Download real-time mass spectrometric analysis of breath

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SCIENCE
Taking your breath away:
real-time mass spectrometric
analysis of breath
Breath is a mixture of nitrogen,
oxygen, carbon dioxide, water
vapour, inert gases, and a small
fraction of trace volatile organic
compounds (VOCs) in the parts
per million (by volume) to parts
per trillion range. Despite the low
concentrations, these trace species
reflect many processes occurring in
the body. Endogenous (internally
produced) VOCs can be produced
anywhere in the body, as the
products of normal metabolism
and those that have altered owing
to disease, and are transported
via the bloodstream to the lungs
where they are exhaled in breath.
The exhaled profile will represent
VOCs originating from the blood,
any VOCs contributed from the
airways, nose or mouth1, and also
those present from exogenous
sources such as inspired air. Analysis
of exhaled breath therefore has
the potential to be a non-invasive
prognostic or diagnostic of a
person’s state of health.
Breath presents an analytical
challenge as, by its very nature, it
is transient, humid and the VOCs
are present at trace levels. Further,
breath is not a homogeneous
sample. The first portion of an
exhaled breath is the ‘dead space’
air which is contained in the airways
and does not participate in gas
exchange. Following this is the
‘alveolar’ air from the deeper, gasexchanging region of the lungs
which is in equilibrium with the
blood. As the alveolar air contains
the highest concentration of bloodbased VOCs, it is the principal target
of most VOC analyses.
Recent work at the University of
Leicester has focussed on the move
to real-time sampling of breath
coupled to direct chemical ionization
time-of-flight mass-spectrometry. A
variant of proton-transfer reaction
mass spectrometry (PTR-MS)2 called
chemical ionization reaction mass
spectrometry (CIR-MS)3 has been
developed and applied to the realtime measurement of breath VOCs.
The modus operandi of PTR-MS is
the chemical ionization, by proton
transfer, of a gaseous sample inside
a drift tube under an electric field.2
The proton source is normally
protonated water, H3O+. The analyte
and H3O+ ions are introduced into
the drift tube, where proton transfer
will occur for those VOCs with a
greater proton affinity than water.
On the basis of proton affinity,
the major constituents of air and
breath (nitrogen, oxygen, carbon
dioxide) show no reaction with
H3O+ but many trace VOCs are
readily protonated, as shown in the
following equation.
VOC + H3O+
Figure 1a) – Breath sampling apparatus connected to the CIR-TOF-MS; b) the
breath sampling apparatus in action.4 It is worth noting that normally the breath
sampling apparatus is kept in an isothermal environment.
VOCH+ +H2O
The fixed length of the drift tube
provides a fixed reaction time for
the ions as they pass along the
tube: the ion residence (and thus
reaction) time can be measured
or calculated. If the proton donor
concentration is largely unchanged
by the addition of an analyte
sample, then measurement of
the (proton donor)/(protonated
acceptor) ion signal ratio allows
the absolute concentration of the
acceptor molecules to be calculated.
From the combination of reaction
kinetics (or calibration) with mass
spectrometry, it is possible to both
identify and quantify individual
organic gases on a relatively short
timescale and with a sensitivity that
can reach well into the pptV mixing
regime. Different proton transfer
agents, such as CH5+ and NH4+, as
well as the charge transfer agents
NO+ and O2+, can be used as reagent
ions, expanding PTR-MS to CIR-MS.3
The mass spectrometer used in all
this work is a time-of-flight mass
spectrometer that benefits from a
mass resolution (m/Δm) in excess
of 1000 Da, a theoretically limitless
mass range, and most significantly
is able to observe all mass channels
simultaneously.
Figure 1 shows the breath sampling
device constructed from off-theshelf components. It consists of
Figure 2 – Real-time signal of acetone (m/z 59) as measured for a single fully
exhaled breath.
Figure 3 – Real-time analysis of a single breath a) acetone showing enhancement
in a subject with diabetes and b) acetonitrile showing enhancement in a subject
who is a smoker.4
a microbial filter, a spirometer to
measure breath volume (see Figure
2) and a pair of two-way valves. In
general, repeated single full exhaled
volumes are sampled as they give
the best VOC profiles from alveolar
air (see Figure 2). Calibration tests
have shown that the sampling
apparatus does not significantly
denude a wide range of VOCs.
Example measurements made with
the PTR-TOF-MS instrument at the
University of Leicester are shown
in Figures 3 and 4. In Figure 3 the
output from real-time analyses
of a single breath are shown for
the compounds acetone and
acetonitrile. Acetone is enhanced
on breath according to blood sugar
levels and is a marker for diabetes.
Acetonitrile is a persistent marker
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SCIENCE
Figure 4 – A breath alcohol experiment showing how the oxidation of ethanol
to acetaldehyde and acetic acid in the body can be tracked by real time breath
sampling.4
Figure 5 – PCA plot of the first principal component (accounting for 59.44% of the
variability in the data) against the second principal component (23.04%) showing
how different bacterial species (see text) can be separated on the basis of 22 m/z
values measured in the culture headspace.
for cigarette smoke inhalation.
Acetonitrile from smoking is
equilibrated among the bodily fluids
(blood, total body water, and urine)
and that excretion occurs via both
exhaled breath and urine. The ability
of PTR-TOF-MS to non-invasively
monitor metabolic processes within
the body is illustrated with the use
of a simple breath alcohol test in
Figure 4. A time series experiment
was conducted where repeated
breath measurements were
made both before and after the
consumption of an alcoholic drink.
There is an initial enhancement
of ethanol from the oral ethanol
followed by a secondary peak from
the blood ethanol. The metabolic
oxidation of the alcohol can then
be followed in time through to
acetaldehyde and acetic acid.
More advanced work has been
undertaken looking at the
signatures of fungal and bacterial
infection. Figure 5 shows a principal
component analysis (PCA) of the
PTR-TOF-MS spectra of volatile
organic compounds released
from plates of bacterial cultures,
namely Haemophilus influenzae,
Streptococcus pneumoniae,
Staphylococcus aureus and
Pseudomonas aeruginosa. The PCA
suggests that you can separate
these individual bacterial species
on the basis of their emitted VOC
components. It is clear that bacteria
Figure 6 – PCA plot of the first principal component (49.94%) against the third
principal component (11.74%) showing the degree of separation between cystic
fibrosis children both with and without bacterial lung infection and healthy
children based on their measured breath VOC profiles (also shown in comparison
to ambient air measurements).
may grow differently within, for
example, the lung environment
compared to that of an agar plate.
Interestingly Figure 6 shows some
data taken from children with and
without Cystic Fibrosis. On the basis
of their breath VOC signatures,
it is possible to separate those in
remission from those that have
active lung infection. This data
set is taken from a small number
of subject (13 individuals) and so
must be treated with caution, but it
nevertheless serves as an illustration
of what can be achieved with
such real-time mass spectrometric
measurements of breath VOCs.
Beyond real-time mass
spectrometric determination of
breath being used alone as a medical
diagnostic, a new holistic approach
is being developed. As part of a new
facility in the emergency room at
the Leicester Royal Infirmary, the
breath measurements described in
this article are being coupled with a
range of cardiovascular non-invasive
diagnostics and advanced patient
spectral imaging. The data from this
instrument suite will be combined
to give an all-round non-invasive
view of patient status and develop
point of care devices.
In summary, CIR-TOF-MS offers
a multitude of possibilities for
fingerprinting mass spectrometric
signatures of VOCs on breath with
attendant potential for insights into
people’s health.
Elements of the article have been
adapted from reference.4
Paul S. Monks*, Kerry A. Willis and
Andrew M. Ellis
Department of Chemistry,
University of Leicester, Leicester,
LE1 7RH
*Author to whom correspondence
should be addressed
References
1. Wang, T.; Pysanenko, A.; Dryahina,
K.; Španěl, P.; Smith, D. Journal of
Breath Research 2008, 2, 1.
2. Blake, R. S.; Monks, P. S.; Ellis, A.
M. Chem. Rev. 2009, 109, 861.
3. Blake, R. S.; Wyche, K. P.; Ellis,
A. M.; Monks, P. S. Int. J. Mass
Spectrom. 2006, 254, 85.
4. Monks, P. S.; Willis, K. A. Education
in Chemistry 2010, In Press.