R eview
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Detection of volatile organic compounds as
biomarkers in breath analysis by different
analytical techniques
Breath is a rich mixture containing numerous volatile organic compounds at trace amounts (ppbv–pptv level) such as:
hydrocarbons, alcohols, ketones, aldehydes, esters or heterocycles. The presence of some of them depends on health
status. Therefore, breath analysis might be useful for clinical diagnostics, therapy monitoring and control of metabolic
or biochemical cell cycle products. This Review presents an update on the latest developments in breath analysis
applied to diagnosing different diseases with the help of high-quality equipment. Efforts were made to fully and
accurately describe traditional and modern techniques used to determine the components of breath. The techniques
were compared in terms of design, function and also detection limit of different volatile organic compounds. GC with
different detectors, MS, optical sensor and laser spectroscopic detection techniques are also discussed.
Manifold metabolic processes occurring within
the human body create a wide variety of volatile
organic compounds (VOCs). A large number of
VOCs may be useful in diagnostics because they
can provide valuable information on health conditions, such as infections or metabolic diseases.
In ancient times, medics sniffed the patients as a
part of examination and particular odors of skin,
sweat, urine and breath were read as a sign of certain diseases. Nowadays, while analysis of body
fluids is fundamental for diagnostics, human
breath analysis is also proposed with increasing
frequency as a tool useful in clinical applications. As VOCs come from a variety of sources,
in order to obtain accurate information it is necessary to establish specific methods of sampling,
sample preparation and sample identification for
each disease.
The first step in the development of a new
b­iomarker is the discovery phase. This is followed by rigorous evaluation of its diagnostic accuracy and then by the evaluation of
the impact the use of biomarker will have on
clinical outcomes. So far, a number of exhaled
biomarkers are still in the discovery phase and
only a few have been evaluated in compliance
with the Standards for Reporting of Diagnostic
Accuracy (STARD) criteria, the internationally
accepted set of requirements for quality of studies concerning diagnostic procedures proposed
by the STARD initiative. STARD lists 25 criteria essential for accuracy and completeness of a
study report, including points such as biomarker
definition, population description, data collection, reporting, methods of calculating, diagnostic accuracy, test reproducibility and technical
details [1].
Exhaled breath analysis is a non-invasive,
painless and nonstressful method proposed for
clinical application [2,3]. However, sampling is
the critical point during breath analysis; therefore, it needs standardized procedures and skilled
staff. Nitrogen, oxygen, carbon dioxide, water,
argon and other products of metabolic processes
within the body are the main components of the
exhaled air. VOCs are related to a person’s diet,
stress level and immune status. Compounds like
acetone, ethane, pentane and isoprene are known
in the medical practice and, if linked with metabolic pathways, provide valuable information
about the state of a patient’s health (Table 1).
The complex metabolic cycle occurring in
the human body produces various compounds.
Some are complex such as proteins and peptides,
and some are simple such as acetone, isoprene
and other VOCs. Strictly speaking the term
simple refers to the VOCs – acetone, isoprene
or acetaldehyde are not the major final products, they are usually intermediate products and
often side compounds of metabolic pathways.
For example, as a result of oxidative stress, polyunsaturated fatty acids are transformed to simple
aldehydes, hydrocarbons or fatty acid hydroxides
(Figure 1).
A similar process occurs in the case of acetone
and isoprene. Acetone is produced by hepatocites
10.4155/BIO.13.183 © 2013 Future Science Ltd
Bioanalysis (2013) 5(18), 2287–2306
Bogusław Buszewski*1,
Damian Grzywinski1,
Tomasz Ligor1,
Tadeusz Stacewicz2 ,
Zygmunt Bielecki3
& Jacek Wojtas3
Department of Environmental
Chemistry & Bioanalytics, Faculty of
Chemistry, Nicolaus Copernicus
University, 7 Gagarin St, 87-100
Torun, Poland
2
Institute of Experimental Physics,
University of Warsaw, 69 Hoza St.,
00-068 Warsaw, Poland
3
Military University of Technology,
2 Kaliskiego St, 00-908 Warsaw,
Poland
*Author for correspondence:
Tel.: +48 56 611 43 08
Fax: +48 56 611 48 37
E-mail: bbusz@chem.uni.torun.pl
1
ISSN 1757-6180
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Buszewski, Grzywinski, Ligor, Stacewicz, Bielecki & Wojtas
Table 1. Simple products of metabolism.
Compound
Metabolic origin
Acetaldehyde
Ethanol metabolism
Acetone
Decarboxylation of acetoacetate and acetyl-CoA
Ethane, ethylene, pentane
Lipid peroxidation
Hydrogen, methane
Gut bacteria
Isoprene
Cholesterol biosynthesis, conversion of dimethylallyl
pyrophosphate, epoxydation by cytochrome
P450-dependent mono-oxygenases
via decarboxylation of excess acetyl-CoA, which
comes from fatty acids and glucose metabolism.
Acetone concentration in breath is connected not
only with glucose metabolism and uncontrolled
diabetes, but also with ventilation, cardiac output, physical exercises or ketonemia. In turn,
isoprene is formed along the mevalonic pathway of cholesterol biosynthesis in the cytosolic
fraction [3,4].
Breath analysis is less popular and less developed than blood and urine analysis and, thus, is
not used for rapid clinical diagnosis. However,
over the years the applications of breath analysis have increased. Recent papers have presented
the correlation between the VOCs and certain
diseases. Nowadays, scientists can identify many
compounds that play an important role in breath
analysis for clinical applications [3,5]. For example, acetone is present in high concentrations in
diabetic patients; dimethylamine and triethylamine in patients with renal insufficiency; and
hydrogen sulfide in patients with liver disease. So
far, biomarkers have been established for a range
of diseases such as lung cancer [1,6,7], breast cancer [8], liver cirrhosis [9], pulmonary tuberculosis
[10,11], diabetes [12] and asthma [13]. In healthcare,
treatment and the food industry the detection of
VOCs has been a research and development target over the last decade, with the most advanced
research f­ocusing on breath analysis.
„„Principles
of study organization
A breath analysis study should be carried out on
the largest possible study population (hereinafter
called the patients) divided into two groups: a
control group of healthy people and a group of
people with the disease. Patients are additionally
classified, for example, according to age, gender,
drinking and smoking habits, risk of disease and
stage of disease. The control group are subjected
to detailed examination in order to exclude
diseases of the tested organ. For example, in
liver cancer studies the control cohort consists
Polyunsaturated fatty acid
Key Terms
Enzymatic oxidation
reactions
Auto-oxidation
Biomarker: Substance used as
an indicator of the biological
state of any system. A
biomarker can be any kind of
molecule indicating the
condition (past or p­resent) of
living organisms. Biomarkers
play an important role in
understanding the r­elationships
between exposure to
environmental chemicals, the
development of chronic human
diseases, immune status or
stress level.
Laser spectroscopy
techniques: A section of
spectroscopy using tunable
l­asers (highly monochromatic
light sources with the possibility
of fine tuning wavelength) as a
light source used for the
e­xcitation of molecules. These
methods enable obtaining of
high selectivity of the excitation,
which allows a wide spectrum of
groups of atoms, molecules,
crystals or plasma to be studied.
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LOO•
•OH
LOOH
HOH
Initiation
Conjugated diene radicals
Fatty acids
hydroperoxides
Prostaglandins
Thromboxanes
O2
Propagation
Peroxyl radicals
Aldehydes
Termination
Hydrocarbons
Fatty acids
hydroperoxides
F2-isoprostanes
Fatty acids hydroxides
Aldehydes
Figure 1. Oxidative stress cycle.
Bioanalysis (2013) 5(18)
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Detection of volatile organic compounds as biomarkers in breath analysis
of people with no history of liver disease and
satisfactory test results for transaminases, liver
synthesis parameters, glucose and iron metabolism, and viral hepatitis markers. In contrast,
the criteria for the control group in studies of
active pulmonary tuberculosis are symptoms and
signs such as cough, sputum production, night
sweats, weight loss or hemoptysis or chest x-ray
abnormalities consistent with active pulmonary
disease [11].
Direct sampling of exhaled air is crucial and
possible to achieve only in case of direct analysis
methods (e-noses, laser spectroscopy techniques
or certain MS techniques). The important point
is end-tidal sampling (alveolar air), which minimizes dead space volume effect. However, it is
still impossible to combine direct sampling with
frequently used GC–MS technique. The choice
of preconcentration method (sorbent tubes for
TD or SPME or direct cryofocusing) is mainly
related to chromatography. Yet preconcentration
is tedious, time-consuming, requires dedicated
devices and carryover problems are observed.
A large number of parameters and the data
from the individual studies must be submitted to statistical calculations. For evaluation
and processing data obtained in chromatographic analysis, different statistical methods
might be used. For this purpose, one can use
chemo­metric calculations or multivariate analysis [14]. Each method provides a large number
of parameters and the best solution is to apply
both. The chemometric calculations and multi­
variate analysis can be done, for example, in
Statistica Data Miner software [15,16]. Other
programs used in statistical analysis are SPSS or
Graphpad Prism [17]. A few different statistical
methods commonly used for data classification
and dimensionality reduction have been also
applied, such as discriminant analysis, canonical analysis and factor analysis. Discriminant
analysis is a supervised method of classification that maximizes the ratio of between-class
variance to the within-class variance in any
particular data set, thereby guaranteeing maximal separability. In clinical research, one can
record different variables related to the health
status of patients to determine which variables
best prophesy whether the patient has a chance
of a complete cure (group 1), partial recovery
(group 2) or no chance for a cure (group 3).
Canonical analysis is based on the estimation
of the relationship between sets of variables. Its
application in medicine can highlight the correlation of various risk factors with the formation
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of a group of disease symptoms. In turn, factor analysis is the classification of the objects
that can be performed in the reduced space
and explained by the reduced set of ­c alculated
factors.
„„Analytical
techniques in breath analysis
GC
GC was the first technique used for breath analysis. It dates back to 1971 when Pauling et al.
applied it to detection of VOCs in breath and
found more than 200 compounds [18]. The GC
method has advanced considerably in the last
40 years and has been widely used in various
environmental, industrial or clinical applications. Compared with other breath analysis techniques, such as the proton transfer reaction–MS
and laser spectroscopy, GC is the simplest one
in terms of apparatus. Technically, GC with a
simple detector (FID or ECD) is not sufficient
for clinical applications. In general, it is used
for qualitative and quantitative analysis of compounds that are typical constituents of breath.
The number of parameters affecting GC analysis
is large (the carrier gas flow rate, temperature,
column length and diameter, and the stationary
phase). However, the necessity for rapid analysis of increasingly complex mixtures of volatile
compounds limits the use of GC.
GC–MS
There are many studies using MS to analyze
exhaled breath [19,20]. The GC–MS system was
successfully used for the analysis of human
breath and, thus, for the diagnosis of certain
diseases, such as lung and breast cancers, diabetes, cystic fibrosis and pulmonary tuberculosis.
Patterson et al. applied the GC–MS technique
to identify biomarkers for breast cancer [8]. 383
various VOCs were monitored in the breath.
Most of them are associated with lipid peroxidation mechanisms. Several compounds, such as
nonenal, hexanal, methacrolein and isoprene,
were considered as breast cancer biomarkers.
The samples were analyzed by thermal desorption followed by GC–MS. Philips et al. used
TD–GC–MS techniques for detection of active
pulmonary tuberculosis in numerous studies of
breath [10,14]. They selected alkanes and alkane
derivatives with cyclohexane and benzene derivatives as potential biomarkers. The amounts of
other compounds: dimethyl sulfide, acetone,
2-butanone and 2-pentanone, increased in the
breath of patients with liver disease. Similar
results were previously obtained by Van den
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Buszewski, Grzywinski, Ligor, Stacewicz, Bielecki & Wojtas
Velde et al. [21]. In these studies, an attempt
was made to find a correlation between specific breath odor compounds and the patients
with liver cirrhosis, which can cause a sweet,
musty aroma of the breath. This very characteristic odor and breath analysis can be helpful in clinical diagnosis. Still, in general breath
tests utilizing GC–MS are not introduced into
clinical practice, except for a few examples
such as the NO x test for asthma, 13C test for
Helicobacer pylori infection, and the alcohol
breath test.
An innovative solution for routine clinical
study of exhaled breath is using dogs, customarily employed to sniff for explosives and drugs.
Consequently, attempts were made to utilize
dogs to detect lung cancer biomarkers in exhaled
breath. The results of such a study have been
compared to GC–MS techniques [22,23].
GC–TOF-MS & GC × GC–TOF-MS
TOF–MS is another type of mass analyzer
for identification of VOCs in exhaled breath
[24]. Here the ion separation occurs due to
differences in their TOF from the ionization
chamber to the detector within a long field-free
vacuum tube. The TOF depends on ion mass,
the length of the flight chamber, accelerating
voltage and the electric charge [23]. The main
advantage of the TOF analyzer is the high
mass resolution and the possibility of obtaining multiple spectra within a short time. The
GC–TOF-MS has been successfully used in
clinical applications for lung cancer detection.
Gaspar and colleagues studied the correlations
of composition of exhaled air in healthy volunteers, smokers and nonsmokers, and in patients
with pretreatment and post-chemotherapy
lung cancer [16]. They focused on linear and
branched C8 to C24 hydrocarbons. Sensitivity
at the level of 0.04 to 8.0 ppbv and RSD below
26% highlight the effectiveness of this method.
Yet, as a result of differences in polarity of the
VOCs, nonpolar compounds are not separated
on a polar column and vice versa. However, a
combination of non-polar stationary phase in
the first chromatography column and polar
stationary phase in the second column eliminates this problem entirely. Consequently, the
researchers have proposed 2D GC. Caldeira
et al. presented the use of GC × GC–TOF-MS
to study the breath of patients with asthma [13].
Separation of a VOCs mixture was performed
using a fused silica capillary column: a non­
polar column (HP-5 stationary phase) was used
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Bioanalysis (2013) 5(18)
in the first GC while a polar column (DB-FFAP
stationary phase) was used in the second GC.
Another application of GC × GC–TOF-MS has
also been described [25]. An equally complicated
system used for detection of VOCs is corona
discharge ion mobility spectrometry with
orthogonal acceleration of TOF-MS (CD IMSoaTOF-MS); Sabo and Matejik monitored 16
VOCs using this method [26].
Selected ion flow tube-MS
The selected ion flow tube MS (SIFT-MS) technique in breath analysis has been described in
detail by several papers [12,27–30]. Thus, only
a brief presentation of the SIFT-MS principle
is included here. Selected precursor ions, such
as H3O+, NO+ and O2+ are formed by electron
impact or a microwave discharge in helium carrier gas and carried by fast flow tube to quantitative mass spectrometer [31]. Figure 2 presents the
full structure of SIFT-MS.
Reactant ions selectively ionize the volatile
and trace compounds present within the analyte
– akin to chemical ionization. Absolute concentrations of trace gases can be calculated with a
LOD being typically in the ppbv range, using
the ratios of ion count rates and the known reaction rate constants [28]. Therefore, the SIFT-MS
method has a number of research applications
(Table 2) [32–36].
The SIFT-MS works in two different modes:
full scan (FS) and multiple ion monitoring. In
the FS mode, it is possible to observe all the ions,
which makes it possible to capture the full spectrum and allows a complete identification. What
is more important, the SIFT-MS instrument in
FS mode greatly reduces the speed of measurements, and is not applicable for direct real-time
analysis of exhaled breath. In turn, the multiple
ion monitoring mode registers strictly selected
ions, which significantly increases the sensitivity
of the analysis.
Application of SIFT-MS technology in breath
analysis provides an opportunity to detect a large
number of VOCs, such as ammonia, acetone,
ethanol, methanol, propanol, isoprene, hydrogen
cyanide, formaldehyde and acetaldehyde [30]. For
example, the presence of ammonia in exhaled
breath is probably generated by bacterial or enzymatic activity on the substrates arginine or urea;
thus, it is recognized as the indicator of Helicobacter pylori in the stomach and gastrointestinal
tract [37]. Turner et al. described a quantitative
study on five volunteers to determine changes
occurring in several trace compounds present in
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Detection of volatile organic compounds as biomarkers in breath analysis
| R eview
Pump
Healed sampling
line
He carrier gas
Microwave
resonator
Detection quadrupole
mass spectrometer
Gas discharge
ion source
Injection quadrupole
mass filter
Detection
vacuum pump
Injection
vacuum pump
Figure 2. Selected ion flow tube-MS instrument indicating the main components.
exhaled breath, before and after ingesting 75 g of
glucose [38]. Summarizing, SIFT-MS is used in
several areas of research including single breath
analysis for medical investigation, and for metabolism and environmental tests. Practically, the
SIFT-MS technique is applied for quantitative
analysis, and online measurement of breathing is
an important advantage of such instrumentation.
„„Proton
transfer reaction & ion-molecule
reaction-MS (IMR-MS)
Proton transfer reaction-MS (PTR–MS) is a
technique very similar to SIFT-MS. The main
difference is the use of a precursor ion to transfer
a proton to gaseous compounds introduced to
the precursor within the system [39]. As a result
the protonated molecules are accelerated and
Table 2. Applications of selected ion flow tube-MS to detect volatile organic
compounds in different research areas.
Type of research
Characteristic volatile organic
compounds
Exhaust gases: respiratory irritants, asthma
Aliphatic and aromatic hydrocarbons,
aldehydes, alcohols and acetone
Hydrogen sulfide, methanethiol,
dimethyl sulfide
Ammonia, acetone, isoprene, ethanol,
acetaldehyde, propanol, methanol
Acetone
Ethanol, acetaldehyde, ammonia,
acetone
Ammonia
Formaldehyde, nitric oxide
Methanol, ethanol, acetone,
acetaldehyde
Hydrogen cyanide
Acetone, isoprene, methyl nitrate
Acetone
Ammonia, ethanol
Rumen gas
Distribution of metabolites in breath in healthy
population
Volatile compounds in urinary headspace
Ethanol metabolism
Monitoring haemodialysis
Infection and tumour in urinary headspace
Cancer cells in vitro
Bacterial cultures associated with cystic fibrosis
Diabetes mellitus
Diabetics
Halitosis (oral malodour)
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Ref.
[32]
[32]
[32]
[32]
[32]
[32]
[32]
[32]
[32]
[33]
[34,35]
[36]
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Ion
source
Proton transfer
reaction-drift tube
Mass analyzer
Pump
Pump
Quadrupole mass spectrometer
H3O+
H3O
H2O
vapor
+
H3O
Ion beam
+
H3O+
Analyte
Pump
Secondary electron
multiplier
Figure 3. Proton transfer reaction-MS instrument.
then reach the detector. An important aspect
of utilizing H3O+ is lower proton affinity of air
components such as NO, O2 , CO, CO2 and
N2 as compared with H 2O molecules [2]. Major
disadvantages are the presence of interfering
substances. Keck et al. described the effects
of CO2 in breath samples [40]. Their research
showed that CO2 caused a pressure increase in
the PTR-MS drift tube (~1% increase for 5%
CO2). This aspect can be taken into account
and reduced in the calibration stage of PTR‑MS
instruments.
Charge exchange cell
Octopole
separation
device
Primary
ion source
Quadrupole
mass filter
Secondary electron
multiplier
Vacuum
system
Data set
Gas inlet
system
Figure 4. Ion molecule reaction-MS.
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Bioanalysis (2013) 5(18)
The measuring system of PTR-MS consists
of several components: the ion source, where
H3O+ ions are produced; the drift tube responsible for the proton transfer reaction; a detection system (mass analyzer) and the last component, containing a secondary ion multiplier [40].
The scheme of PTR is presented in Figure 3.
In the PTR-MS technique, the substances
are identified by product ions with particular
m/z. The ions of many compounds have an
identical m/z; therefore, the identification of
unknown compounds must be achieved by other
t­echniques [41].
PTR-MS is becoming a common, fast and
sensitive method for the analysis of volatile
organic compounds in human exhaled breath
at ppbv [42] or even pptv [43] levels. Kushch
et al. used the PTR-MS technique to measure
variations in human breath isoprene concentrations related to age, gender, BMI and total
serum cholesterol [44]. In turn, Schwarz and coworkers tried to find correlations between the
physiological state of patients and the concentrations of acetone in human breath [45]. The
results reported in both studies emphasize that
the analysis of breath is a very complex process,
requiring extensive knowledge of procedure
analysis. It also depends on many factors, for
example, the individual charac­teristics of each
human. Similarly to SIFT-MS, PTR-MS might
be used in monitoring of diabetes mellitus
future science group
Detection of volatile organic compounds as biomarkers in breath analysis
patients [33]. Both methods are non-invasive,
highly selective and, more importantly, enable
real-time measurement.
Another MS technique briefly described in
the paper is ion-molecule reaction MS (IMRMS), which has been successfully used for breath
analysis in liver disease [46]. According to Millonig et al. this system provides a highly sensitive analysis for online and offline sampling of
organic and inorganic compounds in exhaled
| R eview
breath [46]. A schematic diagram of IMR-MS is
shown in Figure 4, and the detailed apparatus are
described in the Millonig article [46].
In brief, an IMR-MS analyzer consists of the
ion source, two octopole separation systems,
quadrupole mass separator, secondary electron multiplier and gas inlet system. It utilizes
krypton, xenon or atomic mercury gas as an ion
source. This type of ionization might be used for
the detection of different molecules of the same
Table 3. Summary of clinical applications of GC–MS and other MS techniques in recent research.
Type of
measurement
techniques
Analyte
Diseases
LOD
GC–MS
Acetaldehyde
Ethanol
Acetone, carbon disulfide, 2-propanol, ethyl
alcohol, ethyl acetate
Carbon disulfide, dimethyl sulfide, acetone,
2-butanone, 2-pentanone
Oxetane, dodecane, cyclohexane, benzene,
decane, tridecane, heptane and derivatives
1-butanol, 3-hydroxy-2-butanone
Dimethyl sulphide, butane, butanal
Alcoholism
Bacterial infection
Stomach cancer
50.5 ppbv
1224.5 ppbv
0.6–2.1 ppbv
[47]
Liver cirrhosis
0.08–765.13 ppbv
[21]
Active pulmonary
tuberculosis
Lung cancer
Lung cancer
–
[10]
[48]
Pentanal, hexanal, octanal, nonanal
Lung cancer
Cyclohexane
Malignant pleural
mesothelioma
2.18 and 1.29 ng/l
0.30, 0.18, 0.32 nmol/l,
respectively
0.002, 0.000, 0.011,
0.033 nmol/l, respectively
33.08 ng/l
GC–TOF-MS
Linear and branched hydrocarbons C8 – C24
Lung cancer
0.04–8.00 ppbv
[16]
GC × GC–TOF-MS
Nonane, 2,2,4,6,6-pentamethylheptane,
decane, 3,6-dimethyldecane, dodecane,
tetradecane
Asthma
–
[13]
Selected ion flow
tube–MS
Acetone
Ammonia
Acetone, formaldehyde, acetaldehyde,
hexanoic acid, hydrogen sulphide, hydrogen
cyanide, methyl phenol
Ammonia, acetone, methanol, ethanol,
isoprene, propanol, acetaldehyde, hydrogen
cyanide, formaldehyde
Methyl thiocyanate
Diabetes
–
Gastro-esophageal
cancer
~600 ppbv
80–110 ppbv
1.9 ppbv for hydrogen sulphide
to 296 for acetone
–
2 ppbv for formaldehyde to
620 ppbv for ammonia
[30]
Cystic fibrosis
8 ppbv
[52]
Hydrogen cyanide
Cystic fibrosis
8.9 ppbv
[53]
Hydrogen cyanide
Cystic fibrosis
13.5 ppbv
[54]
Isoprene, acetone, methanol
Lung cancer
Acetone
Formaldehyde,propanol, isoprene
–
Primary lung cancer
105.2, 627.5, 142.0 ppbv,
respectively
~600 ppbv
3.0, 94.1, 81.8 ppbv,
respectively
5.8–274.9 ppbv
26.9 ppbv
–
Proton transfer
reaction-MS
Ion molecule
reaction-MS
future science group
Isoprene
–
2-methylbutanal
Emphysema
Acetaldehyde, endogenous ethanol, isoprene, Liver diseases
methane, hydrogen sulphide
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Ref.
[15]
[15]
[49]
[50]
[51]
[35]
[37]
[12]
[6]
[45]
[41]
[44]
[55]
[46]
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Key Terms
Electronic nose: Type of
equipment used for
identifi­c ation and quantification
of, for example, volatile organic
c­ompounds or odors. The
name is connected with the
mimicking of the human
­olfactory system/nose.
Optical sensors:
Opto­electronic detectors that
­convert chemical information
into useful analytical signals
(absorption spectrum).
Chemical information can be
derived from a chemical
reaction, determination of the
component, or the physical
properties of the system.
molecular weight, for example, acetaldehyde or
carbon dioxide, and undoubtedly this is a big
advantage of this method of excitation.
Table 3 summarizes all the above-mentioned
techniques used in breath analysis and provides an opportunity to compare important
parameters and data [47–55].
Electronic nose
GC and MS have become very useful analytical methods within the last few years. However, the main disadvantages of GC–MS that
limit their medical use are long analysis time,
the demand for qualified technicians and high
cost. To overcome these disadvantages, different
measurement techniques were developed, based
on high-performance electronic noses.
The task of an electronic nose (e-nose) is to
simulate functioning of the human olfactory
system, which allows detection and identification of various volatile compounds or odors.
The human nose consists of a large number of
olfactory receptors, which generate electrical signals as a result of specific interaction with the
respective odor receptors [56]. A single neuron
corresponds to different odors, and the interaction of many neurons serves to identify and
classify smells. The idea of imitating the olfactory system has been used in attempts to create
an e-nose. As in the olfactory system, the odor
detection by an e-nose is achieved through the
use of selective electronic sensors. The number
of sensors is increasing with the development of
electronic technology. Selective sensors used in
e-noses are mainly optical sensors, piezoelectric sensors, metal oxide semiconductors and
conducting film polymers (Table 4) [57].
Although scientists have many sensitive breath
analysis techniques at their disposal, more and
more attention is focused on the development of
a handheld e-nose. The main advantages of using
an e-nose are low cost, rapid analysis, ease of use
and miniaturization of the equipment. However,
this method has a significant drawback: to be
successfully used, the electronic nose has to be
trained on a group of patients to recognize the
definite odor/biomarkers to create, for example,
the cancer prediction model, and then on a second group to validate the model. Currently there
are several e-noses with different selective sensors
on the market [58]. Dragonieri et al. used a commercial e-nose (Cyranose 320) for breath analysis
in patients with malignant pleural mesothelioma
[59]. In other applications, Cyranose 320 was used
for breath analysis in patients with bronchogenic
carcinoma [60], asthma [61,62] and lung cancer
[63,64]. A very extensive and thorough work presented by Wilson and Baietto [65] fully describes
the biomedical applications of electronic noses
(Table 5) [66–75].
Optoelectronic sensors – a great alternative
to an e-nose
Optical sensors (OS) are widely used in many
fields of science because the data output can be
precisely measured and defined [57]. OS have
been used for detection of VOCs in exhaled
breath [66]. Although the OS are typically more
Table 4. Commercially available electronic noses.
Selectivity sensors
Type of model
Manufacturer
Ref.
Gas sensor array
i-PEN, PEN3
GEMINI
EOS 835, EOS Ambiente
Cyranose® 320
MOSES II
Fox 2000, 3000, 4000, rq box, prometheus
Air Quality Module
OMD 1.10
QCS
FF2, GFD1
Artinose
Airsense analytics
Alpha MOS
Sacmi
Smith Group
GSG Mess- und Analyseneräte
Alpha mos
Appliedsensor
Dr. Foedisch AG
Gerstel GMBH & Co. KG
RST-Rostock
Sysca AG
Chemsensing
Illumina
Scentrak
Microsensor systems Inc.
Scensive technologies Ltd
[201]
Metal oxide semiconductor
Colorimetric
Fluorescence
Onose
Electrochemical
Conducting polymers
Cw sentry 3g
Bloodhound st214
[202]
[203]
[204]
[205]
[202]
[206]
[207]
[208]
[209]
[210]
[211]
[212]
[213]
[214]
[215]
Data taken from [58].
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Bioanalysis (2013) 5(18)
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Detection of volatile organic compounds as biomarkers in breath analysis
| R eview
Table 5. Selected applications of e-nose in breath analysis.
Sensor type
Diseases
Analyte
LOD
Optical sensors
–
Ethane, pentane, heptane,
octane, decane, benzene,
toluene, styrene
Hydrogen sulfide, butyric
acid, valeric acid
Acetone
0.8 pmol l , for
heptane, to 9.5 pmol l-1,
for decane
–
[66]
–
[68]
–
[69]
–
[70]
–
[71]
[62]
Quartz microbalance coated with metalloporphyrin Halitosis
complex with different metals
Quartz microbalance coated with a thin film
Diabetes
prepared on a ceramic wafer (Al2O3) with Pt and Au
Quartz crystal microbalance with conducting
–
polymers
Ref.
-1
Quartz crystal microbalance with a thin film of
polyaniline
Quartz crystal microbalance coated with
molecularly imprinted polymers
–
–
Acetic acid, butyric acid,
ammonia, dimethyl amine,
benzene, chlorobenzene
Ethanol, methanol,
1-propanol, 2-propanol
Toluene, p-xylene
Quartz crystal microbalance coated by molecular
films of metalloporphyrins
Asthma
Volatile organic compounds
–
Metal oxide semiconductor
Metal oxide chemiresistors
Diabetes
Homeostatic
balance control
Prostate cancer,
breast cancer
Asthma
Malignant pleural
mesothelioma
Bronchogenic
carcinoma
Chronic obstructive
pulmonary disease
Acetone
Acetone, ammonia,
methanol
1,2,4-trimethylbenzene,
2-ethylhexanol, n-octane
Volatile organic compounds
Volatile organic compounds
Low to 20 ppbv
–
Chemiresistors based on gold nanoparticles
Cyranose® 320 (with 32 polymer sensors)
complex than other sensors, they provide different measurement possibilities [57]. Additionally, the interaction of electromagnetic radiation
with matter occurs in a wide frequency range.
The main parts of the construction of OS are
a light source, optical elements (mirror, lenses,
prisms, diffraction gratings, etc.) and detectors
[66]. Interaction between the light source (often
LED) and volatile molecules results in effects
that can be measured by absorbance [58], reflectance [76] and refractive index [66,77–83] investigation. Other effects regarding colorimetric signals
[58,84] or chemiluminescence [85] have also been
observed.
Optical fiber detectors of chemical compounds are interesting solutions. A typical
optical fiber consists of two layers: a core that
is used to transduct the light signal and cladding (­Figure 5). In chemical detectors the core
(deprived of cladding) is covered by a chemically
selective receptor layer, which is responsible for
the change of chemical information concerning
the state of a sample. It results in the change
in the absorption spectrum of the layer. Therefore, the evanescent light wave that propagates
future science group
[67]
[72,73]
[74]
–
[75]
–
–
[59]
Volatile organic compounds
–
[60]
Volatile organic compounds
–
[63]
[61]
above the core surface (e.g., in the receptor layer)
is absorbed. This generates the changes in the
spectrum of light transmitted through the fiber,
generating an analytical signal [66].
The output signals are detected by different
sensors, including photodiodes, charge coupled
devices [86,87] and complementary metal oxide
semiconductors [58].
The most direct way is to measure the
absorbance of the detected analyte in a specific frequency range. Detection of gases such
as hydrogen, oxygen or hydrocarbons is one of
many good examples [76]. However, the system
is insensitive in detection of other compounds
at low concentrations. A simpler solution is to
measure the color change of an indicator such as
metalloporphyrins. A thin-film layer containing
dye molecules is used as the sensitive indicator
in colorimetric sensors. Dye color changes as a
result of the impact of chemical molecules on the
film, and the RGB value of the color is analyzed
by computer software [57,58].
In many cases, the results obtained in the tests
utilizing optical sensors are comparable to results
obtained by other methods, such as GC–MS [66].
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Buszewski, Grzywinski, Ligor, Stacewicz, Bielecki & Wojtas
Analyte
(volatile organic compounds)
Core
er layer)
(transduc
Cladding
Sensitive thin film (receptor layer)
Figure 5. Scheme of an optical fiber.
All in all, optical sensors are a great alternative
to creating new laboratory equipment, as they
are characterized by short analysis time, high
sensitivity, immunity to electrical and magnetic
interferences, low cost of analysis and small size
of the apparatus. A typical example of sensor
technology in breath test is capnography, which
is used in general anesthesia.
Silva et al., in a large number of scientific studies, suggested applying optical fibers to detect
VOCs [66,77,78,83]. Eight VOCs – ethane, pentane,
heptane, octane, decane, benzene, toluene and
styrene – were monitored in human breath for
clinical diagnosis. Detection limits ranging from
0.8 pmoll-1 for heptane and 9.5 pmoll-1 for decane, coupled with linear range and stability of the
analytical signal, perfectly show that the optical
fiber might be successfully used in de­termination
of volatile molecules in the air [66].
Quartz crystal microbalance
Interaction of some sensors with the relevant
analytes can lead to mass changes. This phenomenon was termed the piezoelectric effect
[57,88]. The use of a piezoelectric crystal initiated
the development of a microbalance mass sensor.
In such devices the signal is generated by the
adsorption of the analyte molecules to the sensor
surface [69].
Coatings of the sensor surface play an important role in the detection of specific chemicals.
The most commonly used include polymeric
fibers [89]. Khot et al. applied a microbalance
made of two silver electrodes placed centrally
on both sides of a crystal and a binder Cr [90].
The surface of the microbalance was coated with
regular poly(3-hexyl thiophene).
The selectivity and sensitivity of quartz crystal microbalance (QCM) dependent on receptor
surface coverage, as well as on its thickness and
method of deposition. The main advantages
of mass sensors are simple design, small size
and low power input. However, disadvantages
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Bioanalysis (2013) 5(18)
such as low specificity, short sensor service life
and difficulties in designing sensor elements
significantly limit the use of QCM. Khot
et al. [90], Lu et al. [69] and Andreeva et al. [91]
applied QCM coated with different polymers
for detecting VOCs. A chemical sensor coated
with thin film of b-cyclodextrin for detecting
benzene, toluene and xylene was developed [92].
A sensor system with five piezoelectric detectors
(four for measuring and one as the reference)
has been developed by Xu et al. for chemical
vapor identification [93]. Moreover, piezoelectric
sensors have been used to detect amines and
acetaldehyde [94]. Fleischer et al. proposed to
measure acetone concentrations in breath of
diabetes patients using eight QCMs [68]. Quartz
microbalance might be applied to monitor the
concentrations of hydrogen sulfide, butyric acid
and valeric acid in exhaled breath. These three
compounds are postulated to be biomarkers of
halitosis [67]. Pennazza proposed a prototype
e-nose consisting of seven quartz microbalances. The surface of each microbalance was
coated with different metalloporphyrin complexes of the following metals: copper, cobalt,
zinc, manganese, iron, tin and chromium.
Metal oxide semiconductors
Metal oxide semiconductors (MOS) are another
example of sensor systems used to detect important breath gases (CO and NOx) [95]. NOx is an
important marker for investigation of asthma
and its therapy control. For these sensors the
receptor layer is made of metal oxides (Figure 6).
ZnO [96], WO3 [72,97], TiO2, In2O3 and CuO [74]
are the metal oxides that are used for selective
detection of volatile compounds.
The presence of VOCs changes the conductivity of oxide on the semiconductor surface as
a result of a redox reaction [74]. The presence
of reducing gases such as hydrogen or hydro­
carbons reduces the density of oxygen atoms,
leading to an increase in conductivity. Conversely, increasing oxygen concentration in a
gas mixture leads to a decrease in conductivity.
The selectivity of MOS can be determined by
metal oxide electronic structure [98]. There are
two groups of electronic structures: transitionmetal oxides and nontransition metal oxides.
The most important parameters of MOS
that are responsible for conductivity change
are surface-modification and microstructures
of receptor layers, reduction reactions, temperature and humidity [98]. Konvalina and
Haick studied the influence of humidity on a
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Detection of volatile organic compounds as biomarkers in breath analysis
nano-film-coated receptor layer in laboratory
and real-world applications [75]. Prabhakar et
al. applied porous calcium chloride to adsorb
water and to control humidity [99]. This solution
made it possible to reduce the relative humidity
in breath samples from 95 to 29%. However,
compared to the mass and optical sensors, the
metal oxide semiconductors are characterized by
lower sensitivity and selectivity. Righettoni et al.
proposed to use a thin film of Si-doped WO3 for
detection of acetone in diabetes patients’ breath
[72]. This type of chemiresistor enables fast measurement of low acetone concentrations, down
to 20 ppbv (at up to 90% relative humidity and
325–500°C operating temperature). Although
the study was carried out under ideal conditions
(without water vapor influence), one can conclude that the chemiresistive detectors can be
a great alternative in rapid clinical trials. Luo
et al. performed a similar study using a film of
gold nanoparticles 2 nm in diameter for selective
detection of acetone in human breath [73].
| R eview
MOS film
(receptor layer)
Pt or Au electrodes
Heater
+
–
Alumina substrates
Analyte
+
–
MOS film e.g., ZnO
Heater
Al2O3 substrates
A
Figure 6. Metal oxide semiconductor sensors.
MOS: Metal oxide semiconductor.
Polymer sensors
Polymer surfaces are another example of sensors
used in electronic noses. Volatile compounds,
gases and odors selectively adsorbed on the surface result in a change of conductivity that can be
monitored. The choice of a polymer depends on
the analyte, its physicochemical properties and
structure. The number of polymers that are suitable for breath molecule detection is systematically growing, mostly through numerous modifications. Polypyrrole, polythiophene, polyindol,
polyaniline and polyfuran are the most popular
conducting organic polymers, which have been
used to detect volatile molecules of exhaled air
[100–102]. Silva et al. applied a sensitive film of
poly(methyl[3,3,3-trif luoropropyl] siloxane)
to detect volatile chemical compounds [66,79].
Dragonieri et al. used a commercial electronic
nose (Cyranose 320) consisting of 32 different
polymer sensors to detect malignant pleural
mesothelioma markers [59]. Kukla et al. reported
application of three polymer films (polyaniline,
polypyrrole and poly-3-methylthiophene) for the
analysis of nine VOCs [100]. The results show that
the highest value of response signal factor to analyte was achieved with poly-3-methylthiophene
[100]. On the other hand, Strand et al. suggest
using polypyrrole for measurements of VOCs in
breath because it is easy to produce and exhibits
high sorption efficiency [103]. However, the real
applicability of polymeric sensors is difficult to
define as their use is complicated.
future science group
Laser spectroscopy technique
Laser absorption spectroscopy is based on interaction between light and the medium. The
absorption level is determined by measurement
of radiation attenuation passing through the
medium (Figure 7). This attenuation is described
as a decrease in radiation power registered by a
detector. There are also other detection techniques using different light-material interactions
that cause, for example, temperature changes,
acoustic wave generation and modulation, generation of the electric current or charge in the
medium (optogalvanic spectroscopy), and so on.
Various quantum transitions that take place
between energy levels of a molecule determine
its absorption spectrum. Quantum transitions
between the electronic states usually correspond
to the short wavelength range (UV) [104]. Small
wavelength scale fluctuations of the cross-section within this band reflect the transitions
Absorber
Photoreceiver
Photodetector
Laser
I<Io
Io
Preamplifier
Signal processing
system
L
Figure 7. The absorption technique.
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Buszewski, Grzywinski, Ligor, Stacewicz, Bielecki & Wojtas
100
Absorption (%)
80
NO (1 ppmv)
H2O (500 ppmv)
Selected
absorption line
60
40
20
0
5242
5250
5258
5263 5266
5274
5282
Wavelength (nm)
Figure 8. Selection of absorption line for nitric oxide detection.
between molecular electro-vibronic levels. In
normal conditions, when collisional and Doppler broadening takes place, these individual
transitions are poorly distinguishable.
At the IR wavelengths, the absorption bands
correspond to transitions between molecular
vibronic levels of ground electronic state [105].
This absorption band is a good fingerprint of the
compound (see the example in Figure 8).
Table 6. LOD for major biomarkers of metabolic disorders in breath.
Laser spectroscopic
techniques
Biomarkers
LOD
Calos
Calos
Calos
Calos
Calos
Crds
Crds
Crds
Crds
Crds
Crds
Pas
Pas
Pas
Tdlas
Tdlas
Tdlas
Tdlas
Tdlas
Tdlas
Tdlas
Tdlas
Tdlas
Carbon dioxide
Carbonyl sulphide
Ethane
Formaldehyde
Nitric oxide
Ammonia
Acetone
Carbon dioxide
Methylamine
Dimethylamine
Nitric oxide
Ammonia
Carbon dioxide
Formaldehyde
Acetaldehyde
Ammonia
Ammonia
Carbon monoxide
Carbon dioxide
Carbonyl sulphide
Ethane
Formaldehyde
Nitric oxide
3.778 %
4.9 ppmv
270 pptv
2 ppbv
7 pptv
914 ppbv
0.2 ppbv
3 ppmv
2.3 ppmv
10 ppmv
0.7 ppbv
100 ppmv
7 ppbv
3 ppbv
80 ppbv
50 ppbv
3 ppbv
0.5 ppmv
0.5 ppmv
1.2 ppbv
0–12 ppbv
1.2 ppmv
2 ppbv
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Bioanalysis (2013) 5(18)
Ref.
The spectrum-based procedure of absorption line selection is illustrated below. The
pro­cedure is explained using the case of NO x
detection. Such analyses take into consideration
the radiation sources (quantum cascade lasers)
and interference of water vapour. For NO, H2O
is the main compound that can interfere with
the measurement, hence why the wavelength of
5.282 µm was chosen [106].
Intensity of radiation passing through the
absorber (Figure 8) can be determined using the
Lambert–Beer law:
I^ mh = I0 ^ mh exp6- a^ T, mh L @
[117]
Equation 1
[119]
where I0(l) is the intensity of incident radiation
at the wavelength of l, L is the length of the optical path in the absorber, T is the temperature of
the medium, a denotes the absorption coefficient
determined by the concentration of absorbing
molecules N, and the absorption cross-section.
The relation between the absorption
coefficient and the line strength S(T ) is given by:
[116]
[120]
[121]
[122]
[123]
[124]
[125]
[126]
[127]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
[134]
[135]
[136]
[137]
[138]
a^ m,Th = NS(T)g^ m - m0h
Equation 2
where g(l - l0 ) denotes the normalized line
shape:
3
0
# g^ m - m0h dm = 1
Equation 3
which includes the effects of line broadening
due to Doppler and collision phenomena. In
practice, the characteristic parameter for a given
gas is absorption cross-section described by:
v^ m,Th = S^ Th g^ m - m0h
Equation 4
future science group
Detection of volatile organic compounds as biomarkers in breath analysis
Its value can be determined in a laboratory;
however, for many compounds the parameters
of their spectra are available in commercial
databases.
Values of these parameters depend on the
temperature T, which is also related to elastic
and non-elastic collision effects. Therefore, S(T )
is well determined for a certain medium (air
usually), as well as its composition (humidity)
and pressure.
For breath molecules the typical absorption
cross-section values are from s ~10-20 to ~10 ‑18
cm 2 . For medical applications the detection
limit should be at the level of a single ppmv
(1013 cm-3) or lower, even at the level of parts of
ppbv (N < 1010 cm-3) (Table 6). In the most profitable circumstances the absorption coefficients
are a = N ×s << 10 -4 cm-1 and the described
detection limits are not reached. Due to that,
high-sensitivity laser spectroscopy techniques
must be applied.
One of these methods is tuneable diode laser
absorption spectroscopy (Figure 9). In this setup,
a small sinusoidal modulation of the laser emission frequency is applied. Thereby, the absorption signal is also modulated, and the signal at
the detector has the time-dependent form. With
a lock-in amplifier operated at twice the modulation frequency, the second derivative of the
absorption spectrum is analyzed.
The main advantage of tuneable diode laser
absorption spectroscopy over conventional spectrophotometry is that this method offers selectivity and possibility of in situ measurements.
The detection limit is of 10-4 –10-6 cm-1. However,
higher sensitivity can be obtained with the use of
other techniques, such as multi-pass cell spectroscopy, cavity ring-down spectroscopy (CRDS),
and photo-acoustic spectroscopy (PAS).
CRDS is the most sensitive method of
absorption measurement. Its idea is presented
in F­i gure 10 [107–110]. A radiation pulse with
| R eview
Photoreceiver
Laser
L
Frequency
modulator
Lock-in
amplifier
PC
computer
Figure 9. Tuneable diode laser absorption spectroscopy.
intensity of Io is introduced to the optical cavity
(resonator). The cavity is built of two mirrors
characterized by very high reflectivity R. Due
to multiplication of reflections inside the cavity,
the radiation is trapped.
Changes of the radiation intensity in the
cavity can be described by the equation:
dl = - I0 c c^1 - R h + ac m
L
dt
Equation 5
Solution of this equation shows that the radiation intensity decreases exponentially in the
cavity. The radiation quenching is measured by
registration of the radiation leaking through one
of the mirrors:
I ^ t h = I 0 e;
6^1 - R h + aL @c
E
L
t
= I0 e- x
A
Equation 6
where tA is a decay of radiation in the cavity
(called cavity ring down time) determined from
the formula:
xA =
L
c6^1 - R h + aL @
Equation 7
For the ‘clear’ cavity (a = 0) the formula is:
x0 =
L
c^1 - R h
Equation 8
R
R
Optical cavity
Signal
(arbitrary units)
L
Laser pulse
τA
τO
Time (µs)
Figure 10. Cavity ring down spectroscopy.
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Buszewski, Grzywinski, Ligor, Stacewicz, Bielecki & Wojtas
CW laser
OI
OM
Photoreceiver
Optical cavity
A/D
converter
Figure 11. Continuous-wave cavity ring down spectroscopy setup.
CW: Continuous-wave; OI: Optical isolator; OM: Optical modulator.
Comparing both decay times, the absorption
coefficient and the absorber concentration can
be found:
a = Nv = 1 c 1 - 1 m
c xA x0
Equation 9
Using this technique, sensitivity better than
aL ~10 ‑9 cm-1 can be obtained. Cavity mirrors
of very high reflectivity R (often exceeding the
value of 99.99%) provide multiple reflections
of laser beam.
Spectral tuning is especially important for
the selective and sensitive detection of molecules at their rovibronic transitions. This
technique (called continuous-wave cavity ring
down spectroscopy [CW-CRDS]) was used
for gas detection in 1997 [111,112]. The schematics of the CW-CRDS setup is shown in
Figure 11. The use of CW lasers was possible
due to application of a laser beam modulator.
With this method, a detection limit of an
Absorption α
Acoustic
resonator
Laser beam
power P
Modulated P or λ
at f or f/2
Broadband
microphone
Figure 12. Concept of photoacoustic spectroscopy.
2300
Bioanalysis (2013) 5(18)
absorption coefficient at the level of 10 -14 cm-1
was obtained [113].
A modification of CRDS setup is the cavityenhanced absorption spectroscopy technique.
Due to the off-axis introduction of a laser beam
to the cavity in this technique, the reflected light
is spatially separated by n-beams, where n is the
number of the laser beam trips inside the cavity
[114]. The free-spectral range for such an off-axis
arrangement can be n times less than the freespectral range for an on-axis one. Due to that,
either the dense mode structure of low finesses
occurs or the mode structure establishment does
not happen at all. In this way, sharp resonances
of the cavity are avoided, so there is no problem
with laser modes and matching narrow absorption lines. This provides an opportunity to avoid
the problem of matching the laser line to cavity
and molecule resonances.
The cavity leak-out spectroscopy is a CW
variant of CRDS [115–117]. After an optical excitation of the cavity, the laser power is turned
off. The concentration of investigated gas is
determined based on measurement of the subsequent power decay of the radiation observed
by a photodetector. As usually, the gas sample is
placed in the cavity.
In the PAS, conversion of light energy into an
acoustic wave is utilized [118]. In this setup, when
the modulated light is absorbed in the medium,
the gas temperature is periodically changed and
the acoustic wave with modulation frequency is
observed (Figure 12). The wave is detected with
a very sensitive microphone.
Among the so-called in-situ methods, PAS
belongs to the most popular ones. The absorber
concentration in the investigated sample influences the level of photoacoustic signal. Its
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Detection of volatile organic compounds as biomarkers in breath analysis
| R eview
Executive summary
Breath analysis
„„
Analysis of a large number of volatile organic compounds in the search for biomarkers.
„„
Search for correlation between the volatile organic compounds and certain diseases.
„„
Future use as a rapid diagnostic test along blood and urine testing.
GC
„„
First technique used in breath analysis; due to the complexity of mixtures using GC somewhat limited.
GC–MS
„„
The most popular among breath analysis methods.
„„
GC–MS system successfully used for diagnosis of certain diseases, such as lung and breast cancers, diabetes, cystic fibrosis and
pulmonary tuberculosis.
„„
383 volatile organic compounds monitored in the breath using GC-MS.
„„
Sensitivity of the method at the level of a few ppb for the various components of exhaled air.
GC–TOF-MS
„„
Type of mass spectrometer to another mass analyzer (TOF) in which the ion separation occurs due to the differences in their TOF from
the ionization chamber to the detector within a long field-free vacuum tube.
„„
Factors influencing the identification: mass of ions, the length of the flight chamber, accelerating voltage and the electric charge.
„„
Main advantages of the TOF analyzer: high MS resolution and possibility of obtaining multiple spectra within a short time.
Selected ion flow tube-MS
„„
Uses a type of chemical ionization.
„„
Selected precursor ions formed by electron impact or a microwave discharge in helium carrier gas.
Proton transfer reaction-MS
„„
Technique very similar to SIFT-MS.
„„
Main difference: using of precursor ion to transfer a proton to gaseous compounds introduced to the precursor within the system.
„„
PTR-MS becoming a common, fast and sensitive method for the analysis of volatile organic compounds in human exhaled breath at ppb
or even ppt levels.
Ion-molecule reaction-MS
„„
IMR-MS analyzers consist of the ion source, two octopole separations systems, quadrupole mass separator, secondary electron multiplier
and gas inlet system.
„„
Krypton, xenon or atomic mercury gas used as a ion source.
„„
Successfully used for breath analysis in liver disease.
Electronic nose
„„
Optoelectronic sensors are used for detection of volatile organic compounds in exhaled breath, in environmental applications, in
industrial atmosphere and biological samples.
„„
Interaction between the light source and samples results in effects measurable by absorbance, reflectance, fluorescence, refractive
index, colorimetric signals and chemiluminescence.
„„
Quartz crystal microbalance: changes in adsorbed mass can be determined by detecting the resonant frequency of piezoelectric crystal.
„„
Metal oxide semiconductors: receptor layer made of metal oxides (ZnO, WO3, TiO2, In2O3 and CuO).
„„
Presence of volatile organic compounds changes the conductivity of oxide on the semiconductor surface as a result of a redox reaction.
„„
Important parameters of MOS: type of receptor layers, reduction reactions, temperature and humidity.
„„
Polymer sensors: number of polymers suitable for breath molecule detection is systematically growing, mostly through numerous
modifications.
„„
Choice of a polymer depends on the analyte, its physicochemical properties and structure.
Laser spectroscopy technique
„„
Laser absorption spectroscopy based on interaction of the light and the medium; this attenuation is described as decrease in radiation
power registered by a detector.
„„
Detection techniques use light-material interactions that cause temperature changes, acoustic wave generation and modulation,
generation of the electric current or charge in the medium.
„„
The main laser spectroscopy techniques described, that is, CALOS, CEAS, CRDS, PAS and TDLAS.
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Buszewski, Grzywinski, Ligor, Stacewicz, Bielecki & Wojtas
amplitude registered by the microphone is
given by:
A^ T, mh \ Po Na^ T, mh L m h
vV
Equation 10
where Po denotes average laser power, m is the
modulation coefficient of radiation, f is the frequency of modulation, V is the gas volume, and
h is the microphone efficiency. The sensitivity of
the PAS is approximately several ppbv.
Conclusion & future perspective
This paper discusses the theme of breath analysis as a rapid, useful and non-invasive diagnostic
method in a variety of clinical applications. It
predominantly focuses on the latest research
reports. Numerous international studies provide more and more information on potential
biomarkers of disease states. The known biomarkers point to a correlation between the components of exhaled air and disease. An increased
level of concentrations of many volatile organic
compounds in exhaled breath indicates the
onset or progression of a disease; for example,
the presence of acetone in the breath, associated with the smell of apples, may occur in the
course of untreated or poorly treated diabetes.
Therefore, VOCs provide valuable information
on health conditions, such as infection or metabolic diseases. Odors of bodily waste products
were used for centuries in diagnosis of certain
diseases; analysis of body fluids is fundamental
for present-day clinical chemistry and diagnosis. Today, as a result of emerging technologies
such as electronics, robotics and optics, there
are numerous techniques available. Standard
methods such as GC–MS and modern methods
such as PTR-MS and SIFT-MS were succinctly
described in the text. New developments, for
example, electronic noses or laser spectroscopy techniques, were also included here. This
wide range of sensitive analytical methods for
detection of biomarkers in the breath at the
ppbv or pptv levels can be applied to help the
process of medical diagnosis.
All of these analytical methods are presented
only in a positive aspect. However, the actual
use of the equipment for routine analysis poses
a lot of problems. Therefore, the GC–MS
method requires preconcentration of the sample, no real-time measurements are possible and
a single analysis is time-consuming. Although
SIFT, PTR and IMR-MS can be successfully
used both for qualitative and quantitative analysis, their disadvantages include lack of complete
profile recognition, being time-consuming,
impossibility of real-time measurements, no
single VOC identification, limited number of
components detectable, and no differentiation
of isomeric and isobaric ions. In turn, the actual
usefulness of sensors is difficult to assess, mainly
due to the lack of critical evaluation by professionals in this field. Additionally, sensor technology is regarded as a black box and the signal
is changeable in time
Unfortunately, a large number of breath tests
and the search for correlations between a disease
state and biomarkers are mostly only speculations and scientific attempts to solve this problem. Therefore, further studies are needed to
gather valuable knowledge in this field. Perhaps
one day breath analysis will be used as a rapid
diagnostic test, such as blood or urine tests.
Financial & competing interests disclosure
This research was supported by the National Centre for
Research and Development (project SENSORMED Nr
PBS1/A3/0/2012). The authors have no other relevant
affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the
manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
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201 Airsense analytics.
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202 Alpha MOS.
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203 Sacmi.
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204 Smith Group.
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205 GSG Mess- und Analyseneräte.
www.gsg-analytical.com
206 Appliedsensor.
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207 Dr. Foedisch AG.
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208 Gerstel GMBH & Co. KG.
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209 RST-Rostock.
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210 Sysca AG.
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211 Chemsensing.
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212 Illumina.
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213 Scentrak.
www.cogniscentinc.com
214 Microsensor systems Inc.
www.microsensorsystems.com
215 Scensive technologies Ltd.
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AJ, Peverall R, Ritchie GaD. 3.5-μm high-
Bioanalysis (2013) 5(18)
future science group