U.S. patent application number 11/935518 was filed with the patent office on 2008-03-13 for spectroscopic breath analysis.
Invention is credited to Graham Hancock, Robert Peverall, Grant Andrew Dedman Ritchie.
Application Number | 20080064975 11/935518 |
Document ID | / |
Family ID | 9920540 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064975 |
Kind Code |
A1 |
Hancock; Graham ; et
al. |
March 13, 2008 |
SPECTROSCOPIC BREATH ANALYSIS
Abstract
Methods and apparatus for the analysis of exhaled breath by
spectroscopy are disclosed. An optical cavity containing the
exhaled breath, typically comprising a pair of opposing high
reflectivity mirror, is used to implement a cavity enhanced
absorption technique. Pairs of .sup.12CO.sub.2 and .sup.13CO.sub.2
absorption lines suitable for use in spectroscopic breath analysis
are also disclosed.
Inventors: |
Hancock; Graham; (Oxford,
GB) ; Peverall; Robert; (Oxford, GB) ;
Ritchie; Grant Andrew Dedman; (Oxford, GB) |
Correspondence
Address: |
WESTMAN CHAMPLIN & KELLY, P.A.
SUITE 1400
900 SECOND AVENUE SOUTH
MINNEAPOLIS
MN
55402-3319
US
|
Family ID: |
9920540 |
Appl. No.: |
11/935518 |
Filed: |
November 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10486675 |
Feb 12, 2004 |
7300408 |
|
|
PCT/GB02/03826 |
Aug 16, 2002 |
|
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11935518 |
Nov 6, 2007 |
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Current U.S.
Class: |
600/532 ;
250/339.13; 356/437; 73/23.3 |
Current CPC
Class: |
G01N 33/497 20130101;
A61B 5/083 20130101; G01N 21/39 20130101; H01J 49/04 20130101 |
Class at
Publication: |
600/532 ;
250/339.13; 356/437; 073/023.3 |
International
Class: |
A61B 5/08 20060101
A61B005/08; G01J 3/00 20060101 G01J003/00; G01N 21/84 20060101
G01N021/84; G01N 33/497 20060101 G01N033/497 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 16, 2001 |
GB |
0120027.8 |
Claims
1-22. (canceled)
23. A method of using cavity enhanced absorption spectroscopy to
quantify one or more isotopically-labelled carbon compounds in a
sample, the method comprising the steps of: passing at least a
portion of said sample into an optical cavity; illuminating said
sample in said optical cavity with radiation emitted by an optical
source, wherein the optical cavity is arranged to allow radiation
emitted from the optical source to be repeatedly reflected and
retrace its path to excite a plurality of cavity modes; and
measuring a wavelength-dependent reduction in the intensity of
radiation in said optical cavity caused by variations in cavity
ringdown time caused by absorption of said radiation in said
optical cavity by said one or more isotopically-labelled carbon
compounds, thereby to quantify said one or more
isotopically-labelled carbon compounds.
24. A method according to claim 1 wherein said one or more
isotopically-labelled carbon compounds are compounds of carbon
13.
25. A method according to claim 1 wherein said one or more
isotopically-labelled carbon compounds comprise bacterial
metabolites.
26. A method according to claim 3 wherein said one or more
isotopically-labelled carbon compounds comprise bacterial
metabolites of urea.
27. A method according to claim 1 wherein said one or more
isotopically-labelled carbon compounds comprise enzyme
metabolites.
28. A method according to claim 1 wherein said one or more
isotopically-labelled carbon compounds comprise compounds
indicative of fat digestion.
29. A method according to claim 1 wherein said one or more
isotopically-labelled carbon compounds comprise carbon dioxide.
30. A method according to claim 1 wherein said one or more
isotopically-labelled carbon compounds comprise volatile organic
compounds.
31. A method according to claim 1 wherein said one or more
isotopically-labelled carbon compounds comprise at least one of the
group comprising: methane, alkanes, pentanes, methylpentane,
formaldehyde.
32. A method according to claim 1 wherein said sample is
breath.
33. A method of using cavity enhanced absorption spectroscopy to
quantify one or more nitrogen compounds in a sample, the method
comprising the steps of: passing at least a portion of said sample
into an optical cavity; illuminating said sample in said optical
cavity with radiation emitted by an optical source, wherein the
optical cavity is arranged to allow radiation emitted from the
optical source to be repeatedly reflected and retrace its path to
excite a plurality of cavity modes; and measuring a
wavelength-dependent reduction in the intensity of radiation in
said optical cavity caused by variations in cavity ringdown time
caused by absorption of said radiation in said optical cavity by
said one or more nitrogen compounds, thereby to quantify said one
or more nitrogen compounds.
34. A method according to claim 11 wherein said sample is
breath.
35. A method according to claim 11 wherein said one or more
nitrogen compounds comprise at least one of nitric oxide and
ammonia.
36. A method of using cavity enhanced absorption spectroscopy to
quantify isotopically-labelled water in a sample, the method
comprising the steps of: passing at least a portion of said sample
into an optical cavity; illuminating said sample in said optical
cavity with radiation emitted by an optical source, wherein the
optical cavity is arranged to allow radiation emitted from the
optical source to be repeatedly reflected and retrace its path to
excite a plurality of cavity modes; and measuring a
wavelength-dependent reduction in the intensity of radiation in
said optical cavity caused by variations in cavity ringdown time
caused by absorption of said radiation in said optical cavity by
said isotopically-labelled water, thereby to quantify said
isotopically-labelled water.
37. A method according to claim 14 wherein said sample is
breath.
38. A method of using cavity enhanced absorption spectroscopy to
detect the presence of bacteria in a sample, the method comprising
the steps of: contacting said sample with a metabolizable compound;
collecting gas produced by said sample; passing at least a portion
of said gas into an optical cavity; illuminating said gas in said
optical cavity with radiation emitted by an optical source, wherein
the optical cavity is arranged to allow radiation emitted from the
optical source to be repeatedly reflected and retrace its path to
excite a plurality of cavity modes; and measuring a
wavelength-dependent reduction in the intensity of radiation in
said optical cavity caused by variations in cavity ringdown time
caused by absorption of said radiation in said optical cavity by
metabolites of said compound, thereby to detect the presence of
said bacteria.
39. A method according to claim 16 wherein said metabolizable
compound is an isotopically-labelled carbon compound
40. A method according to claim 16 wherein said metabolizable
compound is an isotopically-labelled compound of carbon 13.
41. A method according to claim 16 wherein said metabolizable
compound is isotopically-labelled urea.
42. A method according to claim 16 wherein said metabolites of said
compound comprise bacterial metabolites of urea.
43. A method according to claim 16 wherein said gas is collected
from breath.
44. A method of using cavity enhanced absorption spectroscopy to
detect the presence in a sample of at least one compound indicative
of one of: explosives, nerve gas, natural gas deposits, and oil
deposits, the method comprising the steps of: passing at least a
portion of said sample into an optical cavity; illuminating said
gas in said optical cavity with radiation emitted by an optical
source, wherein the optical cavity is arranged to allow radiation
emitted from the optical source to be repeatedly reflected and
retrace its path to excite a plurality of cavity modes; and
measuring a wavelength-dependent reduction in the intensity of
radiation in said optical cavity caused by variations in cavity
ringdown time caused by absorption of said radiation in said
optical cavity by said at least one compound, thereby to perform
said detection.
Description
[0001] The present invention relates to apparatus for analysis of
exhaled air by spectroscopy, and to methods of operation and uses
of such apparatus. Particular embodiments of the invention may be
used, for example, to measure the amount of volatile organic
compounds present in human breath. Other embodiments may be used to
measure .delta..sup.13C in exhaled breath pursuant to the .sup.13C
urea breath test.
[0002] Human and animal breath contains hundreds of different trace
volatile organic compounds (VOCs), in addition to the usual large
amounts of H.sub.2O and CO.sub.2. The metabolic pathways leading to
the generation of these VOCs are mostly little understood. However,
much effort has been recently expended correlating the presence of
particular VOCs with particular diseases, and breath analysis may
yet prove to be a useful and routine procedure for assisting
clinicians.
[0003] A clinical procedure which currently makes use of breath
analysis is the .delta..sup.13C urea breath test. This test can be
a helpful tool for clinicians seeking to diagnose the presence of a
Helicobacter pylori infection in the human gut, which is commonly
associated with gastric ulcers and carcinoma. A patient receives an
oral dose of urea having a known enhanced level of the .sup.13C
isotope. Colonies of Helicobacter pylori, which secrete a urease
enzyme, hydrolyse the [.sup.13C] urea to .sup.13CO.sub.2 and
ammonia. The .sup.13CO.sub.2 enters the bloodstream and is
subsequently exhaled. ".delta..sup.13C" is a parts per thousand
expression of the enhancement in the relative proportions of
.sup.13C and .sup.12C in a sample over a standard or background
level.
[0004] A common technique employed in the measurement of
.delta..sup.13C in exhaled breath is isotope ratio mass
spectrometry (IRMS). This technique distinguishes between
isotopomers of a molecular species, for example
.delta..sup.13CO.sub.2, by the mass/charge ratio of ions of the
species. The technique is limited by the existence of two CO.sub.2
isotopomers with an atomic mass of 45, namely
.sup.13C.sup.16O.sub.2 and .sup.12C.sup.16O.sup.17O, as well as by
sample contamination with .sup.12C.sup.16O.sub.2H. Furthermore, the
technique generally requires high vacuums, low impurity levels, and
expensive and bulky equipment.
[0005] A number of spectroscopic methods have been proposed as
alternative techniques for determining .delta..sup.13CO.sub.2.
These methods exploit the differences in the distributions of
rotational and vibrational energy states between .sup.12CO.sub.2
molecules. A number of such techniques, including nondispersive and
fourier transform infrared techniques are mentioned in "Precision
Trace Gas Analysis by FT-IR Spectroscopy. 2. The .sup.13C/.sup.12C
Isotope Ratio of CO.sub.2", M. B. Esler et al., Analytical
Chemistry 72, No. 1, 2000.
[0006] Methods currently used for detecting volatile organic
compounds in breath analysis were reviewed by W-H Cheng and W-J Lee
in "Technology development in breath microanalysis for clinical
diagnosis", J Lab Clin Med 133, No. 3, 1999. The techniques
mentioned include gas chromatography, mass spectrometry, fourier
transform and nondispersive infrared spectroscopy, the selected ion
flow tube and surface acoustic wave techniques, chemiluminescence
and colorimetry.
[0007] The techniques mentioned above have various disadvantages,
particularly when an inexpensive, compact and robust apparatus for
clinical use is sought. Mass spectrometry requires bulky and
expensive equipment operating with high vacuums and voltages. Gas
chromatography relies on the use of specially prepared separation
capillaries, may be slow, and is insensitive to isotopic
differences. The various infrared spectroscopic techniques are
limited by very low IR absorption rates resulting from low
concentrations of the target molecule in small experimental
volumes, thereby requiring long experiment duration and expensive
detectors and post processing circuitry to yield satisfactory
results.
[0008] The present invention seeks to address these and other
problems of the related prior art. The present invention provides
exhaled breath analysis apparatus for quantifying the presence of
one or more target substances in exhaled breath comprising:
[0009] a cavity enhanced absorption assembly comprising an optical
cavity coupled to an optical source operable to emit radiation and
an optical detector configured to generate a signal in response to
illumination by said radiation;
[0010] a breath collection assembly arranged to pass at least a
portion of said exhaled breath into said optical cavity for
illumination by said radiation; and
[0011] a data processor connected to said optical detector and
adapted to quantify the presence of said one or more target
substances in said optical cavity by the contribution to said
signal made by absorption of said radiation by said target
substance.
[0012] The term "cavity enhanced absorption" is used in this
document to refer to techniques whereby the signal available due to
spectroscopic absorption by a target substance present in an
optical cavity is enhanced through repeated reflection of the
radiation within the cavity. The repeated reflection increases many
times the effective absorption path length of the substances
present within the cavity, so that trace components in gas phase
are much more easily detected and their presence quantified.
[0013] An optical cavity is usually provided by two optically
opposed high reflectivity mirrors (typically greater than 99%), and
is characterised in that light within the cavity repeatedly
retraces some or all of its optical path, leading to resonance,
interference and observable energy density build up. Thus optical
cavities are fundamentally different in nature and construction to
optical multipass cells which are not resonant and in which careful
alignment of mirrors permits a light beam to follow an extended,
but well defined path between the entry and exit windows of the
cell.
[0014] The use of an optical cavity within a cavity enhanced
absorption assembly enables an extended optical path length to be
achieved within a far more compact and lightweight breath analysis
apparatus than could be achieved using an equivalent optical
multipass cell. The resulting apparatus is also easier to set up
and align.
[0015] A number of different cavity enhanced absorption techniques
are known in the art. Some of these are discussed in "Cavity
Enhanced Absorption of Methods at 1.73 .mu.m" by H. R, Barry et
al., Chemical Physics Letters 333 (2001) 285-289. In cavity
ringdown techniques an optical resonance is built up in an optical
cavity before the optical source, typically a pulsed or
intermittently operated continuous wave laser, is turned off. The
decay time of the cavity resonance, which depends on both the
properties of the cavity and the absorptive properties of gas phase
components within it, is then measured.
[0016] Instead of using an intermittent source, a continuous wave
source may be used and the level of resonance continuously
measured. In preferred embodiments of the present invention a
tunable laser, or more particularly a tunable continuous wave laser
diode source is scanned in frequency, using a frequency controller
or sweep generator. By scanning the optical source sufficiently
quickly to limit the overlap between the source frequency and each
natural cavity mode to a timescale shorter than the ringdown time
of the cavity, resonant peaks in the output signal due to natural
cavity modes are largely avoided. The optical source and one of the
cavity mirrors may also be simultaneously modulated to randomise
the occurrence of cavity modes which are then lost when averaging
the signal over a number of frequency scans of the source.
[0017] Instead of scanning, discrete frequencies could in principle
be used, selected to include absorption lines of target
substances.
[0018] The breath collection assembly may include a mouthpiece,
typically with an ambient air inlet value and an outlet value,
although such mouthpieces are often treated as single-use or of
limited life, so that they will not necessarily be supplied with
the apparatus.
[0019] The data processor may typically comprise suitable signal
conditioning circuitry coupled to suitable signal processing
circuitry adapted to digitise the detector output and to pass it to
a digital computer for analysis. Usually with reference to the
control of the optical source, for example by a sweep generator,
the digital computer may be programmed to apply curve or peak
fitting algorithms to the spectral data to quantity the presence of
the target substance or substances.
[0020] The apparatus may be set up to detect spectroscopic
absorption lines of .sup.12CO.sub.2 and .sup.3CO.sub.2, for example
such lines at 1607.634 nm and 1607.501 nm, or at 1627.431 nm and
1627.334 nm, so that it can be used to measure the .delta..sup.13C
of a subjects exhaled breath. In this way a compact, economical and
reliable apparatus for clinicians carrying out the [.sup.13C] urea
breath test or similar procedures may be provided.
[0021] A blind detector, substantially identical to or having
substantially the same noise characteristics as the optical
detector but isolated from the optical cavity, may be provided as a
reference in order to improve the signal to noise ratio. This may
be done by modulating the signal input to a lock-in amplifier
between the optical detector and the blind detector using a Dicke
switch or similar arrangement. The Dicke switch is discussed in
Review of Scientific Instruments 17(7) (1946) p 268.
[0022] The invention also provides a method of quantifying the
presence of one or more target substances in breath exhaled by a
subject, the method comprising the steps of:
[0023] collecting said exhaled breath;
[0024] passing at least a portion of said exhaled breath into the
optical cavity of a cavity enhanced absorption assembly;
[0025] illuminating said exhaled breath in said optical cavity with
radiation emitted by an optical source;
[0026] generating a signal in response to the illumination by said
radiation of an optical detector coupled to said optical cavity;
and
[0027] analysing said signal to quantify therefrom the presence of
said one or more target substances in said optical cavity by the
contribution to said signal made by absorption of said radiation by
said target substances.
[0028] The invention also provides the use of cavity enhanced
absorption in the quantification of one or more target components
of exhaled breath, and the use of cavity enhanced absorption in the
detection of human and/or animal disease by quantification of one
or more components present in breath exhaled by a human or
animal.
[0029] The target components may include .sup.13CO.sub.2,
.sup.12CO.sub.2, and volatile organic compounds associated with
disease such as alkanes (associated with lung cancer), pentanes
(associated with implant rejection, breast cancer, arthritis,
asthma) and formaldehyde (associated with breast cancer)
[0030] Embodiments of the invention will now be described by way of
example only, with reference to the drawings, of which:
[0031] FIG. 1 shows schematically an exhaled breath analysis
apparatus embodying the invention;
[0032] FIG. 2 illustrates signal processing aspects of a first
preferred embodiment; and
[0033] FIG. 3 illustrates signal processing aspects of a second
preferred embodiment.
[0034] Referring now to FIG. 1 there is shown schematically an
exhaled breath analysis apparatus embodying the present invention.
A breath acquisition assembly 10 accepts exhaled breath from a
subject. The breath is passed to a gas handling system 12. The gas
handling system is arranged to pass requited portions of exhaled
breath at appropriate pressure, and if required, controlled
humidity and/or temperature to the optical cavity 102 of a cavity
enhanced absorption (CEA) assembly 100. An optical delivery system
104 feeds electromagnetic radiation generated by an optical source
106 into the optical cavity 102 and an optical detector 108 detects
electromagnetic radiation leaving the optical cavity.
[0035] The electrical signal from the optical detector 108 is fed
to a signal processing arrangement 200, and the resulting data is
fed to an analysis/display arrangement 300.
[0036] The breath acquisition assembly 10 may typically take the
form of a mouthpiece provided with an inlet valve for a subject to
draw in environmental or pre-purified air, and an outlet valve to
pass exhaled air to the gas handling system 20. The gas handling
system may be arranged to sample only alveolar air which has
entered sufficiently deeply into the subjects lungs, and not
dead-space air from the mouth, oesophagus and broncheoles. The gas
handling system may also ensure that exhaled breath passed to the
CEA assembly is at an appropriate pressure and falls within
particular ranges of temperature and humidity. An apparatus
suitable for the collection and handling of exhaled breath prior to
spectroscopic analysis, in particular for the detection of volatile
organic compounds, is described in "Method for the Collection and
Assay of Volatile Organic Compounds in Breath", M Phillips,
Analytical Biochemistry 247, 272-273 (1997).
[0037] The optical source 106 of the CEA assembly 100 may be
provided by a Continuous Wave (CW) laser diode 106, and in
particular by a Distributed FeedBack (DFB) laser diode or an
extended cavity laser diode. It is important that the optical
source remains stable and in single mode operation as it is scanned
in frequency across the spectroscopic absorption peaks of gas phase
species to be detected. Appropriate optical sources will typically
be temperature stabilised, and should be reasonably consistent over
their operational lifetime.
[0038] The optical source 106 is coupled to the optical cavity 102
by an optical delivery system 104, either directly, or by turning
mirrors, optical fibre or both. A Faraday rotator may be used to
isolate the optical source from back reflections from the optical
cavity 102, especially if a laser diode sensitive to optical
feedback is used. The Faraday rotator could be a discrete component
or an in-fibre device. Indeed, the entire optical delivery system
could be fibre based. Frequency mixing or doubling may be employed
to obtain radiation in the desired frequency range.
[0039] The optical cavity 102 is constructed from two or more
opposed high reflectivity mirrors in a stable geometry, typically
separated by a distance of the order of 0.1 to 1.0 m. Mirrors with
a reflectivity of about 99.9% are suitable for this application.
For a linear geometry cavity with a physical length of 0.5 m the
enhanced optical path length due to such mirrors is approximately
500 m. The optical cavity 102 forms part of a vacuum vessel so that
samples are not contaminated with environmental air.
[0040] Light exiting the cavity through one of the mirrors is
detected by the optical detector 108, typically a photo diode, and
preferably an InGaAs photodiode sensitive in the infrared.
[0041] Light injected into the optical cavity 102 by the optical
delivery system 104 undergoes many reflections within the cavity,
thus increasing the path length and hence the total absorption by
gas species present in the cavity. The optical source 106 is
scanned repetitively over the same spectral range to build up a low
noise spectrum within the cavity. Light is coupled into the cavity
whenever a resonance occurs between a source frequency and a cavity
mode. The mode structure of the cavity can be made as congested as
possible by mis-aligning the mirrors of the cavity slightly so that
many higher order modes can be excited. Coincidences between the
frequency of the optical source 106 and the cavity modes can be
further randomised by oscillating the cavity length or by
superimposing a jitter on the frequency scan of the source.
[0042] Preferably, over a single frequency scan of the optical
source, several tens of free spectral ranges of the cavity are
covered, and many cavity modes are sequentially excited. The time
constant of the optical detector 108 can be arranged such that
adjacent cavity modes are no longer discretely observed, so that in
a single frequency scan a relatively smooth signal is obtained.
Hundreds of sequential scans can be averaged together to increase
the signal to noise ratio and to let any randomisation processes
smooth mode structures which would otherwise be apparent in the
data.
[0043] Absorption by a target substance in gas phase within the
optical cavity 102 is detected by a decrease in the signal output
by the optical detector 108. At a particular frequency, the average
intensity of the signal is proportional to the ringdown time of the
cavity, and thus is inversely proportional to optical losses of the
cavity. It is important, when using this technique, that
significant radiation fields do not build up inside the cavity. If
they do, then rapidly fluctuating output spikes may occur at the
optical detector output as cavity modes come into resonance with
the optical source, which are detrimental to the smooth output
signal otherwise achieved by rapid frequency scanning of the
optical source 106.
[0044] In some embodiments the signal processing arrangement may
take the form shown schematically in FIG. 2. The signal from the
optical detector, following amplification and other signal
conditioning steps if required, is passed to an analogue to digital
converter 202 which digitises the optical detector signal and
averages over a number of frequency scans of the optical source
106. This averaging and other post processing may be carried out by
a digital signal processor 204. The precise specifications of the
analogue to digital converter 202 are not critical for the present
application. A 10 bit or 12 bit A/D converter should provide
sufficient accuracy for the present application. An 8 bit A/D
converter is likely to be insufficient. The sampling rates
available in such devices far exceed the frequency scan rates
likely to be used for the optical source, which may be 20-30
Hz.
[0045] If the signal to noise ratio in the optical detector signal
is too low for an accurate determination of absorption peak
features then several possible means of enhancement are available.
A digital signal processor 204 may be used to apply low pass
fourier transform filtering or Savitzky-Golay smoothing (which
could also be carried out by software in the analysis/display
arrangement 300.
[0046] The modulation of an optical source is frequently used in
optical engineering to improve the signal to noise ratio of a
subsequently detected signal. However, in the present application,
modulation of the optical source, for example by means of a
pre-cavity acoustic optic modulator, tends to alter the way in
which cavity resonance builds up in a non-linear manner. A more
suitable arrangement is to modulate the detector by switching
between it and another device of identical or very similar noise
characteristics.
[0047] In the lock-in amplifier arrangement of FIG. 3, the detected
signal is modulated between the optical detector 108 and an
adjacent identical but unilluminated optical detector 100, for
example using a Dicke Switch arrangement as discussed in R. H.
Dicke, Rev. Sci. Instrum., 17(7) (1946) p 268. As well as making
good use of the lock-in amplifier made up of an analogue to digital
converter 202 and a real-time digital signal processor 208, this
technique has the added benefit of rejecting ambient noise in the
signal through phase sensitive detection.
[0048] The analysis/display arrangement 300 is preferably provided
by a suitable digital computer, either as a suitable programmed
general purpose personal computer or as a dedicated computer, with
suitable input, output and data storage facilities. The output of
the signal processing arrangement 200 may be easily communicated to
the analysis/display arrangement 300 using methods familiar to the
person skilled in the art. Analysis of the output from the signal
processing arrangement is preferably carried out using software
specifically designed to apply a non-linear curve fitting procedure
to the spectral data with baseline, peak frequency and peak shape
as fitting parameters.
[0049] Specific absorption lines for the determination of
concentration of a target species within the optical cavity may be
selected using high resolution spectral data, available from the
literature or by use of known spectroscopic techniques. Target
spectral lines should be selected such that they do not overlap
with lines of other, non-target species likely to be present, such
as water or methane in the case of analysis of human breath
CO.sub.2.
[0050] If absolute concentration is to be measured then the target
absorption line should be chosen to be in close proximity with a
reference absorption line of a species of known concentration
within the sample. For the analysis of human breath methane is
suitable for this purpose, because it is present in the atmosphere
at a known concentration close to 1.6 parts per million by volume.
Suitably close proximity is within the normal frequency scan range
to be used to measure the target absorption line, typically about 1
cm.sup.-1, but far enough apart for the target and reference lines
not to overlap significantly. A calibrated ratio of the measured
strength of the target and reference absorption lines then provides
the concentration of the target species.
[0051] When isotopic ratios are to be measured, a pair of lines
should be chosen, one from each isotopomer, based on a maximisation
of the following criteria: [0052] (i) the two lines should be in
close proximity, as discussed above; [0053] (ii) the two lines
should be of similar intensity for a naturally occurring isotopic
ratio of the isotopomers; [0054] (iii) the two lines should not be
overlapped by other lines originating from the target molecule or
by structured absorptions from other sample constituents to a
significant or problematic extent; [0055] (iv) the ratio of
intensities of the two lines should not vary significantly with
temperature over any expected experimental temperature fluctuation.
For CO.sub.2 absorption, the lines may originate from rotational
levels in the ground state of CO.sub.2.
[0056] A particular configuration and use of the apparatus
described above will now be discussed. Helicobacter pylori is one
of the most common bacteria found in humans, and its presence has
been linked to the incidence of a variety of stomach diseases
including gastric ulcers and carcinoma. Colonies of this bacterium
in the human stomach can be detected non-invasively by the
measurement of isotopic ratios in CO.sub.2 in the exhaled breath of
patients following ingestion of .sup.13C labelled urea.
[0057] An exhaled breath analysis apparatus as described above may
be constructed or configured to measure the .sup.13C/.sup.12C ratio
by infrared spectroscopy on high overtone absorption bands of
CO.sub.2 using radiation from a laser diode near 6000
cm.sup.-1.
[0058] The diode laser optical source is scanned over a wavelength
range of 0.2 nm which encompasses one absorption line of each of
the isotopomers .sup.13CO.sub.2 and .sup.12CO.sub.2, for example at
1607.501 nm and 1607.634 nm respectively. The transitions giving
rise to these absorption lines are selected so that in a sample of
CO.sub.2 with a naturally occurring isotopic abundance the two
lines have approximately equal absorption intensities.
[0059] Other pairs of .sup.13CO.sub.2, .sup.12CO.sub.2 lines, in
vacuum nanometres, which are also suitable for this purpose
are:
[0060] 1596.978, 1596.869
[0061] 1597.241, 1597.361
[0062] 1597.512, 1597.361
[0063] 1606.997, 1607.142
[0064] 1608.014, 1608.057
[0065] The ratio of the two isotopic species may be measured as a
function of time following the ingestion of .sup.13C labelled urea
by a patient suspected of harbouring a Helicobacter pylori
infection. After ingestion of a standard 100 mg sample of .sup.13C
urea, a change in .delta..sup.13C of +5 after 30 minutes is
considered to be a positive test for the bacterium. The apparatus
may calibrate against an unelevated .sup.13C level using an
internal standard or by sampling ambient atmospheric air.
[0066] The described apparatus does not generally require a
separate reference cell. Wavelength calibration can be accomplished
by frequency scanning over a region which encompasses absorption
lines of various species, including the target line or lines, and
by recognition of the resulting absorption spectrum. For absolute
concentrations absorption levels can be compared with those of a
species of known concentration which is introduced into the sample,
or which is naturally occurring such as methane. For isotopic ratio
measurements, the levels of the two isotopomers present in a
reference sample, such as exhaled breath before ingestion of
.sup.13C labelled urea in the case of a .sup.13C/.sup.12C breath
test, can be used as a relative standard, and the apparatus
calibration can be checked from time to time with such reference,
or preprepared standard samples.
[0067] The apparatus may need to operate with a reduced gas
pressure in the optical cavity 102 in order to increase selectivity
by removing pressure broadening effects. However, such an
arrangement, which can be effected by the gas handling system 12,
should only have a minor effect upon the instrument
sensitivity.
[0068] The apparatus may also be used for CO.sub.2 isotopic
analysis to assist in the measurement of fat digestion in humans,
particularly infants, and observing delayed gastric emptying
associated with diseases such as diabetes and aids, and the
detection of specific enzymes associated with disease by supplying
the enzymes with .sup.13C labelled material.
[0069] The apparatus may also be used, if suitably constructed or
configured, to detect and quantify the presence in exhaled breath
of various other compounds including specific volatile organic
compounds. The first overtones of vibrational transitions of the
C--H bands of such compounds lie conveniently in the wavelength
range of commercially available diode lasers. For example, specific
alkanes have been detected at elevated levels in the breath of lung
cancer patients, in particular methylpentane. Other diseases such
as breast cancer, transplant rejection and asthma have been
associated with elevated levels of pentanes, and formaldehyde has
been observed at elevated levels in breast cancer patients.
[0070] The invention may be used in the detection of isotopically
specialled water (H.sub.2O/HDO), for example in determining human
body fluid status within applications such as dialysis treatment.
The effectiveness of dialysis treatment could also be monitored by
measuring exhaled NH.sub.3. Exhaled nitric oxide (NO) could also be
measured using the invention, for example in the monitoring of
asthma.
[0071] The spectroscopic apparatus described herein may be used for
a variety of applications other than the analysis of exhaled
breath, by providing an appropriate sample collection and/or
injection assembly, and by selecting appropriate target absorption
lines for detection. Other applications include the detection of
explosives and nerve gas, and detecting indications of the
proximity of oil and gas deposits through the isotopic makeup of
methane present in drilling mud.
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