Sensors and Actuators B 202 (2014) 615–621

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Sensors and Actuators B: Chemical
j our nal homepage: www. el sevi er . com/ l ocat e/ snb
Determination of volatile organic compounds as potential markers of
lung cancer by gas chromatography–mass spectrometry versus
trained dogs
Joanna Rudnicka
a
, Marta Walczak
b
, Tomasz Kowalkowski
a
, Tadeusz Jezierski
b
,
Bogusław Buszewski
a,∗
a
Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin St, 87-100 Toru´ n, Poland
b
Institute of Genetics and Animal Breeding of Polish Academy of Sciences, Department of Animal Behaviour, Post˛ epu 36A, Jastrz˛ ebiec, 05-552 Magdalenka,
Poland
a r t i c l e i n f o
Article history:
Received 23 January 2014
Received in revised form 22 April 2014
Accepted 3 June 2014
Available online 7 June 2014
Keywords:
Lung cancer
Volatile organic compounds
Solid phase microextraction
Gas chromatography–mass spectrometry
Canine olfactory
a b s t r a c t
The mainaimof the study was qualitative andquantitative analysis of volatile organic compounds (VOCs)
occurring in biological samples such as exhaled air obtained from 108 patients with lung cancer, 121
healthy volunteers, and 24 persons with other lung diseases. For determination of VOCs in human breath,
solid phase microextraction and gas chromatography–mass spectrometry (SPME/GC–MS) were applied.
Statistical methods such as artificial neural network and chi-squared automatic interaction detector
(CHAID) were applied for data evaluation. The concentration of acetone, isoprene, ethanol, 1-propanol,
2-propanol, hexanal, anddimethyl sulfide were higher inpatients withlung cancer thanincase of healthy
volunteers and persons with other lung diseases.
The second goal was the application of trained dogs for detection of the same breath samples as in
GC–MS research and evaluate the sensitivity and specificity of canine scent detection using 5 station
scent lineup. Among lung cancer patients and complementary samples, overall sensitivity of canine scent
detection was 86%, while specificity was 72%.
There is a possibility that in the future these methods could allow for a fast, painless, and noninvasive
diagnosis of cancer.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Morbidity and mortality caused by cancer diseases seem to be
the most important health problem in modern society. The most
common cancer in the world is lung cancer with 1.6 million new
cases diagnosed every year [1]. Medical progress involved is huge,
however, cancer therapy is not perfect; so early cancer detection is
a desirable goal as it often allows treatment with a lower toxicity
and can lead to longer survival [2,3]. Screening tests are based par-
ticularly on imaging such as X-radiation (X-ray), ultrasonography
(USG), computed tomography (CT), and positron emission tomo-
graphy (PET); however, these methods have limitations in reliably
discriminating between cancer patients and healthy subjects (e.g.,
some benign masses can look like cancer giving false-positive

Corresponding author. Tel.: +48 566114308; fax: +48 566114837.
E-mail addresses: bbusz@chem.umk.pl, bbusz@chem.uni.torun.pl
(B. Buszewski).
anatomical screens). Existing noninvasive screening methods using
proteomic and genetic technologies detecting molecular biomark-
ers may be supplemented by innovative methods such as exhaled
breath analysis [4].
Odorant molecules such as volatile organic compounds (VOCs)
are typically small volatile organic molecules with molecular
mass less than 400Da [5]. Some of the compounds are gener-
ated in the human body during metabolic process, e.g., acetone
formed by decarboxylation of acetoacetate or acetyl-CoA, high-
est concentration of which could be a sign of diabetic disease.
Isoprene is produced along the mevalonic pathway of cholesterol
synthesis in the cytosolic fraction. It is identified as marker of dis-
orders in cholesterol metabolism. Hydrocarbons such as ethane
and pentane are produced by lipid peroxidationof polyunsaturated
fatty acids. Sulfur-containing compounds such as ethyl mercap-
tane, dimethylsulfide, or dimethyldisulfide could be generated by
incomplete metabolismof methionine in the transamination path-
way. Whereas nitrogen-containing compounds (dimethylamine
and trimethylamine) are identified in the breath of patients with
http://dx.doi.org/10.1016/j.snb.2014.06.006
0925-4005/© 2014 Elsevier B.V. All rights reserved.
616 J. Rudnicka et al. / Sensors and Actuators B 202 (2014) 615–621
kidney impairment. Compounds suchas acetone, hexanal, andhep-
tanal detected in blood and breath of patients with lung cancer
couldbe potential markers of cancer [6–8]. Biochemical origin, gen-
eration, or physiological functions of most of VOCs are still not
known.
Volatile organic compounds in human breath occur at the parts
per billion (ppb) level; therefore, as a sampling technique, solid
phase microextraction (SPME) is mainly applied. After sampling of
VOCs, identification of the components is performed most often by
using gas chromatography–mass spectrometry (GC–MS) [7,9,10].
Other promising methods for the detection of VOCs are selected
ion flow tube-mass spectrometry (SIFT-MS) [11], proton trans-
fer reaction-mass spectrometry (PTR-MS) [12], or electronic nose
[13,14].
Medical attention is drawn to the evidence of the dogs detec-
ting malignancies in patients. The first reports about the possibility
of detecting cancer in humans by a dog appeared in the Lancet in
1989, then in 2001; in both cases, dogs with no previous train-
ing have contributed to the detection of melanoma in their owners
[15,16]. This fact has initiated research, the goal of which was to
check whether training of dogs to detect different human cancers
is possible and whether the dogs are able to differentiate odor sam-
ples of healthypersons andfromcancer patients. It has beenapplied
in many studies that have used different kinds of odor sources,
e.g. melanoma tissue samples hidden on the skin of healthy vol-
unteers [17], urine samples for bladder, breast, and prostate cancer
detection [18,19], tumor tissue for detecting ovary cancer [20], and
exhaled breath air for breast and lung cancer detection [21–24].
The ability to explore odor stimuli depends on the size of the
olfactory epithelium and olfactory cells. For example, a German
Shepherd has more than 200 million olfactory cells on an area of
about 170 cm
2
, while a human has about 5 million cells on about 5
cm
2
olfactory epithelium. Olfactory receptors are stimulated only
by gaseous substances floating in the air, which by diffusion gets in
contact with the mucous membrane and dissolves in the thin layer
coveringthemucous membrane. Theabilitytodiscriminatenumer-
ous compounds depends also on the proportion of genes of the
olfactory receptor proteins [25]. It is thought that a dog’s olfaction
is from10 to 100 thousand times more sensitive than human sense
of smell. However, it is difficult to differentiate between sensitivity
and selectivity of olfaction from operant conditioning abilities of
dogs during training. Research carried out by Walker et al. [2006]
showed that the dog odor recognition for amyl acetate is 2 ppt
(parts per trillion) [26].
In this paper, solid phase microextraction technique and gas
chromatography–mass spectrometry (GC–MS) were used for the
analyses of VOCs of exhaled air from lung cancer patients and
healthy persons. Additionally, two trained dogs were used for iden-
tification of the same breath samples as in GC–MS studies.
2. Experimental
2.1. Instrumentation
The GC–MS analysis was performed on 6890N gas chromato-
graph (Agilent Technologies, Waldbronn, Germany) coupled with
mass spectrometer Agilent 5975 Inert XL MSD equipped with CP-
Porabond-Q (Varian Inc., Middelburg, The Netherlands) 25m x
0.25m x 3␮m column. The oven temperature program was as fol-
lows: initial 40

Cheldfor 2min, thenrampedat 10

C/minto140

C
and the ramped at 5

C/min to 270

C and held for 5min. The tem-
perature of the split–splitless injector was 200

C. The MS analyses
were carried out in full-scan mode, with scan mass range of m/z
30–300. Spectra were collected at electron ionization (EI) of 70eV,
both ion source and line transfer temperatures were set to 200

C.
The acquisition of chromatographic data was performed by using
the Chemstation software (Agilent). A manual SPME holder and
carboxen/polydimethylsiloxane (CAR/PDMS) (75␮m) coated fiber
(Supelco, Bellefonte, USA) were used for the SPME method.
Breathsamples werecollectedinto1L Tedlar bags usingabreath
sampler (Medical University of Innsbruck, Austria). The device was
operated in a CO
2
-controlled manner and allowed to collect alveo-
lar exhaled air samples [27].
2.2. Chemical standards
Alkanes, alcohols, aldehydes, and ketones were purchased from
Sigma-Aldrich (Steinheim, Germany). Helium and argon, purity
99.999%, were purchased from B.O.C. (Bydgoszcz, Poland).
2.3. Calibration
Prior to the use, the bulb was cleaned with methanol and dried
in an oven at 60

C for at least 12h. Afterwards, it was purged
with pure argon for 15min. Then, the bulb was evacuated using
a vacuum pump within 30min. Gaseous standards were prepared
by injection of 1–2␮L of each compound into 1 L glass bulb and
its evaporation. Afterwards, the mixture was moved using a gas
syringe to 1 L Tedlar bag filled with 0.5 L of pure argon. During
the sampling, SPME fiber was introduced into the bag through the
septum to obtain concentrations in the range of 3–300 ppb and
exposed to gas mixture.
2.4. Solid Phase Microextraction
Before the first use, the fiber was conditioned in an injector at
200

C for 5h. During exposition, the SPME fiber was introduced
into the bag containing sample of breath, througha silicone septum
and was exposed for 10min, at 25

C. After extraction, the fiber was
withdrawn to the needle, pulled out fromthe bag, and injected into
the GC. The compounds were desorbed in the hot GC injector port
for 2min at 200

C [27].
2.5. Participants and breath samples collection
2.5.1. Basic data for participants researches
Exhaled breath samples were collected from 108 patients with
lung cancer confirmed by histopathological examination, recruited
from Department of Lung Diseases, Collegium Medicum, Nico-
laus Copernicus University, Toru´ n, Poland. Samples of exhaled air
were collected from 76 males, age 39–80 (mean 52±8) and 31
females, age 38–87 (mean 48±9). These included 74 patients with
non-small cell lung cancer (I stage of cancer was 5, II- 12, III-22,
IV-35), 15 patients with small cell lung cancer (stage of cancer-
ED), and 19 patients with not specified histological type. Breath
samples of healthy volunteers were collected from 121 persons,
31 males, age 21–47 (mean 42±8) and 90 females, age 20–73
(mean 51±9). Additionally, breath samples were collected from24
patients with asthma and COPD (8 males, age 53–64 (mean 58±5)
and 16 females, age 25–92 (mean 50±6)). This group of samples
was used to present to the tests using sniffing dogs.
Inorder tofollowethical requirements, all breathsampledonors
were informed about aim of the study and for each participant
the questionnaire was filled out. The experimental procedure was
approved by the Ethical Commission (Nicolaus Copernicus Univer-
sity in Toru´ n, Ludwik Rydygier Collegium Medicum, Bydgoszcz).
2.5.2. Breath sample collection for GC–MS analysis
Before collection of breath, all bags were cleaned by flushing
with argon gas and then filled with argon and heated at 60

C for
12h to remove any contaminations. Afterwards, a 200ml sample in
J. Rudnicka et al. / Sensors and Actuators B 202 (2014) 615–621 617
bags was transferred into second bag. Samples of breath were ana-
lyzed within 3–4hours to prevent loss volatile organic compounds
with bag. Ambient air samples were taken for measurement blank.
2.5.3. Collection of breath samples for testing by dogs
Cylindrical polypropylene tubes 14cm long, outer diameter
3cm (Defencetek, Pretoria, South Africa) with removable inserts
filled with oil-coated polypropylene absorbent were used for
collecting exhaled breath. Breath samples were collected by per-
forming three deep exhalations into sampling tubes. Subsequently,
the tubes were fitted with their end caps and wrapped into sterile
plastic bags and stored in room temperature between the time of
breath sampling and presentation to the dogs. For the test, remov-
able inserts were taken out of the tubes and placed into sterile
glass vessel covered with hole-punched lids to prevent touching
the insert with breath sample from dog’s nose during sniffing.
2.6. Canine detection procedure
The tests using sniffing dogs were carried out at the Department
of Animal Behavior, Institute of Genetics and Animal Breeding,
Polish Academy of Sciences, Jastrzebiec, Poland.
Two male dogs (German shepherds mixed) were used in this
study. Earlier the dogs have completed a special training using
operant conditioning (a reward based training method) in 5 station
scent lineup to distinguish between breath samples collected form
melanoma, lung and breast cancer patients from those of healthy
donors. The details of the canine training were given elsewhere
[23].
The tests were conducted in a laboratory condition, in room
isolated from external stimuli. During the tests only the experi-
menter and the dog handler were present in the laboratory room.
Five pots, heavy enough to prevent pushing by dogs during sniff-
ing, were placed on the floor in a lineup approximately 1m apart.
The glass boxes with breath samples were placed into pots by the
experimenter. One target sample containing breath sample from
patient with cancer was placed in a randomly selected position in
the lineup and the remaining four pots contained complementary
samples from healthy donors, donors with other lung diseases or
synthetic samples. Than the dog handler, who was never aware
of the position of target sample, encouraged the dog to sniff all
pots in the lineup. If the dog sat down in front of the cancer sam-
ple the experimenter gave an acoustic signal using a clicker device
for a correct response of the dog and the dog handler immediately
rewarded the dog with verbal praise and a piece of food. The exper-
imenter was invisible to the dog and observed the trials through
video monitor. Each dog performed daily approximately 10 trials
(walking along the scent lineup and sniffing the samples).
The experimental procedure and keeping conditions for dogs
were approved by the 3
rd
Local Ethical Commission for Animal
Experimentation in Warsaw, Poland.
2.7. Data analysis of experiment with dogs
Rate of correct indications (sensitivity) and rate of false positive
indications (specificity) were calculated as ratio of number of indi-
cations to number of sniffing the cancer and non-cancer samples
respectively, with 50% probability of indication by chance alone.
For estimation of the sensitivity and specificity relationship ROC
(receiver operating characteristic curve) were constructed. The area
under the ROC demonstrated dogs indication accuracy. For differ-
ences between percent of correct indications and false alarms the
Chi-square test was used.
Table 1
Parameters of validation.
Compounds Linarity [ppb] R
2
LOD [ppb] LOQ [ppb]
Acetaldehyde 10.9–217.8 0.998 0.02 0.07
Acetone 16.6–332.9 0.997 1.03 3.08
Acetonitrile 11.7–234.0 0.993 1.12 3.36
Benzene 6.8–136.8 0.999 1.03 3.09
2-Butanone 6.8–136.5 0.999 1.53 4.58
Carbon disulfide 10.2–203.2 0.998 1.22 3.67
Cyclohexane 5.7–113.1 0.997 0.45 1.34
Decane 3.2–63.1 0.992 0.77 2.32
2,3-Dimethylbutane 4.7–93.9 0.997 1.55 4.65
2,5-Dimethylfuran 5.7–114.8 0.999 1.45 4.36
Dimethyl sulfide 8.3–166.4 0.997 1.63 4.90
Ethanol 41.9–418.7 0.991 7.06 21.17
Ethylbenzene 5.0–99.8 0.998 0.83 2.50
Ethyl acetate 6.3–125.1 0.998 1.33 3.98
Ethyl ether 5.9–117.6 0.998 0.78 2.35
Furan 8.3–165.6 0.998 1.69 5.07
Hexanal 5.1–101.8 0.995 0.70 2.11
Heptane 4.2–83.4 0.999 0.96 2.87
4-Heptanone 4.4–87.8 0.998 1.20 3.59
Hexane 4.7–93.5 0.998 1.00 2.99
Isoprene 12.2–244.4 0.997 1.63 4.90
Limonene 3.8–75.5 0.996 1.19 3.58
Methacrolein 7.4–148.2 0.999 1.25 3.75
Methyl acetate 7.7–153.8 0.998 0.82 2.47
2-Methylfuran 6.8–135.5 0.999 1.51 4.52
2-Methylhexane 4.1–82.8 0.998 1.13 3.40
3-Methylhexane 4.2–83.8 0.999 0.53 1.60
2-Methylpentane 4.6–92.6 0.999 1.10 3.30
3-Methylpentane 4.7–94.2 0.991 0.48 1.45
Methyl vinyl ketone 7.5–150.7 0.999 1.88 5.63
Nonane 3.4–68.6 0.998 1.04 3.11
Pentanal 5.8–115.0 0.998 1.38 4.15
Pentane 5.3–106.1 0.996 0.78 2.35
2-Pentanone 5.7–114.8 0.999 1.58 4.74
Pinene 3.9–77.0 0.999 0.83 2.50
Propanal 8.5–169.6 0.997 2.28 6.85
1-Propanol 32.7–327.0 0.996 9.46 28.37
2-Propanol 31.9–319.3 0.997 5.79 17.37
2-Propenal 9.2–182.9 0.994 0.73 2.2
Toluene 5.7–114.8 0.998 1.35 4.05
o-Xylene 5.1–101.2 0.996 1.08 3.25
m-Xylene 5.0–99.1 0.996 1.26 3.78
p-Xylene 5.0–99.1 0.999 1.15 3.45
3. Results and discussion
3.1. Data analysis of measurements SPME/GC–MS
3.1.1. Validation of the method
The linearity, precision and detection limits for selected VOCs
determination in human breath were shown in Table 1. The preci-
sion of the method was determined by performing three analyses.
The value of the relative standard deviation (RSD) was in the range
from 3% to 10% for hydrocarbons, alcohols, aldehydes, ketones
and aromatic compounds. RSD values less than 10% show that the
present method has good repeatability. Acalibration curve was lin-
ear for aliphatic hydrocarbons in the range 3 - 106 ppb, for alcohols
4 - 419 ppb, for aldehydes 5 - 218 ppb, for ketones 4 -333 ppb,
for aromatic compounds 5 -166 ppb, esters 6 -154 ppb, compounds
containingsulfur 8- 203ppb, acetonitrile 12- 234ppb, ethyl ether 6
- 118ppbandterpenes 4- 75ppb. Thelinear correlationcoefficients
were higher than 0.991.
The sensitivity of the method restricts detecting the limits of the
SPME/GC–MS technique. The detection limit (LOD) was defined as
signal-to-noise ratios equaled to three and signal-to-noise ratios
equaled ten as the quantification limits (LOQ). The lowest values of
LOD were obtained for hydrocarbons and aromatic compounds in
the range of 1 - 5 ppb and 2 - 5 ppb, respectively.
618 J. Rudnicka et al. / Sensors and Actuators B 202 (2014) 615–621
Table 2
Concentration range for compounds detected in human breath of healthy volunteers, asthma and COPD patients, cancer patients and indoor air.
Compounds Healthy person Patients with asthma and COPD Patients with lung cancer Indoor air
Concentration
range [ppb]
Median
concentration
[ppb]
Concentration
range [ppb]
Median
concentration
[ppb]
Concentration
range [ppb]
Median
concentration
[ppb]
Concentration
range [ppb]
Median
concentration
[ppb]
Acetaldehyde 3–370 53 10–601 74 9–291 57 4–57 29
Acetone 135–3167 580 171–2625 719 83–7769 1000 6–342 68
Acetonirile 1–2382 224 4–97 34 4–42 14 2–146 42
Benzene 3–23 7 7 7 3–10 5 4–5 4.7
2-Butanone 5–50 7 5–8 6 7–14 9 6–12 7
Carbon disulfide 4–12 7 5–10 7 4–348 18 4–10 8
Cyclohexane 1–96 20 1.3 1.3 3.1 3.1 – –
Decane 4–72 11 3–12 7 3–35 9 6–10 9
2.3-Dimethylbutane 8–127 38 5–6 5.6 4.7–5.2 4.9 4.8 4.8
2.5-Dimethylfuran 5–27 8 4 4 5–30 10 – –
Dimethyl sulphide 5–79 12 – – 5–110 61 – –
Ethanol 23–868 193 43–1234 492 27–5380 1203 21–478 146
Ehylbenzene 3–16 3 3–4 3.2 3–9 4 3 3
Ethyl acetate 4–223 49 4–586 114 4–147 31 4–86 12
Ethyl ether 2–409 63 2–61 15 2–18 6 3–16 9
Furan 2–8 4 – – 8 8 – –
Hexanal 2–8 3 2–8 4 2–14 4 2–8 5
Heptane 3–9 5 3 3 2–4 3 3 3
4-Heptanone – – 6–22 14 4–60 38 – –
Hexane 3–145 18 6–41 25 3–16 10 6–12 9
Isoprene 27–812 190 59–521 236 66–870 280 5–19 10
Limonene 5–880 39 5–22 10 6–91 13 6–18 11
Metacrolein 5–46 19 4–5 4.7 4–9 6 4 4
Methyl acetate 2–22 6 3–28 9 3–39 8 5–13 10
2-Methylfuran 5–44 11 4–8 7 5–29 11 4–7 5
2-Methylhexane 4–13 7 3.4 3.4 3.4 3.4 – –
3-Methylhexane 2–10 4 2 2 2 2 – –
2-Methylpentane 3–297 50 3–23 11 7–21 10 3–23 9
3-Methylpentane 1–179 43 2–34 8 2–5 3 2–12 7
Methylvinylketone 6–15 7 6–7 6.5 7–11 8 6–8 7
Monane 3–59 28 13–18 15 3–132 59 – –
Pentanal 5–11 7 5–6 5.6 4–7 5 5–6 5.3
Pentane 3–664 111 4–71 28 3–23 11 3–16 7
2-Pentanone 5–10 6 19 19 5–39 9 5 5
Pinene 1.5–28 6 2–5 3 2–23 5 3 3
Propanal 1–12 5 9–346 123 1–82 19 – –
1-Propanol 29–116 61 30–293 92 29–424 99 35–324 135
2-Propanol 19–725 169 167–900 523 20–1007 398 34–669 359
2-Propenal 2–12 4 2 2 2–8 4 2.4 2.4
Toluene 5–66 13 5–14 7 4–13 7 5–13 7
o-Xylene 7–110 21 6.8–7.4 7 6–11 8 7–8 7.3
m-Xylene 3–3.5 3 3.06–3.11 3.09 3.1–3.6 3.3 3 3
p-Xylene 3–24 5 2.5–3.5 3 3–8 4 3–5 3.3
3.1.2. Breath analysis
In each research breath sample both acetone and isoprene were
identified. The average concentration of this compounds were
higher in the exhaled air of patients with cancer and COPD and
asthma than in exhaled air from healthy persons. The increase of
concentrations for 1-propanol, 2-propanol, methyl acetate, hex-
anal, dimethyl sulfideandcarbondisulfideweresignificant ingroup
of patients with lung cancer than in case of healthy volunteers
and persons with other lung diseases. The decrease of concentra-
tions were noticedfor pentane, decane, benzene, toluene, o-xylene,
limonene and other compounds which were presented in Table 2.
Furan and dimethyl sulfide were not detected in group of patients
with asthma and COPD, whereas 4-heptanone was not identified
in exhaled air taken from healthy volunteers. Such compounds as
alkanes, ketones, aldehydes are produced in human body as prod-
uct of metabolic processes. Acetone is formed in decarboxylation
of acetoacetate and acetyl-CoA, isoprene is produced along meval-
onic pathway of cholesterol synthesis, hydrocarbons is result of
lipid peroxidation polyunsaturated fatty acids, however other part
have origin exogenous [7].
The chemometric calculations were performed in Statistica
7.1 Data Miner (Statsoft, Krakow, Poland) software running on
Windows XP platform. The peak area of the identified analytes was
used for calculations. For data processing and evaluation, statistical
methods such as artificial neural network (ANN) and chi-squared
automatic interaction detector (CHAID) were applied. Dataset con-
taining 88 parameters (compounds) and 226 cases (patients) was
used to create an artificial neural classifier. 50% of cases were
selected for training the network, 25% to test and 25% for vali-
dation. 1000 different ANN topologies was tested The best model
with 88 input neurons, one hidden layer with 27 neurons and 2
output neurons was chosen. Softmax and logistic formulas ware
used as activation and post synaptic functions of neurons in hidden
layer. The major parameters of the chosen structure were: training
performance 100, test performance 96, validation performance 80.
Applicationthe network gives small error of classification- 4.35%of
incorrectly classified cases. For the model, the ROC was construed
to predict lung cancer with sensitivity=74%, specificity=73% and
AUC=0.97.
Volatile organic compounds identified in human breath were
applied for the construct the CHAID tree (Fig. 2). The tree con-
sist 8 terminal nodes and deep for 5 levels. Dimethyl sulfide is the
main compound which enable to distinguish two groups: patients
with cancer and healthy volunteers. Other compounds, namely
J. Rudnicka et al. / Sensors and Actuators B 202 (2014) 615–621 619
Fig. 1. The CHAID tree performed for healthy person and patients with lung cancer.
1,4-pentadiene, acetate ethyl, methylcyclopentane, 2-propanol,
isobutane and 2,4-dimethylheptane enhance the separation of
breathsamples collectedfromhealthy volunteers andpersons with
lung cancer.
Fig. 1
3.2. Results of experiment with the dogs.
Dogs correctly indicated 86% of sniffed samples collected from
patients with lung cancer. In turn, percentage of indications (false
positive indications) of samples from patients with other lung dis-
eases and healthy volunteers were 42% and 28%, respectively.
Samples frompatients with lung cancer were differentiated sig-
nificantly better (p<0.001) than samples obtained from patients
with other lung diseases (42%). Percent of true positive indications
of cancer samples was also significantly (p<0.001) higher thanper-
cent of falsepositiveindications of samples fromhealthyvolunteers
(28%).
False positive rate of samples from patients with lung diseases
without cancer confirmation was significantly (p<0.001) higher
than false positive rate of samples from healthy volunteers.
The dogs indicated 21% of sniffed synthetic samples prepared
on the basis of exhaled air of cancer patients and 16% of samples
prepared on the basis of exhaled air of the healthy volunteers. This
results was significantly (p<0.001) lower than percentage of indi-
cations of cancer samples or rate of indication of samples from
patients with other lung diseases.
The significant differences were observed between dogs’ ability
to detect particular types of samples (Fig. 2).
When a single binary diagnostic test is used to predict disease or
nodisease, a simple 2-by-2table toasses howwell the test classifies
when the disease state is known.
0
10
20
30
40
50
60
70
80
90
100
l
u
n
g
c
a
n
c
e
r
o
t
h
e
r

l
u
n
g
d
i
s
e
a
s
e
s
h
e
a
l
t
h
y
v
o
l
u
n
t
e
e
r
s
Cygun
Gromit
Fig. 2. Percentage of indications for particular type of samples for two dogs Cygun
and Gromit refers to the names of dogs.
The ROC curve (ROC), a plot of sensitivity vs 1- specificity [28]
is typically used to evaluate clinical utility for both diagnostic and
prognostic models. This curveasses howwell test or model discrim-
inates, or separates individuals into two classes, such as diseased
and non-diseased. In Fig. 3 it was shown an example of receiver
operating characteristic curve (ROC) using the results of sensitivity
of patients withlung cancer versus specificity of healthy volunteers
detection by dogs. The sensitivity (or the probability of a positive
test among those with disease) and the specificity (or the proba-
bility of a negative test among those without disease) can easily
be assessed. In comparing tests, we prefer those that are higher in
both sensitivity and specificity. Because one dog may have higher
sensitivity but lower specificity than another the area under ROC
curve, which indicates the overall accuracy of a diagnostic test is
taken into consideration. In a ideal test, with no false-positive or
false-negative results, the curve forms a right angle inthe upper left
corner of the chart and area under the curve equal 1.0. If accuracy of
620 J. Rudnicka et al. / Sensors and Actuators B 202 (2014) 615–621
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90 100
1-Specificity
S
e
n
s
i
t
i
v
i
t
y
Gromit
Cygun
Fig. 3. Receiver operatingcharacteristic curve(ROC) for samples of exhaledair taken
from patients with lung cancer.
the test deteriorates, the curve becomes rounded. Area under the
curve ROC (AUC) shows the ability of a test or detection method
for distinguishing true and false results, is a measure of the quality
of the method in this way if AUC 0.5 means no predictive ability
and AUC 1 means perfect discrimination. The relative area under
the ROC curve in the case of lung cancer was ranged from 78% to
90%. Considering individual dogs canbe statedthat lung cancer was
detectedthe best by dog Cygunas the area under the ROCcurve was
highest. For the dog, the results of test that predicts cancer disease
with AUC≥90% is considered to be very accurate.
The ROC was construed also in research by Phillips et al. [29] to
predict lung cancer. They accurately identified patients with lung
cancer in a model employing 30 breath VOCs and with sensitivity
=84.5%, specificity =81.0% and AUC =0.90 [29].
4. Conclusions
A combination the solid phase microextraction and gas
chromatography–mass spectrometry (SPME/GC–MS) and sniffer
dogs applicationto detectioncompounds inhumanbreathsamples
were discussed. The technique was applied to determination com-
position breath the 108 patients with lung cancer, 24 asthma/COPD
and 121 volunteers. The total number of compounds identified
in sample of breath equal 88 by GC–MS. Statistical method,
chi-squared automatic interaction detector (CHAID), allowed
indicate dimethyl sulfide, 1,4-pentadiene, acetate ethyl, methylcy-
clopentane, 2-propanol, isobutane and2,4-dimethylheptane which
separated two research groups patients with lung cancer and
healthy persons. The sensitivity detection sample of lung cancer
by trained dogs equal 86%, whereas specificity 28%.
Presented an analytical and canine methods for detection com-
position of exhaled air could be applied as a potential non-invasive
tool for screening of lung cancer.
Acknowledgements
This work was supportedby Grant NN204026238(2010–2013)
and European Social Fund, Polish National Budget, Kujawsko-
pomorskie Voivodship Budget—“Krok w przyszlosc”. The authors
would like to thank Dr. Ewa Trawi ´ nska (Department of Lung Dis-
eases, Toru´ n) for help in contacts with patients.
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Biographies
Joanna Rudnicka received MSc degree at the Nicolaus Copernicus University, Toru´ n,
in 2009 and from 2009 she is PhD student. In 2010 and 2011 she was on scholar-
ship on Innsbruck Medical University in the range CEEPUS. Her research interests
include methods for preparationof samples for trace analysis (SPME), determination
J. Rudnicka et al. / Sensors and Actuators B 202 (2014) 615–621 621
of volatile organic compounds in exhaled breath using gas chromatography–mass
spectrometry (GC–MS), research on the application sniffing dog for the detection
cancer markers.
Marta Walczak received PhD degree at Institute of Genetics and Animal Breeding
of the Polish Academy of Sciences in 2009. Her PhD thesis concerned Operant con-
ditioning of dogs for the detection of odor markers of cancer diseases. Now, she is
a head of the project on the genetic basis of fear related aggression in police dogs
using SNP microarrays, NGS technology and behavioural methods, funded by the
National Science Centre, Poland.
Tomasz Kowalkowski completed his PhD degree at the Nicolaus Copernicus Univer-
sity, Toru´ n, in 2002. In 2010 he received D.Sc. degree in the field of analytical and
environmental chemistry. From 2013 he works as associate professor at Faculty of
Chemistry, Nicolaus Copernicus University. His main scientific interests are related
to chemometric evaluation of complex datasets, separation of particles by field flow
fractionation and transport and fate of pollutants in environment.
Tadeusz Jezierski received his PhD at Institute of Genetics and Animal Breeding of
the Polish Academy of Sciences in 1974. In 1987 he received the Doctor of Sciences
(habilitation) degree and in 1999 he reached a title of professor. He is head of the
Department of Animal Behavior at the Institute of Genetics and Animal Breeding
of the Polish Academy of Science. He was a head of few project concerning dogs
olfactory abilities. He has published 65 scientific papers, including studies on canine
scent detection.
BogusławBuszewski received PhDdegree at the Faculty of Chemical Technology, Slo-
vak Technical University in Bratislava, Czechoslovakia in 1986. In 1992 he reached
the Doctor of Sciences (habilitation) degree. In 1994 he received Nicolaus Coperni-
cus University professor position, in1999 receivedthe title of professor of chemistry
and in 2000 received full professor of the Nicolaus Copernicus University in Toru´ n.
His mainscientific interests are concernedwithenvironmental analysis, chromatog-
raphy and related techniques (HPLC, SPE, GC, CZE, adsorption, sample preparation),
spectroscopy, utilization of waste and sludge and chemometrics.

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