air retained in the respiratory dead space: mouth,
system.
nose, pharynx, trachea and bronchi [51]. Since alveolar air
Ketogenesis or Ketosis involves the production of ke-
normally accounts for two-thirds of the tidal volume, the con-
tone bodies, that is primarily composed of acetoacetate, ␤-
centration of metabolic products in expired air is approxi-
hydroxybutyrate, and acetone. There are many excellent sites
mately two-thirds of that in the alveolar air. During balloon
available on the web that discusses ketogenesis, an excellent
collection, the subject is asked to exhale partially and then
primer is due to King and Marchesini [52]. The production
exhale deeply into the balloon to fill it up (this process is
of ketone bodies occurs from fatty acid oxidation and occurs
at a relatively low rate with normal diet and in the absence of
Table 1 diseases states. The most noted change occurs in untreated
Effects of expected metabolites from human breath on the determination of
or inadequately treated, diabetes mellitus, when diabetic ke-
1.15 mg l−1 acetone in liquid-phase
toacidosis results from a reduced supply of glucose and a
Compound added (mg l−1 ) Normalized peak height
concomitant increase in fatty acid oxidation. Increased pro-
None 100 ± 2 duction of ‘ketone bodies’ also occurs in many continued
Formaldehyde (10) 96.3 ± 2
low-carbohydrate diets, and simply when one is subjected
Formaldehyde (5) 100 ± 2
Methanol (100) 97 ± 1 to starvation. Many diet regimens or weight loss programs
Ethanol (100) 99 ± 1 recommend that urinary level of ‘ketone bodies’ be routinely
MIBK (20) 129 ± 2 tested with commercially available ketone testing strips [53]
MIBK (10) 103 ± 1 to ensure that an increased amount of fat is being metabolized.
Ammonia (100) 102 ± 1
Even in healthy individuals, breath acetone concentration is
198 N. Teshima et al. / Analytica Chimica Acta 535 (2005) 189–199

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