Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVED IONISATION OF GASEOUS SAMPLES
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority from and the benefit of United Kingdom patent
application No. 1503876.3 filed on 6 March 2015, United Kingdom patent
application No.
1503864.9 filed on 6 March 2015, United Kingdom patent application No.
1518369.2 filed
on 16 October 2015, United Kingdom patent application No. 1503877.1 filed on 6
March
2015, United Kingdom patent application No. 1503867.2 filed on 6 March 2015,
United
Kingdom patent application No. 1503863.1 filed on 6 March 2015, United Kingdom
patent
application No. 1503878.9 filed on 6 March 2015, United Kingdom patent
application No.
1503879.7 filed on 6 March 2015 and United Kingdom patent application No.
1516003.9
filed on 9 September 2015.
FIELD OF THE INVENTION
The present invention generally relates to mass spectrometry, and in
particular, to
apparatus for improving the ionisation of a sample. Embodiments relate to
rapid
evaporative ionisation mass spectrometry, mass spectrometers, ion mobility
spectrometers, hybrid ion mobility mass spectrometers, methods of rapid
evaporative
ionisation mass spectrometry ("REIMS"), methods of mass spectrometry, methods
of ion
mobility, methods of hybrid ion mobility mass spectrometry, methods of
electrosurgery and
electrosurgical devices.
BACKGROUND
Rapid evaporative ionization mass spectrometry ("REIMS") is a technology which
has recently been developed for the real-time identification of substrates,
for example for
the identification of biological tissues during surgical interventions. REIMS
analysis of
biological tissues has been shown to yield phospholipid profiles having high
histological
and histopathological specificity, similar to Matrix Assisted Laser Desorption
Ionisation
("mALDr), Secondary Ion Mass Spectrometry ("SIMS") and Desorption Electrospray
Ionisation ("DESI") imaging.
Coupling of REIMS technology with handheld sampling devices has resulted in
iKnife sampling technology, which can provide intra-operative tissue
identification. This
technology allows surgeons to resect target tissues more efficiently, such as
tumours, intra-
operatively by providing information that can assist a surgeon in minimizing
the amount of
healthy tissue removed whilst helping to resect the target tissue. Knife
sampling
technology can also be used by non-surgical operators in non-surgical
procedures to
isolate target matter from an in vitro substrate.
Date Recue/Date Received 2024-04-10
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In a known iKnife sampling system, a mass spectrometric signal is obtained by
subjecting a substrate to alternating electric current at radiofrequency which
causes
localized Joule-heating and the disruption of cells along with desorption of
charged and
neutral particles. The resulting aerosol (e.g., "surgical smoke") is directly
introduced into an
atmospheric interface of an atmospheric pressure ionisation mass spectrometer
for on-line
mass spectrometric analysis. The aerosol contains a sufficient number of
ionised
molecules to allow the direct mass spectrometric fingerprinting of the
biological tissues.
Post-evaporative ionisation of neutrals molecules in the sample may be used to
enhance the ion yield. In this regard, electrospray and corona discharge post-
ionisation
methods were tested. Secondary electrospray ionisation, fused droplet
electrospray
ionisation and extractive electrospray ionisation have been used to increase
the ion yield.
These three techniques are similar in the sense that electrically charged
solvent droplets
are fused with aerosol particles in the gas phase and the resulting fused
droplets undergo
an electrospray-like ionisation process. However, these techniques suffer from
the
delicateness of electrospray setup, the sample carryover effects caused by
DESI-like
phenomena, electrospray-related restrictions on solvent type and flow rates,
and patient
safety considerations in human interventional environments due to the high
voltages
involved in these techniques.
It is also possible to enhance ionisation by facilitating the collision of the
aerosol
particles with a collision surface in the vacuum region of the mass
spectrometer. A
collisional ion generator method was developed and is disclosed in WO
2013/098642
(Medimass) in which the aerosol particles enter the analyser at the
atmospheric interface
and are accelerated into the vacuum region of the analyser in the free jet
regime. The
aerosol particles accelerated by the free jet are then directed onto a
collision surface,
causing the ion yield of the REIMS method to be enhanced.
However, despite this enhancement, a number of problems still remain. For
example, the ionisation yield for this technique remains relatively low. Also,
there may be a
lack of ionisation or suppression of analyte ion formation when
electrosurgical diathermy is
used in a coagulation mode. Also, there may be a lack of ionisation when
tissue having a
high neutral lipid content, such as triglycerides or diglycerides, is being
dissected (e.g.,
adipose tissue or breast tissue).
It is therefore desired to provide an improved method and apparatus.
SUMMARY
According to a first aspect there is provided a method mass spectrometry
and/or ion
mobility spectrometry comprising:
providing an analyte;
supplying a matrix compound to said analyte such that said analyte is diluted
or
dissolved in, or forms first clusters with said matrix; and
colliding said first clusters or first droplets of said diluted or dissolved
matrix with a
collision surface.
Date Recue/Date Received 2024-04-10
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WO 2013/098642 describes a method of generating and analysing an aerosol
sample. The aerosol sample generated comprises droplets that are covered with
polar
lipids. During analysis, the droplets are accelerated by free jet expansion in
the
atmospheric inlet of a mass spectrometer such that the droplets attain a high
velocity and
then impact onto a collision surface. This results in the production of
gaseous ions of the
polar lipid molecules. However, the ionisation yield of this technique is
relatively low.
It has been recognised that the ion yield in this method is relatively low due
to the
poor conversion rate of the droplets into individual molecular species, mostly
caused by the
strong intermolecular bonds between the analyte molecules.
According to embodiments of the present invention, the analyte is diluted by
the
matrix or dissolved into the matrix. For example, the analyte may be in
provided in the
form of droplets, aerosol or liquid and may be fused or coalesced with the
matrix, or
dissolved into the matrix. The matrix may be in the form of droplets, solids,
aerosol or
liquid when in contact with the analyte. Diluting, or dissolving the analyte
in the matrix,
may substantially eliminate or reduce intermolecular bonding between the
analyte
molecules. As such, when the diluted or dissolved analyte droplet is
subsequently collided
with the collision surface it fragments into smaller droplets, wherein any
given smaller
droplet is likely to contain fewer analyte molecules than it would if the
matrix were not
present. This leads to the more efficient generation of analyte ions.
It is thought that ionisation of the analyte predominantly occurs due to ionic
dissociation of the analyte in the solution phase, due to interactions with
counter ions
present in the sample being analysed. Diluting or dissolving the analyte in
the matrix
reduces the concentration of the analyte in each droplet and facilitates ionic
dissociation in
the solution phase, thus ultimately resulting in a greater proportion of the
analyte being
.. ionised. Accordingly, any matrix that dissolves or dilutes the analyte may
be used.
According to the various embodiments described herein, the step of colliding
said
first clusters or droplets with the collision surface may fragment the first
clusters or droplets
into a plurality of second smaller clusters or droplets. However, other
embodiments are
contemplated wherein other forms of droplet dissociation or disintegration are
used such
as, for example, laser irradiation, ultrasonic energy, glow discharge or
photoionization etc.
The step of providing the analyte may comprise providing the analyte in the
form of
a gas-phase analyte, aerosol, vapour, smoke or liquid; and/or said matrix may
be supplied
to said analyte whilst said analyte is in the form of a gas-phase analyte,
aerosol, vapour,
smoke, solid or liquid.
The method may form the first droplets by supplying the matrix to the analyte
whilst
the analyte is in the form of an aerosol, vapour or smoke and whilst the
matrix is in the form
of an aerosol, vapour or solid.
The step of supplying the matrix to the analyte may comprise supplying matrix
molecules to, and intermixing said matrix molecules with, said analyte whilst
said matrix is
.. in a gas phase or in the form of an aerosol, vapour or solid.
The mixture of said analyte and said matrix may be transferred from a high
pressure region to a low pressure region such that said gas phase matrix cools
and
Date Recue/Date Received 2024-04-10
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condenses to a liquid and said analyte dissolves in, or is diluted by, said
liquid matrix so as
to form said first droplets.
Alternatively, the matrix may be supplied to, and intermixed with, the analyte
as a
liquid.
If the analyte and/or matrix is in liquid form then the analyte and/or matrix
may be
converted into droplets or vapour, e.g., by spraying or nebulising. For
example, if the
analyte and matrix are mixed as liquids then the mixture may subsequently be
converted
into the first analyte droplets, e.g., by spraying or nebulising.
The matrix may initially be supplied as a solid (e.g., a powder) and
sublimated or
melted and evaporated so as to form matrix in vapour or gas-phase that is
intermixed with
the analyte. For example, a solid matrix may be mixed with the analyte. If the
analyte is
mixed in liquid form, the mixture may be allowed to dry, e.g., to form
crystals. The mixture
may then be heated to sublimate and/or evaporate the matrix and/or analyte.
Examples of
suitable matrices include MALDI matrices and other matrices, such as:
coumarin; 9-
.. aminoacridine; 2,5-dihydroxybenzoic acid; THAP; CHCA; and quecertin.
The matrix may be selected from the group consisting of: (i) a solvent for
said
analyte; (ii) an organic solvent; (iii) a volatile compound; (iv) polar
molecules; (v) water; (vi)
one or more alcohols; (vii) methanol; (viii) ethanol; (ix) isopropanol; (x)
acetone; (xi)
acetonitrile; (xii) 1-butanol; (xiii) tetrahydrofuran; (xiv) ethyl acetate;
(xv) ethylene glycol;
(xvi) dimethyl sulfoxide; (xvii) an aldehyde; (xviii) a ketone; (xiv) non-
polar molecules; ()oc)
hexane; (xW) chloroform; (xxii) butanol; and (xxiii) propanol.
By way of example, for analytes comprising polar lipids, low molecular weight
alcohols may be used as the matrix (e.g., methanol, ethanol, isopropanol) or
ketones (e.g.,
acetone). These matrices have been shown to enhance the ionisation of species
otherwise detected in the absence of the matrix vapours at lower intensity.
A protic matrix solvent may be used, e.g., for the analysis of lipids or
triglycerides.
Alternatively, a non-protic or aprotic matrix solvent may be used, e.g., for
the analysis of
proteins.
The mixture of analyte and matrix may be a homogeneous or heterogeneous
mixture.
The matrix and/or analyte may be doped with one or more additives for
enhancing
the solvation of the analyte in the matrix or for enhancing the ionisation of
the analyte.
The matrix or analyte may be doped with an acidic or basic additive. For
example,
the matrix may be doped with formic acid, diethylamine.
The matrix may cause derivatisation of the analyte. For example, the matrix
may
cause the derivatisatoin of cholesterol or steroids in the analyte. This may
render the
analyte more easily ionised.
The method may comprise adding a calibrant to the matrix and/or analyte, or
selecting a compound in the matrix and/or analyte as a calibrant, and using
the calibrant to
calibrate the method of mass spectrometry and/or ion mobility spectrometry.
This is
particularly useful in ambient ionisation techniques such as REIMS techniques,
in which it
may be difficult to introduce calibrants according to conventional techniques.
Date Recue/Date Received 2024-04-10
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The method may comprise analysing a lock mass species, e.g. by adding the
lockmass species to the matrix and/or analyte. The lockmass may then be used
to correct
the mass(es) of analyte(s). Alternatively, or additionally, the method may
comprise
analysing a lock mobility species, e.g. by adding the lock mobility species to
the matrix
and/or analyte. The lock mobility species may then be used to correct the
mobilities or drift
time(s) of analyte(s).
The step of colliding said first clusters or first droplets with said
collision surface
may comprise accelerating said first clusters or first droplets onto said
collision surface.
The step of accelerating said first clusters or first droplets onto said
collision surface
may comprise using a pressure differential to accelerate said first clusters
or first droplets
onto said collision surface.
A vacuum pump may be used to create a pressure differential between a first
region and a second region for accelerating the first clusters or first
droplets between the
two regions and onto the collision surface. The apparatus may comprise a mass
spectrometer and/or ion mobility spectrometer having an atmospheric interface
arranged
between the first and second regions, wherein the second region may comprise a
vacuum
chamber that is connected to the vacuum pump and which houses the collision
surface.
The combination of gas flow between the first and second regions (e.g., into
the
spectrometer) and matrix flow may nebulise the matrix, producing droplets of
the
appropriate diameter/volume. The nebulising gas flow may produce a shear force
on the
matrix, producing droplets that enter the sample transfer tube.
The method may comprise evaporating the matrix from the analyte in the second
clusters or droplets, resulting in free analyte ions that are separate from
said matrix.
The step of colliding the first clusters or first droplets with the collision
surface may
evaporate the matrix from the analyte by converting kinetic energy of the
analyte and
matrix into heat.
The step of colliding the first clusters or first droplets causes the second
smaller
clusters or droplets to be generated, at least some of which may have only a
single analyte
molecule therein. This enhances the ionisation process.
The dielectric constant of the matrix may be sufficiently high such that the
solvation
of the analyte in the matrix involves ionic dissociation resulting in solvated
ions of the
analyte present in the condensed phase. In these cases, the impact on the
collision
surface is more likely to produce solvated ions in the gas phase, which may
eventually
yield ions formed by deprotonation (in a negative ion mode, i.e. [M-1-1]-),
ions formed by
protonation (in a positive ion mode, i.e. [M+H]), and/or molecular ions.
Alternatively, or
additionally, ions may be formed by any one of: dehydration, deamidation or
alkaline metal
adducts.
The method may comprising subjecting said analyte or analyte ions to
ionisation
downstream of said collision surface. Optionally, the ionisation is performed
by an
ionisation source selected from the group consisting of: a corona discharge
ionisation
source; a reagent ion Ionisation source; a Photo Ionisation source; a Chemical
Ionisation
source; an Electron Impact ("El") ionisation source; a Field Ionisation ("Fr)
source; a Field
Date Recue/Date Received 2024-04-10
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Desorption ("FD") Ionisation source; an Inductively Coupled Plasma ("ICP")
Ionisation
source; a Fast Atom Bombardment ("FAB") Ionisation source; a Liquid Secondary
Ion
Mass Spectrometry ("LSIMS") Ionisation source; a Desorption Electrospray
Ionisation
("DESI") Ionisation source; a Nickel-63 radioactive Ionisation source; a
Thermospray
Ionisation source; an Atmospheric Sampling Glow Discharge Ionisation ("ASGDI")
Ionisation source; a Glow Discharge ("GD") Ionisation source; an Impactor
Ionisation
source; a Direct Analysis in Real Time ("DART") Ionisation source; a
Laserspray Ionisation
("LSI") source; a Sonicspray Ionisation ("SSI") source; a Matrix Assisted
Inlet Ionisation
("MAII") source; a Solvent Assisted Inlet Ionisation ("SAII") source; a
Desorption
Electrospray Ionisation ("DESI") source; a desorption electroflow focusing
ionisation
("DEFFI") source; a Laser Ablation Electrospray Ionisation ("LAESI") source;
and a Surface
Assisted Laser Desorption Ionisation ("SALDI") source.
The method may comprise trapping the analyte ions in an ion trap and/or
guiding
the analyte ions using an ion guide.
The method may comprise heating said collision surface.
The method may comprise heating said collision surface to a temperature
selected
from the group consisting of: (i) about < 100 C; (ii) about 100-200 C; (iii)
about 200-300
C; (iv) about 300-400 C; (v) about 400-500 C; (vi) about 500-600 C; (vii)
about 600-700
C; (viii) about 700-800 C; (ix) about 800-900 C; (x) about 900-1000 C; (xi)
about 1000-
1100 C; and (xii) about > 1100 C
The method may comprise supplying said matrix to said analyte via a matrix
introduction conduit, and analysing ions of the analyte using an ion analyser
arranged
downstream of an outlet of said matrix introduction conduit.
The distance x between said outlet of said matrix introduction conduit and an
inlet
of said ion analyser may be selected from the group consisting of: (i) about
0.1 to 0.5 mm;
(ii) about 0.5-1.0 mm; (iii) about 1.0-1.5 mm; (iv) about 1.5-2.0 mm; (v)
about 2.0-2.5 mm;
(vi) about 2.5-3.0 mm; (vii) about 3.0-3.5 mm; (viii) about 3.5-4.0 mm; (ix)
about 4.0-4.5
mm; (x) about 4.5-5.0 mm; (xi) about 5.0-5.5 mm; (xii) about 5.5-6.0 mm;
(xiii) about 5 6
mm; (xiv) about 5 5.5 mm; (xv) about 5 5 mm; (xvi) about 5 4.5 mm; (xvii)
about 5 4 mm;
(xviii) about 5 3.5 mm; and (xix) about 5 3 mm.
It is contemplated that the distance x may be selected from the group
consisting of:
about 6.0-6.5 mm; about 6.5-7.0 mm; about 7.0-7.5 mm; about 7.5-8.0 mm; about
8.0-8.5
mm; about 8.5-9.0 mm; about 9.0-9.5 mm; about 9.5-10.0 mm; about 0.1-10 mm;
about
0.1-7.5 mm; about 0.1-5.1 mm; about 0.5-5.1 mm; and about 0.5-5.0 mm.
The ion analyser may comprise a vacuum chamber into which the inlet opens. The
inlet of the ion analyser may be determined to be a region that is at the
pressure of the
vacuum chamber. For example, if the inlet is provided by an elongated tube
then the
distance x may be determined from the position in the tube that is at the
pressure of the
vacuum chamber. Alternatively, or additionally, the inlet may be determined to
be the
entrance and/or exit of an inlet tube or orifice.
Date Recue/Date Received 2024-04-10
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The matrix introduction conduit may have an exit opening substantially facing
or
opposite the inlet of said ion analyser; and/or the exit opening of the matrix
introduction
conduit may be substantially coaxial with the inlet of said ion analyser.
The outlet of the matrix introduction conduit and the inlet of said ion
analyser may
be spaced apart and not connected by a completely enclosed conduit.
The exit of the matrix introduction conduit and the inlet of the ion analyser
may be
interconnected by a sampling tube having an opening in its circumference for
receiving the
analyte through the opening.
The method may comprise delivering the analyte through a sample transfer
conduit,
wherein the analyte is arranged to impact on an upstream side of the sampling
tube and
then flow around the outside of the sampling tube and into said opening in a
downstream
side of the sampling tube.
The outlet of the sample transfer conduit may be spaced apart from the outlet
of the
matrix introduction conduit and/or the inlet of said ion analyser and/or the
sampling tube;
and not connected to these elements by an enclosed conduit.
A longitudinal axis of the sample transfer conduit may be substantially
orthogonal to
a longitudinal axis through the outlet of the matrix introduction conduit
and/or a longitudinal
axis through the inlet of said ion analyser and/or a longitudinal axis of the
sampling tube.
The matrix may be introduced to the analyte at a distance y upstream of the
inlet of
said ion analyser, or a distance y upstream of a vacuum chamber in which the
collision
surface is arranged, wherein y is selected from the group consisting of: (i)
1.5-2.0 mm; (ii)
about 2.0-2.5 mm; (iii) about 2.5-3.0 mm; (iv) about 3.0-3.5 mm; (v) about 3.5-
4.0 mm; (vi)
about 4.0-4.5 mm; (vii) about 4.5-5.0 mm; (viii) about 5.0-5.5 mm; (ix) about
5.5-6.0 mm;
(x) about 6 mm; (xi) about 7 mm; (xii) about 8 mm; (xiii) about 9 mm; (xiv)
about
10 mm; (xv) about 12 mm; (xvi) about 14 mm; (xvii) about 16 mm; (xviii) about
18
mm; (xix) about 20 mm; (xx) about 25 mm; (W) about 30mm.
The analyte may be supplied through a sample transfer conduit and the matrix
may
be supplied directly into the sample transfer conduit; or the matrix may be
supplied through
a matrix introduction conduit and the analyte may be supplied directly into
the matrix
introduction conduit.
The sample transfer conduit and/or matrix introduction conduit may be directly
connected to the inlet of an ion analyser or vacuum chamber in which the
collision surface
is arranged.
Alternatively, the analyte may be supplied through a sample transfer conduit
and
the matrix is supplied to the analyte downstream of an exit of the sample
transfer tube.
For example, a sample transfer conduit may be provided that performs the step
of
providing the analyte and the outlet of the matrix introduction conduit may be
provided at a
location about the circumference of sample transfer conduit. A gas flow may be
arranged
so as to sweep the matrix from the outlet, into the analyte, and to the inlet
of an ion
analyser that analyses the ions.
Date Recue/Date Received 2024-04-10
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The matrix may be supplied through a matrix introduction conduit having an
inner
diameter selected from the group consisting of: (i) about 5 900 pm; (ii) about
5 800 pm; (iii)
about 5 700 pm; (iv) about 5 600 pm; and (v) about 5 500 pm.
Alternatively, the matrix may be supplied through a matrix introduction
conduit
having an inner diameter selected from the group consisting of: (i) about s
450 pm; (ii)
about 5 400 pm; (iii) about 5 350 pm; (iv) about 5 300 pm; (v) about 5 250 pm;
(vi) about 5
200 pm; (vii) about 5 150 pm; (viii) about 5 100 pm; (ix) about 5 50 pm; and
(x) about 5 25
pm.
Matrix introduction conduits having smaller internal diameters tend to produce
better and less noisy the spectra. However, it is contemplated that the matrix
introduction
conduit having an inner diameter 450 pm, a 500 pm, or a 1 mm.
The matrix may be supplied through a matrix introduction conduit and the exit
end
of the matrix introduction conduit may be tapered so as to narrow in a
downstream
direction.
The matrix may be supplied to said analyte by a matrix introduction conduit
and at a
flow rate selected from the group consisting of: 5-50 pl/nnin; (i) about 50-
100 pl/min; (ii)
about 100-150 pl/min; (iii) about 150-200 pl/nnin; (iv) about 200-250 pl/min;
(v) about 250-
300 pl/min; (vi) about 300-350 pl/min; (vii) about 350-400 pl/min; (viii)
about 400-450
pl/min; (ix) about 450-500 pl/min; (x) about 500-550 pl/nnin; (xi) about 550-
600 pl/min; (xii)
about 600-650 pl/min; (xiii) about 650-700 pl/min; (xiv) about 700-750 pl/min;
(xv) about
750-800 pl/min; (xvi) about 800-850 pl/min; (xvii) about 850-900 pl/min;
(xviii) about 900-
950 pl/min; (xix) about 950-1000 pl/min; ()o() about 50 pl/min to 1 ml/min;
(xxi) about 100-
800 pl/min; (xxii) about 150-600 pl/min; and (xxiii) about 200-400 pl/min.
Relatively low matrix flow rates may be used such that the matrix is non-toxic
and/or does not contaminate the instrument.
The method may generate analyte ions and comprise analysing said analyte ions.
The step of analysing said analyte ions may comprise: (i) mass analysing said
analyte ions; (ii) analysing the ion mobility or differential ion mobility of
said analyte ions;
(iii) analysing the ionic cross-sections or collision cross sections of said
analyte ions; (iv)
separating said analyte ions according to their ion mobility or differential
ion mobility; (v)
separating said analyte ions according to their ion mobility or differential
ion mobility prior to
mass analysing said analyte ions; or (vi) excluding or discarding analyte ions
based upon
their ion mobility or differential ion mobility.
The method may comprise analysing the analyte ions with an ion analyser to
obtain
analyte ion data, analysing lockmass, lock mobility or calibration ions, and
calibrating said
ion analyser or adjusting the analyte ion data based upon the data obtained
from analysing
said lockmass, lock mobility or calibration ions.
A lockmass, lock mobility or calibration compound may be in the matrix for
generating the lockmass, lock mobility or calibration ions.
The lockmass, lock mobility or calibration compound/ions may be introduced
into
the matrix introduction conduit, the analyte introduction conduit or may be
supplied in a
separate conduit.
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The method may comprise using a first device to provide said analyte; wherein
said
first device may comprise or forms part of an ambient ion or ionisation
source; or wherein
said first device may generate said aerosol, smoke or vapour from a target to
be analysed
and which contains ions and/or is subsequently ionised by an ambient ion or
ionisation
source or other ionisation source.
The target may comprise native or unmodified target material.
The native or unmodified target may be unmodified (i.e. not modified) by the
addition of a matrix or reagent.
The first device may be arranged and adapted to generate aerosol, smoke or
vapour from one or more regions of said target without said target requiring
prior
preparation.
The first device may comprise or forms part of a device, or an ion source,
selected
from the group consisting of: (i) a rapid evaporative ionisation mass
spectrometry
("REIMS") ion source; (ii) a desorption electrospray ionisation ("DESI") ion
source; (iii) a
laser desorption ionisation ("LDI") ion source; (iv) a thermal desorption ion
source; (v) a
laser diode thermal desorption ("LDTD") ion source; (vi) a desorption electro-
flow focusing
("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") plasma ion
source; (viii) an
Atmospheric Solids Analysis Probe ("ASAP") ion source; (ix) an ultrasonic
assisted spray
ionisation ion source; (x) an easy ambient sonic-spray ionisation ("EASI") ion
source; (xi) a
desorption atmospheric pressure photoionisation ("DAPPI") ion source; (xii) a
paperspray
("PS") ion source; (xiii) a jet desorption ionisation ("JeDI") ion source;
(xiv) a touch spray
("TS") ion source; (xv) a nano-DESI ion source; (xvi) a laser ablation
electrospray ("LAESI")
ion source; (xvii) a direct analysis in real time ("DART") ion source; (xviii)
a probe
electrospray ionisation ("PESI") ion source; (xix) a solid-probe assisted
electrospray
ionisation ("SPA-ESI") ion source; (xx) a cavitron ultrasonic surgical
aspirator ("CUSA")
device; ()xi) a hybrid CUSA-diathermy device; (xxii) a focussed or unfocussed
ultrasonic
ablation device; (xxiii) a hybrid focussed or unfocussed ultrasonic ablation
and diathermy
device; (xxiv) a microwave resonance device; (xm) a pulsed plasma RF
dissection device;
(xxvi) an argon plasma coagulation device; (xxvi) a hybrid pulsed plasma RF
dissection
and argon plasma coagulation device; (xxvii) a hybrid pulsed plasma RF
dissection and
JeDI device; ()mill) a surgical water/saline jet device; (xxix) a hybrid
electrosurgery and
argon plasma coagulation device; and ()ooc) a hybrid argon plasma coagulation
and
water/saline jet device.
The step of using said first device to generate aerosol, smoke or vapour from
one
or more regions of said target further may comprise contacting said target
with one or more
electrodes.
The one or more electrodes may comprise either: (i) a monopolar device,
wherein
said method optionally further comprises providing a separate return
electrode; (ii) a bipolar
device; or (iii) a multi-phase RF device, wherein said method optionally
further comprises
providing a separate return electrode or electrodes.
The one or more electrodes may comprise or form part of a rapid evaporation
ionization mass spectrometry ("REIMS") device.
Date Recue/Date Received 2024-04-10
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The method may comprise applying an AC or RE voltage to said one or more
electrodes in order to generate said aerosol, smoke or vapour.
The step of applying said AC or RE voltage to said one or more electrodes may
comprises applying one or more pulses of said AC or RE voltage to said one or
more
electrodes.
The step of applying said AC or RE voltage to said one or more electrodes may
cause heat to be dissipated into said target.
The step of using said first device to generate aerosol, smoke or vapour from
one
or more regions of said target may comprise irradiating said target with a
laser.
The first device may be arranged and adapted to generate aerosol from one or
more regions of said target by direct evaporation or vaporisation of target
material from
said target by Joule heating or diathermy.
The diathermy may be produced by one of three techniques: ultrasound
(ultrasonic
diathermy); radio frequency diathermy (e.g. shortwave radio frequencies in the
range of, for
example, 1-100 MHz); or microwave diathermy (e.g., in the 915 MHz or 2.45 GHz
bands).
The methods of diathermy differ mainly in their penetration capability.
The step of using said first device to generate aerosol, smoke or vapour from
one
or more regions of said target may comprises directing ultrasonic energy into
said target.
The aerosol may comprise uncharged aqueous droplets, optionally comprising
cellular material.
At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the mass or
matter generated by said first device and which forms said aerosol may be in
the form of
droplets.
The first device may be arranged and adapted to generate aerosol wherein the
Sauter mean diameter ("SMD", d32) of said aerosol is in a range: (i) <5 pm;
(ii) 5-10 pm;
(iii) 10-15 pm; (iv) 15-20 pm; (v) 20-25 pm; or (vi) > 25 pm.
The aerosol may traverse a flow region with a Reynolds number (Re) in the
range:
(i) <2000; (ii) 2000-2500; (iii) 2500-3000; (iv) 3000-3500; (v) 3500-4000; or
(vi) > 4000.
Substantially at the point of generating said aerosol, said aerosol may
comprise
droplets having a Weber number (We) selected from the group consisting of: (i)
< 50; (ii)
50-100; (iii) 100-150; (iv) 150-200; (v) 200-250;(vi) 250-300; (vii) 300-350;
(viii) 350-400;
(ix) 400-450; (x) 450-500; (xi) 500-550; (xii) 550-600; (xiii) 600-650; (xiv)
650-700; (xv) 700-
750; (xvi) 750-800; (xvii) 800-850; (xviii) 850-900; (xix) 900-950; ()o() 950-
1000; and (xW) >
1000.
Substantially at the point of generating said aerosol, said aerosol may
comprise
droplets having a Stokes number (SO in the range: (i) 1-5; (ii) 5-10; (iii) 10-
15; (iv) 15-20;
(v) 20-25; (vi) 25-30; (vii) 30-35; (viii) 35-40; (ix) 40-45; (x) 45-50; and
(xi) > 50.
Substantially at the point of generating said aerosol, said aerosol may
comprise
droplets having a mean axial velocity selected from the group consisting of:
(i) <20 m/s; (ii)
20-30 m/s; (iii) 30-40 m/s; (iv) 40-50 m/s; (v) 50-60 m/s; (vi) 60-70 m/s;
(vii) 70-80 m/s; (viii)
80-90 m/s; (ix) 90-100 m/s; (x) 100-110 m/s; (xi) 110-120 m/s; (xii) 120-130
m/s; (xiii) 130-
140 m/s; (xiv) 140-150 m/s; and (xv) > 150 m/s.
Date Recue/Date Received 2024-04-10
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The target may comprise biological tissue, such as healthy or diseased tissue.
The biological tissue may comprise human tissue or non-human animal tissue.
The biological tissue may comprise in vivo biological tissue.
The biological tissue may comprise ex vivo biological tissue.
The biological tissue may comprise in vitro biological tissue.
The biological tissue may comprise adrenal gland tissue, appendix tissue,
bladder
tissue, bone, bowel tissue, brain tissue, breast tissue, bronchi, coronal
tissue, ear tissue,
esophagus tissue, eye tissue, gall bladder tissue, genital tissue, heart
tissue, hypothalamus
tissue, kidney tissue, large intestine tissue, intestinal tissue, larynx
tissue, liver tissue, lung
tissue, lymph nodes, mouth tissue, nose tissue, pancreatic tissue, parathyroid
gland tissue,
pituitary gland tissue, prostate tissue, rectal tissue, salivary gland tissue,
skeletal muscle
tissue, skin tissue, small intestine tissue, spinal cord, spleen tissue,
stomach tissue,
thymus gland tissue, trachea tissue, thyroid tissue, soft tissue, connective
tissue, peritoneal
tissue, blood vessel tissue, fat tissue, ureter tissue, urethra tissue; grade
I, grade II, grade
III or grade IV cancerous tissue; metastatic cancerous tissue; mixed grade
cancerous
tissue; a sub-grade cancerous tissue; healthy or normal tissue; or cancerous
or abnormal
tissue.
The first device may comprise a point of care ("POC"), diagnostic or surgical
device.
The method may comprise ionising at least some of said aerosol, smoke or
vapour
so as to generate analyte ions.
The method may comprise directing or aspirating at least some of said aerosol,
smoke or vapour into a vacuum chamber of a mass and/or ion mobility
spectrometer.
The method may comprise ionising at least some said aerosol, smoke or vapour
within a or said vacuum chamber of said mass and/or ion mobility spectrometer
so as to
generate a plurality of analyte ions.
The method may comprise causing said aerosol, smoke or vapour to impact upon
the collision surface, optionally located within a vacuum chamber of said mass
and/or ion
mobility spectrometer, so as to generate the plurality of analyte ions.
The method may comprise mass and/or ion mobility analysing said analyte ions
or
ions derived from said aerosol, smoke or vapour in order to obtain mass and/or
ion mobility
spectrometric data.
The method may comprise analysing said spectrometric data in order either: (i)
to
distinguish between healthy and diseased tissue; (ii) to distinguish between
potentially
cancerous and non-cancerous tissue; (iii) to distinguish between different
types or grades
of cancerous tissue; (iv) to distinguish between different types or classes of
target material;
(v) to determine whether or not one or more desired or undesired substances
are present
in said target; (vi) to confirm the identity or authenticity of said target;
(vii) to determine
whether or not one or more impurities, illegal substances or undesired
substances are
present in said target; (viii) to determine whether a human or animal patient
is at an
increased risk of suffering an adverse outcome; (ix) to make or assist in the
making a
diagnosis or prognosis; and (x) to inform a surgeon, nurse, medic or robot of
a medical,
surgical or diagnostic outcome.
Date Recue/Date Received 2024-04-10
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The method may either be part of a surgical method or a non-surgical method.
For
example, the method may be a surgical method in which the sample may be human
or
animal tissue containing the analyte. The sample may be subjected to
electrosurgical
diathermic evaporation, or other forms of rapid evaporation, in order to form
the gas phase
analyte, vapour analyte or aerosol. By way of example only, the device and
method may
be used for the identification of human tissues in breast cancer surgery. By
analysing the
analyte ions it is possible to determine whether or not the tissues are
cancerous.
Alternatively, the method may comprise a non-surgical method. For example,
human or animal tissue that is not part of the human or animal body (i.e.
previously
excised, deposited or removed) may be analysed, or samples or biological
tissues other
than human or animal tissues may be analysed. Again, by analysing the analyte
ions it is
possible to determine the properties or constituents of the sample, such as
whether or not
they contain cancerous tissues.
The method may be used in other non-surgical methods, such as country of
origin
identification, pharmaceutical testing, food safety testing (e.g., dairy),
cosmetics testing,
military applications, air pollution testing, post-mortem analysis, microbe
identification (e.g.,
bacteria), and automated sampling.
The method may be used to analyse non-biological samples and compounds.
The analyte that is formed from the sample may be partially charged and/or may
have a
relatively high organic content.
The step of analysing the spectrometric data may comprise analysing one or
more
sample spectra so as to classify an aerosol, smoke or vapour sample.
Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise unsupervised analysis of the one or more sample
spectra
(e.g., for dimensionality reduction) and/or supervised analysis of the one or
more sample
spectra (e.g., for classification).
Analysing the one or more sample spectra may comprise unsupervised analysis
(e.g., for dimensionality reduction) followed by supervised analysis (e.g.,
for classification).
Analysing the one or more sample spectra may comprise using one or more of:
(i)
univariate analysis; (ii) multivariate analysis; (iii) principal component
analysis (PCA); (iv)
linear discriminant analysis (LDA); (v) maximum margin criteria (MMC); (vi)
library-based
analysis; (vii) soft independent modelling of class analogy (SIMCA); (viii)
factor analysis
(FA); (ix) recursive partitioning (decision trees); (x) random forests; (xi)
independent
component analysis (ICA); (xii) partial least squares discriminant analysis
(PLS-DA); (xiii)
orthogonal (partial least squares) projections to latent structures (OPLS);
(xiv) OPLS
discriminant analysis (OPLS-DA); (xv) support vector machines (SVM); (xvi)
(artificial)
neural networks; (xvii) multilayer perceptron; (xviii) radial basis function
(RBF) networks;
(xix) Bayesian analysis; ()o<) cluster analysis; ()xi) a kemelized method; and
()WO
subspace discriminant analysis; (xxiii) k-nearest neighbours (KNN); (xxiv)
quadratic
discriminant analysis (QDA); (xxv) probabilistic principal component Analysis
(PPCA);
(xxvi) non negative matrix factorisation; (xxvii) k-means factorisation;
()mill) fuzzy c-means
factorisation; and (xxix) discriminant analysis (DA).
Date Recue/Date Received 2024-04-10
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Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise developing a classification model or library using
one or
more reference sample spectra.
Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise performing linear discriminant analysis (LDA)
(e.g., for
classification) after performing principal component analysis (PCA) (e.g., for
dimensionality
reduction).
Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise performing a maximum margin criteria (MMC) process
(e.g.,
for classification) after performing principal component analysis (PCA) (e.g.,
for
dimensionality reduction).
Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise defining one or more classes within a
classification model or
library.
Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise defining one or more classes within a
classification model or
library manually or automatically according to one or more class or cluster
criteria.
The one or more class or cluster criteria for each class may be based on one
or
more of: a distance between one or more pairs of reference points for
reference sample
spectra within a model space; a variance value between groups of reference
points for
reference sample spectra within a model space; and a variance value within a
group of
reference points for reference sample spectra within a model space.
The one or more classes may each be defined by one or more class definitions.
The one or more class definitions may comprise one or more of: a set of one or
more reference points for reference sample spectra, values, boundaries, lines,
planes,
hyperplanes, variances, volumes, Voronoi cells, and/or positions, within a
model space;
and one or more positions within a class hierarchy.
Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise using a classification model or library to classify
one or more
unknown sample spectra.
Analysing the one or more sample spectra so as to classify the aerosol, smoke
or
vapour sample may comprise classifying one or more sample spectra manually or
automatically according to one or more classification criteria.
The one or more classification criteria may comprise one or more of:
a distance between one or more projected sample points for one or more sample
spectra within a model space and a set of one or more reference points for one
or more
reference sample spectra, values, boundaries, lines, planes, hyperplanes,
volumes,
Voronoi cells, or positions, within the model space being below a distance
threshold or
being the lowest such distance;
a position for one or more projected sample points for one or more sample
spectra
within a model space being one side or other of one or more reference points
for one or
Date Recue/Date Received 2024-04-10
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more reference sample spectra, values, boundaries, lines, planes, hyperplanes,
or
positions, within the model space;
a position for one or more projected sample points for one or more sample
spectra
within a model space being within one or more volumes or Voronoi cells within
the model
space; and
a probability or classification score being above a probability or
classification score
threshold or being the highest such probability or classification score.
The mass and/or ion mobility spectrometer may obtain data in negative ion mode
only, positive ion mode only, or in both positive and negative ion modes.
Positive ion mode
spectrometric data may be combined or concatenated with negative ion mode
spectrometric data. Negative ion mode can provide particularly useful spectra
for
classifying aerosol, smoke or vapour samples, such as aerosol, smoke or vapour
samples
from targets comprising lipids.
Ion mobility spectrometric data may be obtained using different ion mobility
drift
gases, and/or dopants may be added to the drift gas to induce a change in
drift time of one
or more species. This data may then be combined or concatenated.
The first aspect of the present invention also provides apparatus for
performing
mass and/or ion mobility spectrometry comprising:
an analyte inlet for receiving analyte;
a matrix inlet for receiving a matrix compound;
a mixing region for mixing said analyte with said matrix compound such that,
in use,
said analyte is diluted by, dissolves in, or forms first clusters with said
matrix;
a collision surface; and
a device arranged and adapted to cause said first clusters or first droplets
of the
diluted or dissolved analyte to collide with said collision surface.
The device may be arranged and adapted to cause the first clusters or first
droplets
to collide with the collision surface and fragments into a plurality of second
smaller clusters
or droplets.
The apparatus may be configured to provide the analyte in the form of a gas-
phase
analyte, aerosol, vapour, smoke or liquid; and/or to supply the matrix to said
analyte whilst
said analyte is in the form of a gas-phase analyte, aerosol, vapour, smoke or
liquid.
The apparatus may be configured to supply the matrix to the analyte whilst the
analyte is in the form of an aerosol, vapour or smoke and whilst the matrix is
in the form of
an aerosol, vapour, or solid particles.
The apparatus may be configured to supply matrix molecules to, and intermix
said
matrix molecules with, said analyte whilst said matrix is in a gas phase or in
the form of an
aerosol, vapour or solid particles.
The apparatus may comprise a high pressure region and a low pressure region
and
may be configured to transfer the mixture of said analyte and said matrix from
the high
pressure region to the low pressure region such that, in use, said gas phase
matrix cools
and condenses to a liquid and said analyte dissolves in said liquid matrix so
as to form said
first droplets.
Date Recue/Date Received 2024-04-10
- 15 -
Alternatively, the apparatus may be configured to supply the matrix to, and
intermixed with, the analyte as a liquid.
If the analyte and/or matrix is in liquid form then the apparatus may be
configured to
convert the analyte and/or matrix into droplets or vapour, e.g., by spraying
or nebulising.
For example, if the analyte and matrix are mixed as liquids then the mixture
may
subsequently be converted into the first analyte droplets, e.g., by spraying
or nebulising.
The matrix may initially be supplied as a solid (e.g., a powder) and
sublimated or
melted and evaporated so as to form matrix in vapour or gas-phase that is
intermixed with
the analyte to form clusters of analyte and matrix.
The matrix may be selected from the group consisting of: (i) a solvent for
said
analyte; (ii) an organic solvent; (iii) a volatile compound; (iv) polar
molecules; (v) water; (vi)
one or more alcohols; (vii) methanol; (viii) ethanol; (ix) isopropanol; (x)
acetone; (xi)
acetonitrile; (xii) 1-butanol; (xiii) tetrahydrofuran; (xiv) ethyl acetate;
(xv) ethylene glycol;
(xvi) dirnethyl sulfoxide; (xvii) an aldehyde; (xviii) a ketone; (xiv) non-
polar molecules; (xx)
hexane; ()oci) chloroform; ()ocii) butanol; and ()o:iii) propanol.
By way of example, for analytes comprising polar lipids, low molecular weight
alcohols may be used as the matrix (e.g., methanol, ethanol, isopropanol) or
ketones (e.g.,
acetone). These matrices have been shown to enhance the ionisation of species
otherwise detected in the absence of the matrix vapours at lower intensity.
A protic matrix solvent may be used, e.g., for the analysis of lipids or
triglycerides.
Alternatively, a non-protic or aprotic matrix solvent may be used, e.g., for
the analysis of
proteins.
The mixture of analyte and matrix may be a homogeneous or heterogeneous
mixture.
The matrix and/or analyte may be doped with one or more additives for
enhancing
the solvation of the analyte in the matrix or for enhancing the ionisation of
the analyte.
The matrix or analyte is doped with an acidic or basic additive. For example,
the
matrix may be doped with formic acid, diethylamine.
The matrix may cause derivatisation of the analyte. For example, the matrix
may
.. cause the derivatisation of cholesterol or steroids in the analyte. This
may render the
analyte more easily ionised.
A calibrant may be added to the matrix and/or analyte, or a compound in the
matrix
and/or analyte may be selected as a calibrant, and used to calibrate the
method of mass
spectrometry and/or ion mobility spectrometry. This is particularly useful in
ambient
ionisation techniques such as REIMS techniques, in which it may be difficult
to introduce
calibrants according to conventional techniques.
The apparatus may be configured to accelerate said first clusters or first
droplets
onto said collision surface.
The apparatus may be configured to accelerate said first clusters or first
droplets
onto said collision surface using a pressure differential.
Date Recue/Date Received 2024-04-10
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The apparatus may be configured to create a pressure differential between a
first
region and a second region for accelerating the first clusters or first
droplets between the
two regions and onto the collision surface.
The apparatus may comprise a mass and/or ion mobility spectrometer having an
atmospheric interface arranged between the first and second regions, wherein
the second
region may comprise a vacuum chamber that is connected to the vacuum pump and
which
houses the collision surface.
The apparatus may be configured to evaporate the matrix from the analyte in
the
second droplets so as to provide analyte ions that are separate from said
matrix.
The step of colliding the first clusters or first droplets with the collision
surface may
evaporate the matrix from the analyte by converting kinetic energy of the
analyte and
matrix into heat.
The step of colliding the first clusters or first droplets causes the second
smaller
clusters or droplets to be generated, at least some of which may have only a
single analyte
molecule therein. This enhances the ionisation process.
The step of evaporating said matrix from said analyte may result in charge
transfer
to or from said analyte so as to ionise said analyte to form analyte ions.
The apparatus may comprise ion trap for trapping analyte ions and/or an ion
guide
for guiding analyte ions.
The apparatus may comprise a heater for heating said collision surface.
The heater may be configured to heat said collision surface to a temperature
selected from the group consisting of: (i) about < 100 C; (ii) about 100-200
C; (iii) about
200-300 C; (iv) about 300-400 C; (v) about 400-500 C; (vi) about 500-600
C; (vii) about
600-700 C; (viii) about 700-800 C; (ix) about 800-900 C; (x) about 900-1000 C;
(xi) about
1000-1100 C; and (xii) about > 1100 C
The apparatus may comprise a matrix introduction conduit for supplying said
matrix
to said analyte, and an ion analyser arranged downstream of an outlet of said
matrix
introduction conduit for analysing ions of the analyte.
The distance x between said outlet of said matrix introduction conduit and an
inlet
of said ion analyser may be selected from the group consisting of: (i) about
0.1 to 0.5 mm;
(ii) about 0.5-1.0 mm; (iii) about 1.0-1.5 mm; (iv) about 1.5-2.0 mm; (v)
about 2.0-2.5 mm;
(vi) about 2.5-3.0 mm; (vii) about 3.0-3.5 mm; (viii) about 3.5-4.0 mm; (ix)
about 4.0-4.5
mm; (x) about 4.5-5.0 mm; (xi) about 5.0-5.5 mm; (xii) about 5.5-6.0 mm;
(xiii) about 5 6
mm; (xiv) about 5 5.5 mm; (xv) about 5 5 mm; (xvi) about 5 4.5 mm; (xvii)
about 5 4 mm;
(xviii) about 5 3.5 mm; and (xix) about 5 3 mm.
The ion analyser may comprise a vacuum chamber into which the inlet opens. The
inlet of the ion analyser may be determined to be a region that is at the
pressure of the
vacuum chamber. For example, if the inlet is provided by an elongated tube
then the
distance x may be determined from the position in the tube that is at the
pressure of the
vacuum chamber. Alternatively, or additionally, the inlet may be determined to
be the
entrance and/or exit of an inlet tube or orifice.
Date Recue/Date Received 2024-04-10
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The matrix introduction conduit may have an exit opening substantially facing
or
opposite the inlet of said ion analyser; and/or wherein the exit opening of
the matrix
introduction conduit is substantially coaxial with the inlet of said ion
analyser.
The outlet of the matrix introduction conduit and the inlet of said ion
analyser may
be spaced apart and not connected by a completely enclosed conduit.
The exit of the matrix introduction conduit and the inlet of the ion analyser
may be
interconnected by a sampling tube having an opening in its circumference for
receiving the
analyte through the opening.
The apparatus may comprise a sample transfer conduit for delivering the
analyte,
wherein the sample transfer conduit may be arranged such that, in use, the
analyte
impacts on an upstream side of the sampling tube, flows around the outside of
the
sampling tube and into said opening in a downstream side of the sampling tube.
The outlet of the sample transfer conduit may be spaced apart from the outlet
of the
matrix introduction conduit and/or the inlet of said ion analyser and/or the
sampling tube;
and not connected to these elements by an enclosed conduit.
A longitudinal axis of the sample transfer conduit may be substantially
orthogonal to
a longitudinal axis through the outlet of the matrix introduction conduit
and/or a longitudinal
axis through the inlet of said ion analyser and/or a longitudinal axis of the
sampling tube.
The matrix may be introduced to the analyte at a distance y upstream of the
inlet of
said ion analyser, or a distance y upstream of a vacuum chamber in which the
collision
surface is arranged, wherein y is selected from the group consisting of: (i)
1.5-2.0 mm; (ii)
about 2.0-2.5 mm; (iii) about 2.5-3.0 mm; (iv) about 3.0-3.5 mm; (v) about 3.5-
4.0 mm; (vi)
about 4.0-4.5 mm; (vii) about 4.5-5.0 mm; (viii) about 5.0-5.5 mm; (ix) about
5.5-6.0 mm;
(x) about 6 mm; (xi) about 7 mm; (xii) about 8 mm; (xiii) about 9 mm; (xiv)
about
10 mm; (xv) about 12 mm; (xvi) about 14 mm; (xvii) about 16 mm; (xviii) about
18
mm; (xix) about 20 mm; (xx) about 25 mm; (W) about 30mm.
The apparatus may comprise a sample transfer conduit for supplying the analyte
and matrix introduction conduit for supplying the matrix directly into the
sample transfer
conduit; or may comprise a matrix introduction conduit for supplying the
matrix and a
sample transfer conduit for supplying the analyte directly into the matrix
introduction
conduit.
The sample transfer conduit and/or matrix introduction conduit may be directly
connected to the inlet of an ion analyser or vacuum chamber in which the
collision surface
is arranged.
Alternatively, the analyte may be supplied through a sample transfer conduit
and
the matrix is supplied to the analyte downstream of an exit of the sample
transfer tube. For
example, a sample transfer conduit may be provided that performs the step of
providing the
analyte and the outlet of the matrix introduction conduit may be provided at a
location
about the circumference of sample transfer conduit. A gas flow may be arranged
so as to
sweep the matrix from the outlet, into the analyte, and to the inlet of an ion
analyser that
analyses the ions.
Date Recue/Date Received 2024-04-10
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The apparatus may comprise a matrix introduction conduit for supplying the
matrix,
wherein the matrix introduction conduit has an inner diameter selected from
the group
consisting of: (i) about 5 450 pm; (ii) about 5 400 pm; (iii) about 5 350 pm;
(iv) about 5 300
pm; (v) about s 250 pm; (vi) about s 200 pm; (vii) about s 150 pm; (viii)
about 5 100 pm;
(ix) about 5. 50 pm; and (x) about 5 25 pm.
Matrix introduction conduits having smaller internal diameters tend to produce
better and less noisy the spectra.
The apparatus may comprise a matrix introduction conduit for supplying the
matrix,
wherein the exit end of the matrix introduction conduit is tapered so as to
narrow in a
downstream direction.
The apparatus may comprise a pump configured to supply said matrix to the
analyte through a matrix introduction conduit at a flow rate selected from the
group
consisting of: 5-50 pl/min; (i) about 50-100 pl/min; (ii) about 100-150
pl/min; (iii) about 150-
200 pl/min; (iv) about 200-250 pl/min; (v) about 250-300 pl/min; (vi) about
300-350 pl/min;
.. (vii) about 350-400 pl/min; (viii) about 400-450 pl/min; (ix) about 450-500
pl/min; (x) about
500-550 pl/min; (xi) about 550-600 pl/rnin; (xii) about 600-650 pl/min; (xiii)
about 650-700
pl/min; (xiv) about 700-750 pl/min; (xv) about 750-800 pl/min; (xvi) about 800-
850 pl/min;
(xvii) about 850-900 pl/min; (xviii) about 900-950 pl/min; (xix) about 950-
1000 pl/min; (xx)
about 50 pl/min to 1 ml/min; NO about 100-800 pl/min; (xxii) about 150-600
pl/min; and
.. (xxiii) about 200-400 pl/min.
Relatively low matrix flow rates may be used such that the matrix is non-toxic
and/or does not contaminate the instrument.
The apparatus may comprise a mass and/or ion mobility analyser for analysing
analyte ions.
The apparatus may comprise a first device for providing said analyte; wherein
said
first device comprises or forms part of an ambient ion or ionisation source;
or wherein said
first device is configured to generate a or said aerosol, smoke or vapour from
a target to be
analysed and which contains ions and/or is subsequently ionised by an ambient
ion or
ionisation source or other ionisation source.
The target may comprise native or unmodified target material.
The native or unmodified target material is unmodified by the addition of a
matrix or
reagent.
The first device or apparatus is arranged and adapted to generate aerosol,
smoke
or vapour from one or more regions of said target without said target
requiring prior
preparation.
The first device or apparatus may comprise or form part of a device, or an ion
source, selected from the group consisting of: (i) a rapid evaporative
ionisation mass
spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionisation
("DESI") ion
source; (iii) a laser desorption ionisation ("LDI") ion source; (iv) a thermal
desorption ion
source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a
desorption electro-
flow focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge
("DBD") plasma ion
source; (viii) an Atmospheric Solids Analysis Probe ("ASAP") ion source; (ix)
an ultrasonic
Date Recue/Date Received 2024-04-10
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assisted spray ionisation ion source; (x) an easy ambient sonic-spray
ionisation ("EASI")
ion source; (xi) a desorption atmospheric pressure photoionisation ("DAPPI")
ion source;
(xii) a paperspray ("PS") ion source; (xiii) a jet desorption ionisation
("JeDI") ion source;
(xiv) a touch spray ("TS") ion source; (xv) a nano-DESI ion source; (xvi) a
laser ablation
electrospray ("LAESI") ion source; (xvii) a direct analysis in real time
("DART") ion source;
(xviii) a probe electrospray ionisation ("PESI") ion source; (xix) a solid-
probe assisted
electrospray ionisation ("SPA-ESI") ion source; ()o() a cavitron ultrasonic
surgical aspirator
("CUSA") device; (x) a hybrid CUSA-diathermy device; (xxii) a focussed or
unfocussed
ultrasonic ablation device; (xxiii) a hybrid focussed or unfocussed ultrasonic
ablation and
__ diathermy device; (xxiv) a microwave resonance device; (x) a pulsed plasma
RF
dissection device; ()ocvi) an argon plasma coagulation device; (xxvi) a hybrid
pulsed plasma
RF dissection and argon plasma coagulation device; (xxvii) a hybrid pulsed
plasma RF
dissection and JeDI device; (mcviii) a surgical water/saline jet device;
(xxix) a hybrid
electrosurgery and argon plasma coagulation device; and (xxo() a hybrid argon
plasma
coagulation and water/saline jet device.
The first device or apparatus may comprise one or more electrodes and may be
arranged and adapted to generate aerosol, smoke or vapour from one or more
regions of
said target by contacting said target with said one or more electrodes.
The one or more electrodes may comprise either: (i) a monopolar device,
wherein
optionally a separate return electrode is provided; (ii) a bipolar device; or
(iii) a multi-phase
RF device, wherein optionally at least one separate return electrode is
provided.
The one or more electrodes may comprise a rapid evaporation ionization mass
spectrometry ("REIMS") device.
The apparatus may comprise a device arranged and adapted to apply an AC or RF
voltage to said one or more electrodes in order to generate said aerosol,
smoke or vapour.
The device for applying said AC or RF voltage to said one or more electrodes
may
be arranged to apply one or more pulses of said AC or RF voltage to said one
or more
electrodes.
The application of said AC or RF voltage to said one or more electrodes may
cause
heat to be dissipated into said target.
The first device or apparatus may comprise a laser for irradiating said
target.
The first device or apparatus may be arranged and adapted to generate aerosol
from one or more regions of said target by direct evaporation or vaporisation
of target
material from said target by Joule heating or diathermy.
The first device or apparatus may be arranged and adapted to direct ultrasonic
energy into said target.
The aerosol may comprise uncharged aqueous droplets, optionally comprising
cellular material.
At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the mass or
matter generated by said first device or apparatus and which forms said
aerosol may be in
the form of droplets.
Date Recue/Date Received 2024-04-10
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The first device may be arranged and adapted to generate aerosol wherein the
Sauter mean diameter ("SMD", d32) of said aerosol is in a range: (i) <5 pm;
(ii) 5-10 pm;
(iii) 10-15 pm; (iv) 15-20 pm; (v) 20-25 pm; or (vi) > 25 pm.
The aerosol may traverse a flow region with a Reynolds number (Re) in the
range:
(i) <2000; (ii) 2000-2500; (iii) 2500-3000; (iv) 3000-3500; (v) 3500-4000; or
(vi) > 4000.
Substantially at the point of generating said aerosol, said aerosol may
comprise
droplets having a Weber number (VVe) selected from the group consisting of:
(i) < 50; (ii)
50-100; (iii) 100-150; (iv) 150-200; (v) 200-250;(vi) 250-300; (vii) 300-350;
(viii) 350-400;
(ix) 400-450; (x) 450-500; (xi) 500-550; (xii) 550-600; (xiii) 600-650; (xiv)
650-700; (xv) 700-
750; (xvi) 750-800; (xvii) 800-850; (xviii) 850-900; (xix) 900-950; ()o() 950-
1000; and (x) >
1000.
Substantially at the point of generating said aerosol, said aerosol may
comprise
droplets having a Stokes number (Sk) in the range: (i) 1-5; (ii) 5-10; (iii)
10-15; (iv) 15-20;
(v) 20-25; (vi) 25-30; (vii) 30-35; (viii) 35-40; (ix) 40-45; (x) 45-50; and
(xi) > 50.
Substantially at the point of generating said aerosol, said aerosol may
comprise
droplets having a mean axial velocity selected from the group consisting of:
(i) <20 m/s; (ii)
20-30 m/s; (iii) 30-40 m/s; (iv) 40-50 m/s; (v) 50-60 m/s; (vi) 60-70 m/s;
(vii) 70-80 m/s; (viii)
80-90 m/s; (ix) 90-100 m/s; (x) 100-110 m/s; (xi) 110-120 m/s; (xii) 120-130
m/s; (xiii) 130-
140 m/s; (xiv) 140-150 m/s; and (xv) > 150 m/s.
The target may comprise biological tissue.
The biological tissue may comprise human tissue or non-human animal tissue.
The biological tissue may comprise in vivo biological tissue.
The biological tissue may comprise ex vivo biological tissue.
The biological tissue may comprise in vitro biological tissue.
The biological tissue may comprise adrenal gland tissue, appendix tissue,
bladder
tissue, bone, bowel tissue, brain tissue, breast tissue, bronchi, coronal
tissue, ear tissue,
esophagus tissue, eye tissue, gall bladder tissue, genital tissue, heart
tissue, hypothalamus
tissue, kidney tissue, large intestine tissue, intestinal tissue, larynx
tissue, liver tissue, lung
tissue, lymph nodes, mouth tissue, nose tissue, pancreatic tissue, parathyroid
gland tissue,
pituitary gland tissue, prostate tissue, rectal tissue, salivary gland tissue,
skeletal muscle
tissue, skin tissue, small intestine tissue, spinal cord, spleen tissue,
stomach tissue,
thymus gland tissue, trachea tissue, thyroid tissue, soft tissue, connective
tissue, peritoneal
tissue, blood vessel tissue, fat tissue, ureter tissue, urethra tissue; grade
I, grade II, grade
III or grade IV cancerous tissue; metastatic cancerous tissue; mixed grade
cancerous
tissue; a sub-grade cancerous tissue; healthy or normal tissue; or cancerous
or abnormal
tissue.
The first device or apparatus may comprise a point of care ("POC"), diagnostic
or
surgical device.
The collision surface may be substantially spherical, coil-shaped, helical,
spiral-
shaped, cylindrical, tubular, rod-shaped, hemispherical, teardrop-shaped,
plate-shaped,
concave, dish-shaped or conical; or wherein the collision surface is the inner
surface of a
hollow collision assembly.
Date Recue/Date Received 2024-04-10
- 21 -
The present invention provides a mass and/or ion mobility spectrometer
comprising
apparatus as described herein.
The spectrometer may comprise a spectrometer main housing or assembly and the
source housing may be connected, in use, to said spectrometer main housing.
The spectrometer may comprise an ion trap and/or an ion guide; optionally
wherein
the ion guide is configured to apply an electric field that separates ions
from neutral
species.
The spectrometer may comprise an analyser for analysing analyte ions.
The analyser may comprise: (i) a mass analyser for mass analysing said analyte
.. ions; (ii) an ion mobility or differential ion mobility analyser; (iii) an
analyser for analysing
the ionic cross-sections or collision cross sections of said analyte ions;
(iv) a separator for
separating said analyte ions according to their ion mobility or differential
ion mobility; (v) a
separator for separating said analyte ions according to their ion mobility or
differential ion
mobility prior to mass analysing said analyte ions; or (vi) a device arranged
and adapted to
exclude or discard analyte ions based upon their ion mobility or differential
ion mobility.
The apparatus and spectrometer may be arranged and configured to perform any
one of the methods described herein.
The present invention also provides a method of surgery or electrosurgery
comprising any of the method steps described herein, wherein the method
comprises:
contacting biological tissue with a surgical or electrosurgical tool and
activating said
tool so as to generate smoke, fumes, liquid, gas, surgical smoke, aerosol or
vapour
containing said analyte;
aspirating said smoke, fumes, liquid, gas, surgical smoke, aerosol or vapour;
mixing said matrix with said aspirated smoke, fumes, liquid, gas, surgical
smoke,
.. aerosol or vapour;
causing said aspirated smoke, fumes, liquid, gas, surgical smoke, aerosol or
vapour
in combination with said matrix to impact upon said collision surface located
within a
vacuum chamber of a mass and/or ion mobility spectrometer in order to form
analyte ions;
and
mass and/or ion mobility analysing said analyte ions.
The present invention also provides a surgical or electrosurgical apparatus
comprising:
a surgical or electrosurgical tool comprising one or more electrodes;
a device arranged and adapted to activate said tool when said tool is in
contact, in
use, with biological tissue so as to generate analyte, smoke, fumes, liquid,
gas, surgical
smoke, aerosol or vapour;
a device arranged and adapted to aspirate said analyte, smoke, fumes, liquid,
gas,
surgical smoke, aerosol or vapour;
a device arranged and adapted to mix a matrix with said aspirated analyte,
smoke,
.. fumes, liquid, gas, surgical smoke, aerosol or vapour; and
a mass and/or ion mobility spectrometer comprising: (i) a collision surface
located
within a vacuum chamber of said spectrometer wherein, in use, analyte, smoke,
fumes,
Date Recue/Date Received 2024-04-10
- 22 -
liquid, gas, surgical smoke, aerosol or vapour is arranged to impact upon said
collision
surface so as to form analyte ions; and (ii) a mass and/or ion mobility
analyser for mass
and/or ion mobility analysing said analyte ions.
The surgical or electrosurgical apparatus may be configured to perform any of
the
.. method described herein.
Although the mixture of the matrix and analyte have been described above as
impacting on the collision surface, it has been found that there may be
improvements in
ionisation of the analyte without impacting the mixture on the collision
surface. For
example, techniques other than colliding the mixture with a collision surface
may be used
to atomise the mixture. Accordingly, the step of colliding the first clusters
or first droplets of
the diluted or dissolved matrix with the collision surface is not an essential
feature of the
invention.
Therefore, from a second aspect the present invention also provides a method
mass and/or ion mobility spectrometry comprising:
providing an analyte;
supplying a matrix compound to said analyte such that said analyte is diluted
by,
dissolved in, or forms first clusters with said matrix; and
fragmenting or disintegrating said first clusters or first droplets of said
diluted or
dissolved matrix into a plurality of second smaller clusters or droplets.
The first clusters or first droplets may be fragmented or disintegrated by
applying
laser irradiation, by applying ultrasonic energy, by a glow discharge
technique or by
photoionization.
The method of the second aspect may comprise any of the features described in
relation to the first aspect of the invention, except wherein the mixture is
not collided on the
collision surface.
For example, the method may comprise subjecting analyte in, or derived from,
said
second smaller clusters or droplets to ionisation. Optionally, said ionisation
is performed
by an ionisation source selected from the group consisting of: a corona
discharge
ionisation source; a reagent ion Ionisation source; a Photo Ionisation source;
a Chemical
Ionisation source; an Electron Impact ("El") ionisation source; a Field
Ionisation ("FI")
source; a Field Desorption ("FD") Ionisation source; an Inductively Coupled
Plasma ("ICP")
Ionisation source; a Fast Atom Bombardment ("FAB") Ionisation source; a Liquid
Secondary Ion Mass Spectrometry ("LSIMS") Ionisation source; a Desorption
Electrospray
Ionisation ("DESI") Ionisation source; a Nickel-63 radioactive Ionisation
source; a
Thernnospray Ionisation source; an Atmospheric Sampling Glow Discharge
Ionisation
("ASGDI") Ionisation source; a Glow Discharge ("GD") Ionisation source; an
Impactor
Ionisation source; a Direct Analysis in Real Time ("DART") Ionisation source;
a Laserspray
Ionisation ("LSI") source; a Sonicspray Ionisation ("SSI") source; a Matrix
Assisted Inlet
Ionisation ("MAII") source; a Solvent Assisted Inlet Ionisation ("SAII")
source; a Desorption
Electrospray Ionisation ("DESI") source; a desorption electroflow focusing
ionisation
("DEFFI") source; a Laser Ablation Electrospray Ionisation ("LAESI") source;
and a Surface
Assisted Laser Desorption Ionisation ("SALDI") source.
Date Recue/Date Received 2024-04-10
- 23 -
The second aspect of the invention also provides, an apparatus for performing
mass and/or ion mobility spectrometry comprising:
a mixing region for mixing analyte with a matrix compound such that, in use,
said
analyte is diluted by, dissolved in, or forms first clusters with said matrix;
and
a device arranged and adapted to fragment or disintegrate said first clusters
or first
droplets of said diluted or dissolved matrix into a plurality of second
smaller clusters or
droplets.
The apparatus may comprise an analyte inlet for receiving analyte and/or a
matrix
inlet for receiving a matrix compound.
The apparatus according to the second aspect may comprise any of the features
described in relation to the first aspect, except wherein the apparatus need
not comprise
the collision surface or the device arranged and adapted to cause said first
clusters or first
droplets of the diluted or dissolved analyte to collide with the collision
surface.
The invention also provides a mass and/or ion mobility spectrometer comprising
such an apparatus.
The invention also provides a method of surgery or electrosurgery comprising
the
method of the second aspect, wherein the method comprises:
contacting biological tissue with a surgical or electrosurgical tool and
activating said
tool so as to generate smoke, fumes, liquid, gas, surgical smoke, aerosol or
vapour
containing said analyte;
aspirating said smoke, fumes, liquid, gas, surgical smoke, aerosol or vapour;
mixing said matrix with said aspirated smoke, fumes, liquid, gas, surgical
smoke,
aerosol or vapour;
fragmenting or disintegrating first clusters or droplets of the mixture to
form a
plurality of second smaller clusters or droplets;
forming analyte ions from the second clusters or droplets; and
mass and/or ion mobility analysing said analyte ions.
The invention also provides a surgical or electrosurgical apparatus comprising
the
apparatus of the second aspect, wherein the surgical or electrosurgical
apparatus
comprises:
a surgical or electrosurgical tool comprising one or more electrodes;
a device arranged and adapted to activate said tool when said tool is in
contact, in
use, with biological tissue so as to generate smoke, fumes, liquid, gas,
surgical smoke,
aerosol or vapour containing the analyte;
a device arranged and adapted to aspirate said analyte, smoke, fumes, liquid,
gas,
surgical smoke, aerosol or vapour;
a device arranged and adapted to mix a matrix with said aspirated analyte,
smoke,
fumes, liquid, gas, surgical smoke, aerosol or vapour; and
a device arranged and adapted to fragment or disintegrate first clusters or
droplets
of the mixture to form a plurality of second smaller clusters or droplets;
a device arranged and adapted to ionise the analyte to form analyte ions from
the
second clusters or droplets; and
Date Recue/Date Received 2024-04-10
- 24 -
a mass and/or ion mobility analyser for analysing said analyte ions.
According to an aspect there is provided a method of Rapid Evaporative
Ionisation
Mass Spectrometry ("REIMS"). The method includes providing an analyte;
supplying a
matrix compound to the analyte such that the analyte dissolves in the matrix
and forms first
dissolved analyte droplets; and colliding the first dissolved analyte droplets
with a collision
surface or a gas such that the first dissolved analyte droplets fragment into
a plurality of
second smaller dissolved analyte droplets.
The collisional ion generator REIMS technique described in the background
section
involves generating a sample of aerosol droplets that are comprised of aqueous
droplets
covered with polar lipids. The aqueous droplets are accelerated by the free
jet expansion
in the atmospheric inlet of a mass spectrometer such that the high velocity
droplets impact
onto a collision surface or other gaseous particles, producing gaseous ions of
the polar
lipid molecules. However, the ionisation yield of this technique is relatively
low.
It has been recognised that the ion yield in the conventional method is
relatively low
due to the poor conversion rate of the droplets into individual molecular
species mostly
caused by the strong intermolecular bonds between the analyte molecules.
An embodiment involves dissolving the analyte in a matrix thereby
substantially
eliminating the intermolecular bonding between the analyte molecules. As such,
when the
dissolved analyte is subsequently collided with the collision surface or a gas
so as to
.. fragment into droplets, any given droplet is likely to contain fewer
analyte molecules than it
would if the matrix were not present.
The approach according to an embodiment therefore leads to the more efficient
generation of ions when the matrix in each droplet is evaporated.
The step of colliding the first dissolved analyte droplets with the collision
surface or
gas causes the step of evaporating the matrix from the analyte by converting
kinetic energy
of the analyte and matrix into heat.
The step of colliding the first dissolved analyte droplets may cause the
smaller
dissolved analyte droplets to be generated, at least some of which may have
only a single
analyte molecule therein. This enhances the ionisation process.
The analyte may, for example, comprise a polar lipid and the vapour or aerosol
may
comprise aqueous droplets covered with the polar lipids.
The analyte may comprise triglycerides.
The analyte to which the matrix is supplied may comprise ionised analyte
molecules.
The method may further comprise the step of generating the gas phase analyte,
vapour analyte, aerosol, or liquid from a sample to be analysed.
The gas phase analyte, vapour analyte or aerosol may be generated by heating
the
sample containing the analyte, may by diathermic evaporation of the sample.
The method may either be part of a surgical method or a non-surgical method.
For
.. example, the method may be a surgical method in which the sample may be
human or
animal tissue containing the analyte. The sample may be subjected to
electrosurgical
diathermic evaporation, or other forms of rapid evaporation, in order to form
the gas phase
Date Recue/Date Received 2024-04-10
- 25 -
analyte, vapour analyte or aerosol. By way of example only, the device and
method may
be used for the identification of human tissues in breast cancer surgery. By
analysing the
analyte ions it is possible to determine whether or not the tissues are
cancerous.
Alternatively, the method may comprise a non-surgical method. For example,
human or animal tissue that is not part of the human or animal body (i.e.
previously
excised, deposited or removed) may be analysed, or samples or biological
tissues other
than human or animal tissues may be analysed. Again, by analysing the analyte
ions it is
possible to determine the properties or constituents of the sample, such as
whether or not
they contain cancerous tissues.
The embodiment may be used in other non-surgical methods, such as country of
origin identification, pharmaceutical testing, food safety testing (e.g.
dairy), cosmetics
testing, military applications, air pollution testing, post-mortem analysis,
microbe
identification (e.g. bacteria), and automated sampling.
The method may be used to analyse non-biological samples and compounds.
The analyte that is formed from the sample may be partially charged and/or may
have a relatively high organic content.
The method may further comprise evaporating the matrix from the analyte in the
second smaller dissolved analyte droplets so as to provide analyte ions that
are
substantially separate from the matrix.
The step of evaporating the matrix from the analyte may result in separation
of the
ionically dissociated analyte ions from the matrix so as to form gas phase
analyte ions.
After the step of evaporating the matrix from the analyte, the method may
further
comprise trapping analyte ions in an ion trap and/or guiding analyte ions
using an ion
guide.
The method may further comprise analysing the analyte ions.
The step of analysing the analyte ions may further comprises: (i) mass
analysing
the analyte ions; (ii) analysing the ion mobility or differential ion mobility
of the analyte ions;
(iii) analysing the ionic cross-sections or collision cross sections of the
analyte ions; (iv)
separating the analyte ions according to their ion mobility or differential
ion mobility; (v)
separating the analyte ions according to their ion mobility or differential
ion mobility prior to
mass analysing the analyte ions; or (vi) excluding or discarding analyte ions
based upon
their ion mobility or differential ion mobility.
The matrix may be supplied to the analyte whilst the analyte is in gas phase,
vapour
form, aerosol form or in liquid phase.
The step of supplying a matrix compound to the analyte may comprise supplying
matrix molecules to, and intermixing the matrix molecules with, the analyte
whilst the matrix
is in a gas phase.
The mixture of the analyte and the matrix may be transferred from a high
pressure
region to a low pressure region such that the gas phase matrix cools and
condenses to a
liquid and the analyte dissolves in the liquid matrix so as to form the first
dissolved analyte
droplets.
Date Recue/Date Received 2024-04-10
- 26 -
The matrix may be selected from the group consisting of: (i) a solvent for the
analyte, smoke, fumes, liquid, gas, surgical smoke, aerosol or vapour; (ii) an
organic
solvent; (iii) a volatile compound; (iv) polar molecules; (v) water; (vi) one
or more alcohols;
(vii) methanol; (viii) ethanol; (ix) isopropanol; (x) acetone; and (xi)
acetonitrile.
The step of colliding the first dissolved analyte droplets with the collision
surface or
the gas further may comprise accelerating the first dissolved analyte droplets
onto the
collision surface or into the gas.
The step of accelerating the first dissolved analyte droplets into the
collision surface
or the gas may comprise using a pressure differential to accelerate the first
dissolved
analyte droplets onto the collision surface or into the gas.
The method may further comprise analysing the analyte ions using a mass
spectrometer comprising an atmospheric interface adjacent a vacuum chamber,
wherein
the first dissolved analyte droplets are accelerated into the collision
surface or gas by a
pressure difference across the atmospheric interface.
The method may further comprise heating the collision surface (or
alternatively a
collision gas).
The method may further comprise heating the collision surface (or
alternatively a
collision gas) to a temperature selected from the group consisting of: (i)
about < 100 C; (ii)
about 100-200 C; (iii) about 200-300 C; (iv) about 300-400 C; (v) about 400-
500 C; (vi)
about 500-600 C; (vii) about 600-700 C; (viii) about 700-800 C; (ix) about
800-900 C; (x)
about 900-1000 C; (xi) about 1000-1100 C; and (xii) about > 1100 C
The matrix may be supplied to the analyte by a matrix introduction conduit.
The method may further comprise analysing analyte ions using an ion analyser
arranged downstream of an outlet of the matrix introduction conduit.
The distance x between the outlet of the matrix introduction conduit and an
inlet of
the ion analyser may be selected from the group consisting of: (i) about 0.1
to 0.5 mm; (ii)
about 0.5-1.0 mm; (iii) about 1.0-1.5 mm; (iv) about 1.5-2.0 mm; (v) about 2.0-
2.5 mm; (vi)
about 2.5-3.0 mm; (vii) about 3.0-3.5 mm; (viii) about 3.5-4.0 mm; (ix) about
4.0-4.5 mm;
(x) about 4.5-5.0 mm; (xi) about 5.0-5.5 mm; (xii) about 5.5-6.0 mm; (xiii)
about 6.0-6.5
mm; (xiv) about 6.5-7.0 mm; (xv) about 7.0-7.5 mm; (xvi) about 7.5-8.0 mm;
(xvii) about
8.0-8.5 mm; (xviii) about 8.5-9.0 mm; (xix) about 9.0-9.5 mm; (xx) about 9.5-
10.0 mm; (W)
about 0.1-10 mm; ()ocii) about 0.1-7.5 mm; (xxiii) about 0.1-5.1 mm; (xxiv)
about 0.5-5.1
mm; and (m) 0.5-5.0 mm.
The matrix may be supplied to the analyte by a matrix introduction conduit at
a flow
rate selected from the group consisting of: (i) about 50-100 pl/min; (ii)
about 100-150
pl/min; (iii) about 150-200 pl/min; (iv) about 200-250 pl/min; (v) about 250-
300 pl/min; (vi)
about 300-350 pl/min; (vii) about 350-400 pl/min; (viii) about 400-450 pl/min;
(ix) about 450-
500 pl/min; (x) about 500-550 pl/min; (xi) about 550-600 pl/min; (xii) about
600-650 pl/min;
(xiii) about 650-700 pl/min; (xiv) about 700-750 pl/min; (xv) about 750-800
pl/min; (xvi)
about 800-850 pl/min; (xvii) about 850-900 pl/min; (xviii) about 900-950
pl/min; (xix) about
950-1000 pl/min; (xx) about 50 pl/min to 1 ml/min; (xW) about 100-800 pl/min;
()xii) about
150-600 pl/min; and (xxiii) about 200-400 pl/min.
Date Recue/Date Received 2024-04-10
- 27 -
An outlet of the matrix introduction conduit may be opposite or coaxial with
an inlet
of an ion analyser.
The method may further comprise mass and/or ion mobility analysing analyte
ions
with an ion analyser to obtain analyte ion data, and wherein the method
further comprises
analysing lockmass, lock mobility or calibration ions, and may calibrating the
ion analyser
or adjusting analyte ion data based upon the data obtained from analysing the
lockmass,
lock mobility or calibration ions.
According to another aspect there is provided a method of mass and/or ion
mobility
spectrometry, comprising a method as described above.
According to another aspect there is provided apparatus for performing rapid
evaporative ionisation mass spectrometry ("REIMS") comprising:
an analyte inlet for receiving analyte;
a mixing region for mixing the analyte with a matrix compound such that the
analyte
dissolves, in use, in the matrix and forms first dissolved analyte droplets;
a collision surface or a gas; and
a device arranged and adapted to cause the first dissolved analyte droplets to
collide with the collision surface or the gas such that the first dissolved
analyte droplets
fragment into a plurality of second smaller dissolved analyte droplets.
The apparatus may further comprise a device arranged and adapted to evaporate
the matrix from the analyte in the second smaller dissolved analyte droplets
so as to
provide analyte ions that are separate from the matrix.
The apparatus may further comprise a device arranged and adapted to evaporate
the matrix from the analyte so as to cause charge transfer to or from the
analyte so as to
ionise the analyte to form analyte ions.
The apparatus may further comprise an ion trap and/or an ion guide.
The apparatus may further comprise a device which is arranged and adapted
after
the matrix has been evaporated, in use, from the analyte, to trap analyte ions
in the ion trap
and/or to guide analyte ions using the ion guide.
The apparatus may further comprise an analyser for analysing the analyte ions.
The analyser may comprise: (i) a mass analyser for mass analysing the analyte
ions; (ii) an ion mobility or differential ion mobility analyser; (iii) an
analyser for analysing
the ionic cross-sections or collision cross sections of the analyte ions; (iv)
a separator for
separating the analyte ions according to their ion mobility or differential
ion mobility; (v) a
separator for separating the analyte ions according to their ion mobility or
differential ion
mobility prior to mass analysing the analyte ions; or (vi) a device arranged
and adapted to
exclude or discard analyte ions based upon their ion mobility or differential
ion mobility.
The matrix may be supplied, in use, to the analyte whilst the analyte is in
gas
phase, vapour form, aerosol form or in liquid phase.
The apparatus may further comprise a device arranged and adapted to supply
matrix molecules to, and to intermix the matrix molecules with, the analyte
whilst the matrix
is in a gas phase.
Date Recue/Date Received 2024-04-10
- 28 -
The apparatus may further comprise a device which is arranged and adapted to
transfer the mixture of the analyte and the matrix from a high pressure region
to a low
pressure region such that the gas phase matrix cools and condenses to a liquid
and the
analyte dissolves in the liquid matrix so as to form the first dissolved
analyte droplets.
The matrix may be selected from the group consisting of: (i) a solvent for the
analyte, smoke, fumes, liquid, gas, surgical smoke, aerosol or vapour; (ii) an
organic
solvent; (iii) a volatile compound; (iv) polar molecules; (v) water; (vi) one
or more alcohols;
(vii) methanol; (viii) ethanol; (ix) isopropanol; (x) acetone; and (xi)
acetonitrile.
According to another aspect there is provided a mass and/or ion mobility
spectrometer comprising apparatus as described above.
The spectrometer may further comprise a device which is arranged and adapted
to
accelerate the first dissolved analyte droplets into the collision surface or
the gas.
The spectrometer may further comprise a device arranged and adapted to
maintain
a pressure differential to accelerate the first dissolved analyte droplets
into the collision
surface or the gas.
The spectrometer may further comprise an analyser which is arranged to analyse
the analyte ions, wherein the spectrometer further comprises an atmospheric
interface
adjacent a vacuum chamber, wherein the first dissolved analyte droplets are
accelerated
into the collision surface or the gas by a pressure difference across the
atmospheric
interface.
The spectrometer may further comprise a heater for heating the collision
surface (or
alternatively a collision gas).
The heater may be arranged to heat the collision surface (or alternatively a
collision
gas) to a temperature selected from the group consisting of: (i) about < 100
C; (ii) about
100-200 C; (iii) about 200-300 C; (iv) about 300-400 C; (v) about 400-500
C; (vi) about
500-600 C; (vii) about 600-700 C; (viii) about 700-800 C; (ix) about 800-
900 C; (x) about
900-1000 C; (xi) about 1000-1100 C; and (xii) about > 1100 C
The spectrometer may further comprise a matrix introduction conduit for
supplying
the matrix to the analyte.
The spectrometer may further comprise an ion analyser for analysing the
analyte
ions, wherein the ion analyser is arranged downstream of an outlet of the
matrix
introduction conduit.
The distance x between the outlet of the matrix introduction conduit and an
inlet of
the ion analyser may be selected from the group consisting of: (i) about 0.1
to 0.5 mm; (ii)
about 0.5-1.0 mm; (iii) about 1.0-1.5 mm; (iv) about 1.5-2.0 mm; (v) about 2.0-
2.5 mm; (vi)
about 2.5-3.0 mm; (vii) about 3.0-3.5 mm; (viii) about 3.5-4.0 mm; (ix) about
4.0-4.5 mm;
(x) about 4.5-5.0 mm; (xi) about 5.0-5.5 mm; (xii) about 5.5-6.0 mm; (xiii)
about 6.0-6.5
mm; (xiv) about 6.5-7.0 mm; (xv) about 7.0-7.5 mm; (xvi) about 7.5-8.0 mm;
(xvii) about
8.0-8.5 mm; (xviii) about 8.5-9.0 mm; (xix) about 9.0-9.5 mm; ()o() about 9.5-
10.0 mm; ()o<i)
about 0.1-10 mm; (xxii) about 0.1-7.5 mm; (xxiii) about 0.1-5.1 mm; (xxiv)
about 0.5-5.1
mm; and ()c<v) about 0.5-5.0 mm.
Date Recue/Date Received 2024-04-10
- 29 -
The spectrometer may further comprise a pump for supplying the matrix to the
analyte via a matrix introduction conduit at a flow rate selected from the
group consisting
of: (i) about 50-100 pl/min; (ii) about 100-150 pl/min; (iii) about 150-200
pl/min; (iv) about
200-250 pl/min; (v) about 250-300 pl/min; (vi) about 300-350 pl/min; (vii)
about 350-400
pl/min; (viii) about 400-450 pl/min; (ix) about 450-500 pl/min; (x) about 500-
550 pl/min; (xi)
about 550-600 pl/min; (xii) about 600-650 pl/min; (xiii) about 650-700 pl/min;
(xiv) about
700-750 pl/min; (xv) about 750-800 pl/min; (xvi) about 800-850 pl/min; (xvii)
about 850-900
pl/min; (xviii) about 900-950 pl/min; (xix) about 950-1000 pl/min; ()x) about
50 pl/min to 1
ml/min; (xW) about 100-800 pl/min; ()ocii) about 150-600 pl/min; and (xxiii)
about 200-400
pl/min.
An outlet of the matrix introduction conduit may be opposite or coaxial with
an inlet
of an ion analyser.
The mass spectrometer may further comprise a mass analyser for analysing the
analyte ions to obtain analyte ion data, and wherein the mass analyser may be
further
arranged to analyse lockmass, lock mobility or calibration ions, and to
calibrate the ion
analyser or adjust analyte ion data based upon the data obtained from
analysing the
lockmass, lock mobility or calibration ions.
According to another aspect there is provided a method of electrosurgery
comprising:
contacting biological tissue with an electrosurgical tool and activating the
electrosurgical tool so as to generate analyte, smoke, fumes, liquid, gas,
surgical smoke,
aerosol or vapour;
aspirating the analyte, smoke, fumes, liquid, gas, surgical smoke, aerosol or
vapour;
mixing a matrix with the aspirated analyte, smoke, fumes, liquid, gas,
surgical
smoke, aerosol or vapour;
causing the aspirated analyte, smoke, fumes, liquid, gas, surgical smoke,
aerosol or
vapour in combination with the matrix to impact upon a collision surface (or
alternatively a
collision gas) may located within a vacuum chamber of a mass spectrometer in
order to
form analyte ions; and
mass and/or ion mobility analysing the analyte ions.
According to another aspect there is provided an Electrosurgical apparatus
comprising:
a rapid evaporative ionisation mass spectrometry ("REIMS") electrosurgical
tool
comprising one or more electrodes;
a device arranged and adapted to activate the electrosurgical tool when the
electrosurgical tool is in contact, in use, with biological tissue so as to
generate analyte,
smoke, fumes, liquid, gas, surgical smoke, aerosol or vapour;
a device arranged and adapted to aspirate the analyte, smoke, fumes, liquid,
gas,
surgical smoke, aerosol or vapour;
a device arranged and adapted to mix a matrix with the aspirated analyte,
smoke,
fumes, liquid, gas, surgical smoke, aerosol or vapour; and
Date Recue/Date Received 2024-04-10
- 30 -
a mass and/or ion mobility spectrometer comprising: (i) a collision surface
(or
alternatively a collision gas) may located within a vacuum chamber of the mass
spectrometer wherein, in use, analyte, smoke, fumes, liquid, gas, surgical
smoke, aerosol
or vapour is arranged to impact upon the collision surface (or alternatively a
collision gas)
so as to form analyte ions; and (ii) a mass analyser for mass analysing the
analyte ions.
According to an embodiment the matrix may initially be supplied as a solid
e.g.
powder and sublimated or melted and evaporated so as to form matrix in vapour
or gas-
phase that is intermixed with the analyte.
Alternatively, the matrix may be supplied to, and intermixed with, the analyte
as a
liquid, aerosol or vapour. If the analyte and/or matrix is in liquid form then
the mixture of
analyte and matrix may need to be subsequently converted into the first
dissolved analyte
droplets e.g. by spraying.
The dielectric constant of the matrix may be sufficiently high such that the
solvation
of the analyte involves ionic dissociation resulting in solvated ions of the
analyte present in
the condensed phase. In these cases, the impact on the collision surface (or
alternatively
a collision gas) is more likely to produce solvated ions in the gas phase,
which may
eventually yield ions formed by deprotonation (in a negative ion mode, i.e. [M-
I-1]-), ions
formed by protonation (in a positive ion mode, i.e. [M+H]), and/or molecular
ions.
lsopropanol is a particularly advantageous matrix to use, e.g., for lipid
species.
By way of example, for analytes comprising polar lipids, the matrix may be or
may
comprise, low molecular weight alcohols (e.g. methanol, ethanol, isopropanol)
or ketones
(e.g. acetone). These matrices have been shown to enhance the ionisation of
all or certain
species otherwise detected in the absence of the matrix vapours at lower
intensity.
The mixture of analyte and matrix may be a homogeneous or heterogeneous
mixture.
Voltages may be applied to the ion trap or ion guide so as to trap or guide
the ions
respectively. The ions may then be delivered from the ion trap or ion guide to
an ion
analyser for analysing the mass and/or ion mobility of the ions.
The ions may be separated according to ion mobility prior to being mass
analysed.
Ions may then be excluded or discarded based upon their ion mobility.
Any one of the above mentioned ranges may be combined with any one of the
ranges in the list of ranges for distance x.
The inlet of the ion analyser may be an aperture or orifice that separates a
vacuum
chamber of the ion analyser from a higher pressure region upstream of the ion
analyser.
For example, the inlet may be an atmospheric pressure interface.
According to an alternative embodiment the matrix introduction conduit may
deliver
matrix directly into a sample transfer conduit that performs the step of
providing the
analyte.
Alternatively, a sample transfer conduit may be provided that performs the
step of
providing the analyte, and the outlet of the matrix introduction conduit may
be provided at a
location about the circumference of sample transfer conduit. A gas flow may be
arranged
Date Recue/Date Received 2024-04-10
- 31 -
so as to sweep the matrix from the outlet to the inlet of the ion analyser
that analyses the
ions.
The device for evaporating the sample may comprise an electrosurgical tool
such
as a diathermic device.
The device may have an end, point or region for inserting onto a sample to
evaporate the sample and wherein the analyte inlet is adjacent the end, point
or region.
The apparatus may comprise a source of the matrix compound for supplying the
matrix compound to the conduit.
The accelerating means may comprise a vacuum pump for creating a pressure
differential between a first region and a second region for accelerating the
first dissolved
analyte droplets between the two regions and onto the collision surface (or
alternatively a
collision gas).
The apparatus may comprise a mass spectrometer having an atmospheric interface
arranged between the first and second regions, wherein the second region may
comprise a
vacuum chamber that is connected to a vacuum pump and which houses the
collision
surface (or alternatively a collision gas).
The apparatus may comprise an ion trap or ion guide for trapping or guiding
the
analyte ions.
The ion analyser may comprise a mass and/or ion mobility analyser or
spectrometer.
The apparatus may be arranged and configured to perform any one of the methods
described herein.
The mixing region may be provided upstream of the inlet to the ion analyser,
or the
mixing region may be provided at least in part downstream of the ion analyser.
The inlet of the ion analyser may be an aperture or orifice that separates a
vacuum
chamber of the ion analyser from a higher pressure region upstream of the ion
analyser.
For example, the inlet may comprise an atmospheric pressure interface.
The matrix introduction conduit may deliver matrix directly into a sample
transfer
conduit that performs the step of providing the analyte.
Alternatively, a sample transfer conduit may be provided that performs the
step of
providing the analyte and the outlet of the matrix introduction conduit may be
provided at a
location about the circumference of sample transfer conduit. A gas flow may be
arranged
so as to sweep the matrix from the outlet to the inlet of the ion analyser
that analyses the
ions.
The apparatus may comprise a source of the lockmass, lock mobility or
calibration
compound or ions.
The lockmass, lock mobility or calibration compound/ions may be introduced
into
the matrix introduction conduit, the analyte introduction conduit or may be
supplied in a
separate conduit.
According to the embodiments, aerosol particles containing the analyte (or gas
phase analyte molecules) may be introduced into a mass spectrometer together
with a
volatile matrix compound, which may be an organic solvent. The volatile matrix
compound
Date Recue/Date Received 2024-04-10
- 32 -
may be introduced to the analyte as a solid (e.g., powder), liquid, aerosol or
vapour. The
mixture of analyte and matrix may be drawn into the mass spectrometer by a
pressure
differential across the inlet to the spectrometer. The lower pressure inside
the mass
spectrometer results in the gas that entrains the analyte and matrix
expanding, causing a
temperature drop in the free jet region. This causes gaseous or vapourised
analyte and/or
matrix to condense such that the analyte dissolves in the matrix. The role of
the matrix
compound may be to produce aerosol particles containing the matrix in excess
of the
analyte molecules and incorporating the analyte molecules in solvated form.
The solvation
substantially eliminates the intermolecular secondary binding forces between
the analyte
molecules, since each dissolved analyte molecule is fully surrounded by the
matrix
molecules. The separation of analyte molecules in condensed phase increases
the
probability that when the aerosol particles impact upon the collision surface
they will form
clusters that each contain only a single analyte molecule. The matrix molecule
may have a
high dielectric constant and/or a high vapour pressure.
BRIEF DESCRIPTION OF THE DRAWINGS
Various embodiments will now be described, by way of example only, and with
reference to the accompanying drawings in which:
Fig. 1 illustrates a method of rapid evaporative ionisation mass spectrometry
("REIMS") wherein an RF voltage is applied to bipolar forceps resulting in the
generation of
an aerosol or surgical plume which is captured through an irrigation port of
the bipolar
forceps and is then transferred to a mass spectrometer for ionisation and mass
analysis;
Fig. 2 shows an embodiment in which the analyte and matrix may be provided in
the gas or vapour phase;
Fig. 3 shows another embodiment in which the analyte and matrix may be
provided
in the liquid phase;
Fig. 4A shows a mass spectrum obtained without the use of a matrix and Fig. 4B
shows a mass spectrum obtained using a matrix;
Fig. 5A shows an embodiment of a mass spectrometer interface comprising a
Venturi device for introducing analyte aerosol and matrix into a mass
spectrometer, Fig. 5B
shows an expanded view of Fig. 5B, and Fig. 5C shows a close up of the
sampling device
in the interface;
Fig. 6 shows how the ion signal detected using the embodiment of Fig. 5 varies
depending on the distance between the outlet of the matrix conduit and the
inlet of the ion
analyser;
Fig. 7 shows how the ion signal detected using the embodiment of Fig. 5 varies
depending on flow rate of the matrix;
Figs. 8A-8I show mass spectra obtained using different isopropanol matrix flow
rates;
Fig. 9A shows a mass spectrum obtained using a lockmass compound and Fig. 9B
shows a mass spectrum obtained without using a lockmass compound;
Date Recue/Date Received 2024-04-10
- 33 -
Fig. 10 shows the results of a principle component analysis on data obtained
over
different days, both with and without the use of lockmass ions;
Fig. 11 shows the results of a principle component analysis on data obtained
for
different tissue types, both with and without the use of lockmass ions;
Fig. 12A shows an embodiment wherein the collision surface is spherical and
Fig.
12B shows an embodiment wherein the collision surface is coil-shaped;
Fig. 13A shows a mass spectrum obtained using a collision surface that is not
heated and Fig. 13B shows a mass spectrum obtained using a heated collision
surface;
Fig. 14A shows a mass spectrum obtained when analysing a sample wherein an
IPA matrix is introduced upstream of a heated collision surface, Fig. 14B
shows a mass
spectrum obtained from the same analysis except when a matrix is not used, and
Fig. 14C
shows a mass spectrum obtained from the same analysis but when the collision
surface is
not heated and a matrix is not used;
Fig. 15A shows the total ion current detected for several different distances
between the exit of a matrix introduction conduit having an inner diameter of
250 pm
and the entrance to the mass spectrometer vacuum chamber, and Figs. 15B-15F
show the
mass spectra obtained at the different distances of Fig. 15A;
Fig. 16A shows the total ion current detected for several different distances
between the exit of a matrix introduction conduit having an inner diameter of
100 pm
and the entrance to the mass spectrometer vacuum chamber, and Figs. 16B-161
show the
mass spectra obtained at the different distances of Fig. 16A;
Fig. 17A shows the total ion current detected for several different distances
between the exit of a matrix introduction conduit having an inner diameter of
50 pm
and the entrance to the mass spectrometer vacuum chamber, and Figs. 17B-171
show the
mass spectra obtained at the different distances of Fig. 15A;
Figs. 18A-18C show three spectra obtained for matrix introduction conduits
having
internal diameters of 50 pm, 100 pm and 250 pm, respectively;
Fig. 19A shows the total ion current detected for several different distances
between the exit of a matrix introduction conduit having an inner diameter of
250 pm and
the coaxial entrance to the mass spectrometer inlet tube, and Figs. 19B-19H
show the
mass spectra obtained at the different distances of Fig. 19A;
Fig. 20A shows the total ion current detected for several different distances
between the exit of a matrix introduction conduit having an inner diameter of
100 pm and
the coaxial entrance to the mass spectrometer inlet tube, and Figs. 20B-20G
show the
mass spectra obtained at the different distances of Fig. 20A;
Fig. 21 shows the total ion current detected for several different distances
between
the exit of a matrix introduction conduit having an inner diameter of 50 pm
and the coaxial
entrance to the mass spectrometer inlet tube, and Figs. 21B-21I show the mass
spectra
obtained at the different distances of Fig. 21A;
Fig. 22A shows a mass spectrum obtained in negative ion mode without the
introduction of a matrix into the analyte stream and without the use of a
collision surface,
Fig. 228 shows a mass spectrum obtained with the introduction of a matrix into
the analyte
Date Recue/Date Received 2024-04-10
- 34 -
stream and without the use of a collision surface, and Fig. 22C shows a mass
spectrum
obtained with the introduction of a matrix into the analyte stream and with
the use of a
collision surface;
Fig. 23A shows a mass spectrum obtained in positive ion mode without the
introduction of a matrix into the analyte stream and without the use of a
collision surface,
Fig. 22B shows a mass spectrum obtained with the introduction of a matrix into
the analyte
stream and without the use of a collision surface, and Fig. 22C shows a mass
spectrum
obtained with the introduction of a matrix into the analyte stream and with
the use of a
collision surface;
Fig. 24A shows a mass spectrum obtained from the analysis of normal breast
tissue
without the use of a matrix, and Fig. 24B shows a mass spectrum obtained from
the
analysis of normal breast tissue with the use of a matrix;
Fig. 25 shows a method of analysis that comprises building a classification
model
according to various embodiments;
Fig. 26 shows a set of reference sample mass spectra obtained from two classes
of
known reference samples;
Fig. 27 shows a multivariate space having three dimensions defined by
intensity
axes, wherein the multivariate space comprises plural reference points, each
reference
point corresponding to a set of three peak intensity values derived from a
reference sample
mass spectrum;
Fig. 28 shows a general relationship between cumulative variance and number of
components of a PCA model;
Fig. 29 shows a PCA space having two dimensions defined by principal component
axes, wherein the PCA space comprises plural transformed reference points or
scores,
each transformed reference point corresponding to a reference point of Fig.
27;
Fig. 30 shows a PCA-LDA space having a single dimension or axis, wherein the
LDA is performed based on the PCA space of Fig. 29, the PCA-LDA space
comprising
plural further transformed reference points or class scores, each further
transformed
reference point corresponding to a transformed reference point or score of
Fig. 29;
Fig. 31 shows a method of analysis that comprises using a classification model
according to various embodiments;
Fig. 32 shows a sample mass spectrum obtained from an unknown sample;
Fig. 33 shows the PCA-LDA space of Fig. 30, wherein the PCA-LDA space further
comprises a PCA-LDA projected sample point derived from the peak intensity
values of the
sample mass spectrum of Fig. 32;
Fig. 34 shows a method of analysis that comprises building a classification
library
according to various embodiments; and
Fig. 35 shows a method of analysis that comprises using a classification
library
according to various embodiments.
DETAILED DESCRIPTION
Date Recue/Date Received 2024-04-10
- 35 -
Various embodiments will now be described in more detail below which in
general
relate to generating an aerosol, surgical smoke or vapour from one or more
regions of a
target (e.g., in vivo tissue) using an ambient ionisation ion source.
The aerosol, surgical smoke or vapour is then mixed with a matrix and
aspirated
.. into a vacuum chamber of a mass spectrometer. The mixture is caused to
impact upon a
collision surface causing the aerosol, smoke or vapour to be ionised by impact
ionisation
which results in the generation of analyte ions.
The resulting analyte ions (or fragment or product ions derived from the
analyte
ions) are then mass and/or ion mobility analysed and the resulting mass and/or
ion mobility
spectrometric data may then be subjected to multivariate analysis in order to
determine
one or more properties of the target in real time.
For example, the multivariate analysis may enable a determination to be made
as
to whether or not a portion of tissue which is currently being resected is
cancerous or not.
.. Ambient ionisation ion sources
According to various embodiments a device is used to generate an aerosol,
smoke
or vapour from one or more regions of a target (e.g., in vivo tissue). The
device may
comprise an ambient ionisation ion source which is characterised by the
ability to generate
.. analyte aerosol, smoke or vapour, e.g., from a native or unmodified target.
The aerosol,
smoke or vapour may then be mixed with a matrix and aspirated into a vacuum
chamber of
a mass and/or ion mobility spectrometer. The mixture may be caused to impact
upon a
collision surface causing the aerosol, smoke or vapour to be ionised by impact
ionisation
which results in the generation of analyte ions. The resulting analyte ions
(or fragment or
product ions derived from the analyte ions) may then be mass and/or ion
mobility analysed
and the resulting mass and/or ion mobility spectrometric data may be subjected
to
multivariate analysis or other mathematical treatment in order to determine
one or more
properties of the target, e.g., in real time. For example, the multivariate
analysis may
enable a determination to be made as to whether or not a portion of tissue
which is
.. currently being resected is cancerous or not.
It will be apparent that the requirement to add a matrix or a reagent directly
to a
sample prevents the ability to perform in vivo analysis of tissue and also,
more generally,
prevents the ability to provide a rapid simple analysis of target material.
In contrast, therefore, ambient ionisation techniques are particularly
advantageous
.. since firstly they do not require the addition of a matrix or a reagent to
the sample (and
hence are suitable for the analysis of in vivo tissue) and since secondly they
enable a rapid
simple analysis of target material to be performed. For example, other types
of ionisation
ion sources such as Matrix Assisted Laser Desorption Ionisation ("MALDI") ion
sources
require a matrix or reagent to be added to the sample prior to ionisation.
A number of different ambient ionisation techniques are known and are intended
to
fall within the scope of the present invention. As a matter of historical
record, Desorption
Electrospray Ionisation ("DESI") was the first ambient ionisation technique to
be developed
Date Recue/Date Received 2024-04-10
- 36 -
and was disclosed in 2004. Since 2004, a number of other ambient ionisation
techniques
have been developed. These ambient ionisation techniques differ in their
precise
ionisation method but they share the same general capability of generating gas-
phase ions
directly from native (i.e. untreated or unmodified) samples. A particular
advantage of the
various ambient ionisation techniques which are intended to fall within the
scope of the
present invention is that the various ambient ionisation techniques do not
require any prior
sample preparation. As a result, the various ambient ionisation techniques
enable both in
vivo tissue and ex vivo tissue samples to be analysed without necessitating
the time and
expense of adding a matrix or reagent to the tissue sample or other target
material.
A list of ambient ionisation techniques which are intended to fall within the
scope of
the present invention are given in the following table:
Acronym Ionisation technique
DESI Desorption electrospray ionization
DeSSI Desorption sonic spray ionization
DAPPI Desorption atmospheric pressure
photoionization
EASI Easy ambient sonic-spray ionization
JeDI Jet desorption electrospray ionization
TM-DESI Transmission mode desorption electrospray
ionization
LMJ-SSP Liquid microjunction-surface sampling probe
DICE Desorption ionization by charge exchange
Nano-DESI Nanospray desorption electrospray ionization
EADESI Electrode-assisted desorption electrospray
ionization
APTDCI Atmospheric pressure thermal desorption
chemical ionization
V-EASI Venturi easy ambient sonic-spray ionization
AFAI Air flow-assisted ionization
LESA Liquid extraction surface analysis
PTC-ESI Pipette tip column electrospray ionization
AFADESI Airflow-assisted desorption electrospray
ionization
DEFFI Desorption electro-flow focusing ionization
ESTASI Electrostatic spray ionization
PASIT Plasma-based ambient sampling ionization
transmission
DAPCI Desorption atmospheric pressure chemical
ionization
DART Direct analysis in real time
Date Recue/Date Received 2024-04-10
- 37 -
ASAP Atmospheric pressure solid analysis probe
APTDI Atmospheric pressure thermal desorption
ionization
PADI Plasma assisted desorption ionization
DBDI Dielectric barrier discharge ionization
FAPA Flowing atmospheric pressure afterglow
HAPGDI Helium atmospheric pressure glow discharge
ionization
APGDDI Atmospheric pressure glow discharge
desorption ionization
LTP Low temperature plasma
LS-APGD Liquid sampling-atmospheric pressure glow
discharge
MIPDI Microwave induced plasma desorption
ionization
MFGDP Microfabricated glow discharge plasma
RoPPI Robotic plasma probe ionization
PLASI Plasma spray ionization
MALDESI Matrix assisted laser desorption electrospray
ionization
ELDI Electrospray laser desorption ionization
LDTD Laser diode thermal desorption
LAESI Laser ablation electrospray ionization
CALDI Charge assisted laser desorption ionization
LA-FAPA Laser ablation flowing atmospheric pressure
afterglow
LADES! Laser assisted desorption electrospray
ionization
LDESI Laser desorption electrospray ionization
LEMS Laser electrospray mass spectrometry
LSI Laser spray ionization
IR-LAM ICI Infrared laser ablation metastable induced
chemical ionization
LDSPI Laser desorption spray post-ionization
Plasma assisted multiwavelength laser
PAMLDI
desorption ionization
HALDI High voltage-assisted laser desorption
ionization
PALDI Plasma assisted laser desorption ionization
ESSI Extractive electrospray ionization
PESI Probe electrospray ionization
Date Recue/Date Received 2024-04-10
- 38 -
ND-ESSI Neutral desorption extractive electrospray
ionization
PS Paper spray
Direct inlet probe-atmospheric pressure
DIP-APCI
chemical ionization
TS Touch spray
Wooden-tip Wooden-tip electrospray
CBS-SPME Coated blade spray solid phase
microextraction
TSI Tissue spray ionization
RADIO Radiofrequency acoustic desorption ionization
LIAD-ESI Laser induced acoustic desorption
electrospray ionization
SAWN Surface acoustic wave nebulization
UASI Ultrasonication-assisted spray ionization
SPA-nanoESI Solid probe assisted nanoelectrospray
ionization
PAUSI Paper assisted ultrasonic spray ionization
DPESI Direct probe electrospray ionization
ESA-Py Electrospray assisted pyrolysis ionization
APPIS Ambient pressure pyroelectric ion source
RASTIR Remote analyte sampling transport and
ionization relay
SACI Surface activated chemical ionization
DEMI Desorption electrospray metastable-induced
ionization
REIMS Rapid evaporative ionization mass
spectrometry
SPAM Single particle aerosol mass spectrometry
TDAMS Thermal desorption-based ambient mass
spectrometry
MAI I Matrix assisted inlet ionization
SAI I Solvent assisted inlet ionization
SwiFERR Switched ferroelectric plasma ionizer
Leidenfrost phenomenon assisted thermal
LPTD
desorption
According to an embodiment the ambient ionisation ion source may comprise a
rapid evaporative ionisation mass spectrometry ("REIMS") ion source wherein a
RF voltage
is applied to one or more electrodes in order to generate an aerosol or plume
of surgical
smoke by Joule heating.
Date Recue/Date Received 2024-04-10
- 39 -
However, it will be appreciated that other ambient ion sources including those
referred to above may also be utilised. For example, according to another
embodiment the
ambient ionisation ion source may comprise a laser ionisation ion source.
According to an
embodiment the laser ionisation ion source may comprise a mid-IR laser
ablation ion
source. For example, there are several lasers which emit radiation close to or
at 2.94 pm
which corresponds with the peak in the water absorption spectrum. According to
various
embodiments the ambient ionisation ion source may comprise a laser ablation
ion source
having a wavelength close to 2.94 pm on the basis of the high absorption
coefficient of
water at 2.94 pm. According to an embodiment the laser ablation ion source may
comprise
a Er:YAG laser which emits radiation at 2.94 pm.
Other embodiments are contemplated wherein a mid-infrared optical parametric
oscillator ("0 P0") may be used to produce a laser ablation ion source having
a longer
wavelength than 2.94 pm. For example, an Er:YAG pumped ZGP-OPO may be used to
produce laser radiation having a wavelength of e.g. 6.1 pm, 6.45 pm or 6.73
pm. In some
situations it may be advantageous to use a laser ablation ion source having a
shorter or
longer wavelength than 2.94 pm since only the surface layers will be ablated
and less
thermal damage may result. According to an embodiment a Co:MgF2 laser may be
used
as a laser ablation ion source wherein the laser may be tuned from 1.75-2.5
pm.
According to another embodiment an optical parametric oscillator ("OPO")
system pumped
by a Nd:YAG laser may be used to produce a laser ablation ion source having a
wavelength between 2.9-3.1 pm. According to another embodiment a CO2 laser
having a
wavelength of 10.6 pm may be used to generate the aerosol, smoke or vapour.
According to other embodiments the ambient ionisation ion source may comprise
an ultrasonic ablation ion source, or a hybrid electrosurgical ¨ultrasonic
ablation source
that generates a liquid sample which is then aspirated as an aerosol. The
ultrasonic
ablation ion source may comprise a focused or unfocussed ultrasound.
According to an embodiment the first device for generating aerosol, smoke or
vapour from one or more regions of a target may comprise an tool which
utilises an RF
voltage, such as continuous RF waveform. According to other embodiments a
radiofrequency tissue dissection system may be used which is arranged to
supply pulsed
plasma RF energy to a tool. The tool may comprise, for example, a PlasmaBlade
(RTM).
Pulsed plasma RF tools operate at lower temperatures than conventional
electrosurgical
tools (e.g. 40-170 C c.f. 200-350 C) thereby reducing thermal injury depth.
Pulsed
waveforms and duty cycles may be used for both cut and coagulation modes of
operation
by inducing electrical plasma along the cutting edge(s) of a thin insulated
electrode.
According to an embodiment the first device comprises a surgical water/saline
jet
device such as a resection device, a hybrid of such device with any of the
other devices
herein, an electrosurgery argon plasma coagulation device, a hybrid argon
plasma
coagulation and water/saline jet device.
Other embodiments are contemplated wherein the first device for generating
aerosol, smoke or vapour from the target may comprise an argon plasma
coagulation
("APC") device. An argon plasma coagulation device involves the use of a jet
of ionised
Date Recue/Date Received 2024-04-10
- 40 -
argon gas (plasma) that is directed through a probe. The probe may be passed
through an
endoscope. Argon plasma coagulation is essentially a non-contact process as
the probe is
placed at some distance from the target. Argon gas is emitted from the probe
and is then
ionized by a high voltage discharge (e.g., 6 kV). High-frequency electric
current is then
conducted through the jet of gas, resulting in coagulation of the target on
the other end of
the jet. The depth of coagulation is usually only a few millimetres.
The first device, surgical or electrosurgical tool, device or probe or other
sampling
device or probe disclosed in any of the aspects or embodiments herein may
comprise a
non-contact surgical device, such as one or more of a hydrosurgical device, a
surgical
water jet device, an argon plasma coagulation device, a hybrid argon plasma
coagulation
device, a water jet device and a laser device.
A non-contact surgical device may be defined as a surgical device arranged and
adapted to dissect, fragment, liquefy, aspirate, fulgurate or otherwise
disrupt biologic tissue
without physically contacting the tissue. Examples include laser devices,
hydrosurgical
devices, argon plasma coagulation devices and hybrid argon plasma coagulation
devices.
As the non-contact device may not make physical contact with the tissue, the
procedure
may be seen as relatively safe and can be used to treat delicate tissue having
low
intracellular bonds, such as skin or fat.
Rapid evaporative ionisation mass spectrometry ("REIMS")
Fig. 1 illustrates a method of rapid evaporative ionisation mass spectrometry
("REIMS") wherein bipolar forceps 1 may be brought into contact with in vivo
tissue 2 of a
patient 3. In the example shown in Fig. 1, the bipolar forceps 1 may be
brought into
contact with brain tissue 2 of a patient 3 during the course of a surgical
operation on the
patient's brain. An RF voltage from an RF voltage generator 4 may be applied
to the
bipolar forceps 1 which causes localised Joule or diathermy heating of the
tissue 2. As a
result, an aerosol or surgical plume 5 is generated. The aerosol or surgical
plume 5 may
then be captured or otherwise aspirated through an irrigation port of the
bipolar forceps 1.
The irrigation port of the bipolar forceps 1 is therefore reutilised as an
aspiration port. The
aerosol or surgical plume 5 may then be passed from the irrigation
(aspiration) port of the
bipolar forceps 1 to tubing 6 (e.g. 1/8" or 3.2 mm diameter Teflon (RTM)
tubing). The
tubing 6 is arranged to transfer the aerosol or surgical plume 5 to an
atmospheric pressure
interface 7 of a mass spectrometer 8.
According to various embodiments a matrix comprising an organic solvent such
as
isopropanol may be added to the aerosol or surgical plume 5 at the atmospheric
pressure
interface 7. The mixture of aerosol 3 and organic solvent may then be arranged
to impact
upon a collision surface within a vacuum chamber of the mass spectrometer 8.
According
to one embodiment the collision surface may be heated. The aerosol is caused
to ionise
upon impacting the collision surface resulting in the generation of analyte
ions. The
ionisation efficiency of generating the analyte ions may be improved by the
addition of the
organic solvent (i.e. the matrix).
Date Recue/Date Received 2024-04-10
-41 -
Analyte ions which are generated by causing the aerosol, smoke or vapour 5 to
impact upon the collision surface are then passed through subsequent stages of
the mass
and or ion mobility spectrometer and are subjected to mass and/or ion mobility
analysis in
a mass and/or ion mobility analyser. The mass analyser may, for example,
comprise a
quadrupole mass analyser or a Time of Flight mass analyser.
Fig. 2 shows a schematic of an embodiment. The device may comprise an ion
analyser 207 having an inlet 206, a vacuum region 208, a collision surface 209
and ion
optics 212 such as a Stepwave (RTM) ion guide arranged within the vacuum
region 208.
The device also may comprise a sample transfer tube 202 and a matrix
introduction
conduit 203. The sample transfer tube 202 has an inlet for receiving aerosol
sample 201
(which may correspond to the aerosol or surgical plume 5 described in relation
to Fig. 1)
from a sample being investigated and an outlet that is connected to the inlet
206 of the ion
analyser 207. The matrix introduction conduit 203 has an inlet for receiving a
matrix
compound and an outlet that intersects with the sample transfer tube 202 so as
to allow the
matrix 204 to be intermixed with the aerosol sample 201 in the sample transfer
tube 202. A
T-junction component may be provided at the junction between tubes 202, 203
and 206.
The tubes 202, 203 and 206 may be removably inserted into the T-junction.
A method of operating the device of Fig. 2 will now be described. A sample,
such
as a biological sample, may be subjected to the REIMS technique. For example,
a
diathermic device may be used to evaporate biological tissue from the sample
so as to
form an aerosol, e.g., as described above in relation to Fig. 1. The aerosol
particles 201
are then introduced into the inlet of the sample transfer tube 202. A matrix
compound 204
is introduced into the inlet of the matrix introduction conduit 203. The
aerosol particles 201
and matrix compound 204 are drawn towards the inlet 206 of the ion analyser
207 by a
pressure differential caused by the vacuum chamber 208 being at a lower
pressure than
the inlets to the tubes 202, 203. The aerosol particles 201 may encounter the
molecules of
matrix compound 204 in, and downstream of, the region that the sample transfer
tube 202
intersects with the matrix introduction conduit 203. The aerosol particles 201
intermix with
the matrix 204 so as to form aerosol particles containing matrix molecules
205, in which
both the molecular constituents of the aerosol sample 201 and the matrix
compound 204
are present. The matrix molecules 204 may be in excess compared to the
molecular
constituents of aerosol sample 201.
The particles 205 may exit the sample transfer tube 202 and pass into the
inlet 206
of the ion analyser 207. The particles 205 then enter into the decreased
pressure region
208 and gain substantial linear velocity due to the adiabatic expansion of gas
entering the
vacuum region 208 from the sample transfer tube 202 and due to the associated
free jet
formation. The accelerated particles 205 may impact on the collision surface
209, where
the impact event fragments the particles 205, leading to the eventual
formation of gas
phase ions 210 of the molecular constituents of the aerosol sample 201 and the
formation
of matrix molecules 211. The collision surface 209 may be controlled and
maintained at a
temperature that is substantially higher than the ambient temperature.
Date Recue/Date Received 2024-04-10
-42 -
The matrix 204 may include a solvent for the analyte 201, such that the
analyte 201
may dissolve in the matrix 204, thereby eliminating intermolecular bonding
between the
analyte molecules 201. As such, when the dissolved analyte 205 is then
collided with the
collision surface 209, the dissolved analyte 205 will fragment into droplets
and any given
droplet is likely to contain fewer analyte molecules than it would if the
matrix were not
present. This in turn leads to a more efficient generation of analyte ions 210
when the
matrix in each droplet is evaporated. The matrix may include an organic
solvent and/or a
volatile compound. The matrix may include polar molecules, water, one or more
alcohols,
methanol, ethanol, isopropanol, acetone or acetonitrile. Isopropanol is of
particular
interest.
The matrix molecules 211 may freely diffuse into the vacuum. In contrast, the
gas
phase ions 210 of the molecular constituents of the aerosol sample 201 may be
transferred
by the ion optics 212 to an analysis region (not shown) of the ion analyser
207. The ions
210 may be guided to the analysis region by applying voltages to the ion
optics 212. The
ions are then analysed by the ion analyser, which may comprise a mass
spectrometer 102
or an ion mobility spectrometer, or a combination of the two. As a result of
the analysis,
chemical information about the sample 201 may be obtained.
Fig. 3 shows a schematic of an embodiment that is substantially similar to
that
shown and described in relation to Fig. 2, except that the sample 201 is
delivered by a
fluid/liquid transfer pump or a Venturi pump 240 and the matrix 204 may be
delivered in
liquid form. This allows the matrix compound 204 to be mixed into the aerosol
201 as a
vapour, or as a liquid, prior to introduction into the ion analyser 207.
The Venturi pump 240 may comprise an inlet tube 242 that may be connected to a
device or probe (e.g., a REIMS device or probe as described herein) and may be
configured to transport aerosol particles or liquid from a sample (e.g.,
biologic tissue) to the
Venturi pump 240.
The Venturi pump may comprise a gas inlet 244 that may be arranged and adapted
to introduce a gas (e.g., nitrogen or standard medical air) into the flow path
of the aerosol
particles 201 or liquid being transported into the Venturi pump 240 by the
inlet tube 242.
The Venturi pump 240 may therefore facilitate the aspiration of aerosol
particles 201 or
other gaseous sample containing the analyte. The Venturi pump also comprises
an
exhaust 246 for exhausting the Venturi gas from the system such that it is not
directed into
the vacuum chamber 208 of the mass spectrometer 207.
The Venturi pump 240 may comprise a sample transfer portion or capillary 202
that
may be arranged and adapted to direct the sample and gas mixture produced by
the
Venturi pump 240 towards a junction 248. A matrix introduction conduit 203 is
arranged
and adapted to introduce matrix or a matrix compound 204 into the junction 248
and direct
the flow of the matrix compound 204 towards an inlet tube 206.
The aerosol particles 201 and the matrix 204 may intermix at the junction 248
and
the resulting aerosol particles 205 may be carried into the inlet tube 206 by
the suction
from the vacuum chamber 208. The larger aerosol particles 201 may be too heavy
to be
Date Recue/Date Received 2024-04-10
- 43 -
carried into the inlet tube 206 and may travel past the junction 248 and leave
the apparatus
via the exhaust 246.
Whilst shown as contiguous in Fig. 3, the sample transfer portion 202 may be a
separate component from the junction 248 and inlet tube 206. The junction 248
may
comprise a connector or connecting portion (not shown) for connecting to a
separate
sample transfer portion 202. The connection between the junction 248 and the
sample
transfer portion 206 may be fluidly sealed and/or may comprise a ring clamp.
As described hereinabove, an important feature is the formation of molecular
clusters 205 containing the original analyte aerosol constituents 201 and the
matrix
compound 204, followed by the surface-induced dissociation of these clusters
205. The
benefit of using a matrix 204 in accordance with an embodiment can be seen
from Fig. 4A
and Fig. 4B.
Fig. 4A shows a mass spectrum obtained by subjecting a sample to a REIMS
technique in which an aerosol was generated from a target, the aerosol was
collided with a
heated collision surface and the resulting ions generated therefrom were mass
analysed.
The mass spectrum in Fig. 4B was obtained by subjecting the same sample to the
same
analysis technique except that the aerosol was mixed with a matrix
(isopropanol) before
being collided with the collision surface and then mass analysed. It can be
seen from the
two mass spectra in Figs. 4A and 4B that the use of a matrix substantially
increases the
intensity of ions detected.
It is thought that there are several mechanisms by which the addition of the
matrix
may improve ionisation of the analyte. For example, mechanisms that result in
protonation
or deprotonation of the analyte may occur. Alternatively, or additionally,
reactions that
involve the removal of water and/or ammonia from the analyte may occur.
Alternatively, or
additionally, the adducting of metal ions such as sodium may play a role in
the mechanism.
The most probable mechanisms of protonation or deprotonation are analogous to
MALDI
methods.
It is thought that the dominant mechanism by which the addition of the matrix
improves ionisation of the analyte is by diluting or dissolving the analyte so
as to facilitate
the formation of analyte ions in solution phase by ionic dissociation. The
sample being
analysed may contain counter ions, such as Na, K+, H30+ etc, which interact
with the
analyte so as to facilitate the formation of the solution phase analyte ions.
The resulting
analyte ions may then be separated from the matrix in the gas phase (e.g.,
after collision
with the collision surface and/or evaporation) on collision with the surface,
via desolvation,
or via the so called MALDI lucky survivor mechanism.
It is thought that, dependent upon the matrix characteristics, a possible
mechanism
of matrix enhancement of ionisation in ambient ionisation techniques, such as
REIMS,
involves the formation of matrix (M) ions and analyte (A) ions in solution, as
follows.
A + M < __
Solution
A + M __________________________
Solution
Date Recue/Date Received 2024-04-10
- 44 -
Alternatively, the ionisation of the analyte may occur in the gas phase, e.g.,
as
described in Biochinnica et Biophysica Acta 1458 (2000) 6-27. However, this
mechanism is
thought to be energetically less favourable. The formation of matrix (M) ions
and analyte
(A) ions in the gas phase may proceed as follows:
A + M <
Gas Phase
A + M <
Gas Phase
The ionisation may occur in the gas phase or in the liquid phase. As the
droplets
containing the matrix and analyte molecules rapidly desolvate on contact with
the collision
surface, this brings them into the gas phase in close proximity and high
concentration.
The proton exchange may occur via two different types of transition. As there
is a
potential barrier to the transfer, the proton exchange may occur via an over
barrier
transition or an under barrier (tunnelling) transition. The probability of the
under barrier
transition depends on the form of the barrier and on the energy (E) of the
particle which is
to tunnel. The energy dependence is very strong and is approximately an
exponential of
the form exp(E/LiE), where AE is the energy characteristic for a given
barrier. Accounting
for the probability of the proton to be at the energy level E, the 'pure'
tunnelling probability
is required to be multiplied by the Boltzmann factor exp(-E/kT). Accordingly,
the total
contribution of the level E is proportional to exp(E/AE-E/kT).
Therefore, there are two limiting cases. If AE kT, then the probability tends
to
exp(-E/kT), and the most probable mechanism is quantum behaviour by tunnelling
from the
ground state. Alternatively, if AE kT, then the proton is most likely to be at
the highest
possible level of E, i.e. at the top of the barrier, and so the most probable
mechanism of
transfer is an over barrier transition. For the first limiting case, the
probability of proton
transfer depends strongly on the overlap of its vibrational wave functions,
thus the degree
of ionisation enhancement observed by the introduction of the matrix will be
specific to the
given analyte and given matrix M. This is what is observed, for example, with
reference to
Figs. 4A-4B in which it is clear that the enhancement in ionisation due to the
addition of the
matrix for ions of mass 766.6 Da is greater than that for ions of mass 885.6
Da.
Another possible mechanism , which is again analogous to MALDI mechanisms, is
a two-step process. The first step is the formation of primary matrix (M) ions
in the matrix-
analyte solution, as follows:
2M ____
Solution
According to Knockenmuss (Analyst 2006, 131 966-986), this remains the most
controversial aspect of the two-step process.
The second step of the process may involve ion-molecule reactions in the plume
that forms as the droplets strike the collision surface (which may or may not
be heated), as
follows:
Date Recue/Date Received 2024-04-10
- 45 -
A + [M ¨ Hr
Gas Phase/Clusters
A + [M + Hr _________________________________ > [A + Hr + M
Gas Phase/Clusters
Further desolvation, in the case of cluster formation, may result in
separation of the
charged molecular ions.
Fig. 5A shows another embodiment of a mass spectrometer interface for
introducing the analyte aerosol and matrix into the mass spectrometer. The
instrument
comprises a Venturi pump 501. The Venturi pump 501 comprises a tube 502 that
may be
connected to a device or probe (e.g., a REIMS device or probe as described
herein) and
may be configured to transport aerosol particles from a sample (e.g., biologic
tissue) to the
Venturi pump 501. The Venturi pump 501 may comprise a gas inlet 503 that may
be
arranged and adapted to introduce a gas (e.g., a Venturi gas) into the flow
path of the
aerosol particles being transported into the Venturi pump 501 by the tube 502.
The Venturi
pump 501 may comprise an elongated sample transfer tube 504 that may be
arranged and
adapted to transfer the sample and gas mixture from the tube 502 onto a
sampling device
510 via an outlet end 506 of the sample transfer tube 504.
The sampling device 510 may broadly comprise a hollow tube or whistle 512, a
matrix introduction conduit 530 and an inlet tube 540. The matrix introduction
conduit 530
may be arranged and adapted to introduce a matrix in liquid form through a
channel 534
(Fig. 5B) within the matrix introduction conduit 530. Matrix leaves the matrix
introduction
conduit 530 through an end 534 disposed or located within the whistle 512 and
it may be
nebulised by a gas that is being drawn into the inlet tube 540. The quality of
nebulisation
of the matrix may be controlled and affected by the dimensions and/or relative
distances
between the various parts of the sampling device 510, as described in more
detail below.
The inlet tube 540 leads to an inlet of a ion analyser or mass spectrometer
and may
be arranged and adapted such that a mixture of sample, gas and matrix passes
through an
end 542 of the inlet tube 540 disposed or located within the whistle 512 and
through a
passage 544 to be transferred into a ion analyser or mass spectrometer. In
these
arrangement the collision surface 209 is arranged downstream of the inlet tube
540.
Fig. 5C shows a close-up view of the sampling device 510.
The whistle 512 may be provided in the form of a hollow tube optionally having
a
first side 522 that may be arranged so as to face the outlet end 506 of the
sample transfer
tube 504, and a second, opposite side 524 optionally facing away from the
outlet end 506
of the sample transfer tube 504.
The whistle 512 may comprise a first end 518 that may be located
concentrically
around the inlet tube 540 and may be in sealing engagement therewith. The
whistle may
comprise a second end 520 that may be located concentrically around the matrix
introduction conduit 530 and may be in sealing engagement therewith.
A void, aperture or cut-out 514 may be provided on the second side 524 of the
whistle 512, and the cut-out 514 may form an inlet such that the sample and
gas mixture
flowing past the whistle 512 from the outlet end 506 of the sample transfer
tube 504 may
transfer into the interior of the whistle 512.
Date Recue/Date Received 2024-04-10
- 46 -
The mixture of sample and gas exiting the outlet end 6 of the sample transfer
tube
504 may impact on the first side 522 of the whistle 512, and then travel
around the outside
surface and into the cut-out 514. Once the sample and gas mixture is in the
interior of the
whistle, it may mix with the nebulised matrix emerging from the matrix
introduction conduit
530 before the mixture of sample, gas and matrix is optionally transferred
into the inlet tube
540 through the end 542 of the inlet tube 540. The mixture of sample, gas and
matrix may
then be transferred via the passage 544 to an ion analyser or mass
spectrometer.
Positioning the cut-out 514 on the second side 524 of the whistle 512 means
that
the initial impact of the sample and gas mixture is on a surface that is not
directly exposed
to the vacuum of the mass spectrometer. In various embodiments, therefore, the
sampling
device 510 is arranged and adapted such that the initial impact of the sample
and gas
mixture is on a surface that is not directly exposed to the vacuum of the mass
spectrometer.
The cut-out 514 may have a substantially semi-circular profile when the
whistle 512
is viewed in cross-section (as shown, for example, in Figs. 5A and 5B). This
will mean that
the edge 517 of the cut-out 514 is oval when viewed from a direction facing
the second
side 524 of the whistle 512 (see Fig. 5C). Alternatively, the cut-out 514 may
have a
different shape profile when the whistle 512 is viewed in cross-section, for
example a
square, triangular or irregular shaped profile. The edge 517 of the cut-out
514 may also be
square, triangular or irregular when then whistle 512 is viewed from a
direction facing the
second side 524 of the whistle 12 (see Fig. 5C).
The position and orientation of the whistle 512 can affect the quantity and
quality of
sample that is transferred into the mass spectrometer. The cut-out 514 may
comprise a
centre point 516 which may be in line with a longitudinal centreline 508 of
the sample
transfer tube 504. Fig. 5C shows a view of the second side 524 of the whistle
512 (the
whistle 512 is shown in isolation in Fig. 5C), and the centre point 516 can be
seen as the
centre point of the oval.
The whistle 512 may be oriented such that longitudinal axis 526 of the whistle
lies
coincident with an axis of symmetry of the cut-out 514. The centre point 516
may lie on the
longitudinal axis 526 of the whistle 512 and/or an axis of symmetry of the cut-
out. The axis
of symmetry of the cut-out may comprise the longitudinal axis of symmetry,
wherein the
longitudinal direction may be defined as the direction along the longitudinal
axis 526.
The position of the various parts of the sampling device 510 can also affect
the
quantity and quality of sample that is transferred into the mass spectrometer.
Now referring to Fig. 5B, a distance xis defined as the distance (e.g., the
shortest
distance) between the end 534 of the matrix introduction conduit 530 and the
end 542 of
the inlet tube 540.
A distance y is defined as the distance (e.g., the shortest distance) between
the
centre point 516 of the cut-out 514 and the end 542 of the inlet tube 540.
A distance z is defined as the distance (e.g., the shortest distance) between
the
outlet end 506 of the sample transfer tube 504 and the whistle 512 (e.g., the
first side 522
of the whistle 512).
Date Recue/Date Received 2024-04-10
- 47 -
The diameter a of the matrix introduction conduit 530 can also affect the
quantity
and quality of sample that is transferred into the mass spectrometer, and can
also affect
the nebulisation of the matrix as it leaves the end of the matrix introduction
conduit 530.
The diameter b of the inlet tube 540, and the diameter c of the sample
transfer tube
504 can also affect the quantity and quality of sample that is transferred
into the mass
spectrometer.
The diameters a, b and c may correspond to the diameters at the end 532 of the
matrix introduction conduit 530, the end 542 of the inlet tube and the outlet
end 506 of the
sample transfer tube 504, respectively.
Any or all of the diameters a, b and c may be greater than, less than or
substantially
equal to 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm,
2
mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4
mm,
4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm or 5 mm.
Any or all of the diameters/distances a, b, c, x, y and z may be changed to
optimise
the quantity and quality of sample that is transferred into the mass
spectrometer.
Aspects of the disclosure may extend to methods of optimising the sampling
device
510, comprising identifying one or more parameters associated with the
sampling device,
for example ion abundance or ion signal intensity and changing one or more of
the
distances a, b, c, x, y and z until the one or more parameters are optimised
or at a
maximum or minimum value.
The Venturi pump 501 may be for introducing aerosol particles into the sample
transfer tube 504. The sampling device 510 may be provided for sampling the
aerosol.
The matrix introduction conduit 530 may be arranged to introduce a matrix
(such as
isopropanol) into the sampling device 510 and the inlet tube 540 may be
arranged to direct
a mixture of aerosol particles and matrix onwards to an ion analyser or mass
spectrometer.
The Venturi pump 501 may facilitate the aspiration of aerosol or other gaseous
sample containing the analyte and may be driven by nitrogen or standard
medical air.
Aerosol sampling may be arranged to occur orthogonally to the outlet end 506
of the
Venturi pump 501 as shown from Figs. 1A and 1B. The outlet 532 of the matrix
introduction conduit 530 may be spaced apart from the inlet tube 540 to the
ion analyser or
mass spectrometer by the distance x. The distance x can be modified as
required to
achieve an optimum ion signal intensity.
Altering the value of the distance x can change the velocity of the gas being
drawn
into the inlet tube 540 and can have an effect upon the nebulisation
conditions. If the
nebulisation conditions are less favourable then the matrix droplets may not
be of the
correct size for interacting with the analyte aerosol and/or may not fragment
efficiently
when the aerosol collides with the collision surface.
Fig. 6 shows the intensity of an ion signal obtained by an ion analyser 207
for
different distances x between the outlet 532 of the matrix introduction
conduit 530 and the
inlet 540, when the matrix flow rate was set to about 0.2 ml/min. Fig. 6 shows
the ion
signals for values of x= 0 mm, x = 1 mm, x = 2 mm, x = 4 mm, x = 4.5 mm, x = 5
mm, and
x = 5.2 mm. It can be seen that when the distance x is about 0 mm (i.e. the
outlet of the
Date Recue/Date Received 2024-04-10
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matrix conduit 530 is touching the inlet 540) then no ion signal is detected.
When the
distance x is increased to about 1 mm, an ion signal is detected. When the
distance x is
increased from about 1 mm to about 2 mm, the relative intensity of the ion
signal is
increased. When the distance x is increased from about 2 mm to about 4 mm, the
relative
intensity of the ion signal is increased further. When the distance x is
increased from about
4 mm to about 4.5 mm, the relative intensity of the ion signal decreases. When
the
distance x is further increased from about 4.5 mm to about 5 mm, the relative
intensity of
the ion signal is decreased significantly. When the distance x is increased
from about 5
mm to about 5.2 mm, substantially no ion signal is detected. This shows that
the ion signal
detected can be optimised by selecting an appropriate value of x.
As the matrix 204 leaves the matrix introduction conduit 530 it may be
nebulised by
the gas that is being drawn into the ion analyser inlet 240. It is believed
that altering the
value of the distance x changes the velocity of the gas being drawn into the
ion analyser
inlet 240 and hence affects the nebulisation conditions. If the nebulisation
conditions are
not favourable then the matrix droplets may not be of the correct size for
interacting with
the analyte aerosol 201 and/or may not fragment well when collided with the
collision
surface 209.
The effect of different matrix 204 (e.g., isopropanol) flow rates on the
spectral
appearance was tested.
Fig. 7 shows the intensity of the ion signal obtained by the ion analyser 207
for
different flow rates of matrix 204 when the spacing x between the outlet 232
of the matrix
introduction conduit 230 and the inlet 240 to the ion analyser 207 was set at
about 2.5 mm.
The ion signal was measured at a flow rate about 0.2 ml/min. The flow rate was
then
increased to about 0.4 ml/min, resulting in an increased intensity of the ion
signal. The flow
rate was increased further to about 0.8 ml/min, resulting in a decreased
intensity of the ion
signal. The flow rate was then decreased to about 0.1 ml/min, resulting in a
decreased
intensity of the ion signal. The flow rate was then decreased further to about
0.05 ml/min,
resulting in a further decreased intensity of the ion signal. The flow rate
was then
decreased further to about 0.025 ml/min, resulting in a further decreased
intensity of the
ion signal. The flow rate was then decreased yet further to about 0.01 ml/min,
resulting in
a further decreased intensity of the ion signal. This shows that the ion
signal is not
necessarily improved simply by increasing the flow rate of the matrix 204, but
that the flow
rate may be optimised to produce the optimal ion signal intensity.
Figs. 8A-8I show the effect of different isopropanol flow rates on REIMS
spectral
profiles for Bacteroides fragilis, using flow rates between 0.01 to 0.25
mUmin. Figs. 8A-8I
show spectra for the mass range of 500-900, covering phospholipid analytes.
The effect of
the isopropanol being present in the analysis of this sample is detectable at
very low rates,
e.g., from 0.02 mL/min, and is clearly visible from the appearance of m/z 590
(cerannide
species) and m/z 752 (a-Galactosylceramide) in the spectra. These species were
found to
.. increase in their relative abundance when further increasing the
isopropanol flow rate.
Date Recue/Date Received 2024-04-10
- 49 -
Although the matrix has been described in Fig. 5 as being introduced opposite
the
inlet 240 to the ion analyser 207 and downstream of the sample transfer tube
504, it may
alternatively be introduced into the sample transfer tube 504.
Alternatively, the matrix may be introduced at a location around the
circumference
.. of the transfer tube 504 and may be swept towards and into the inlet 240 to
the ion
analyser 207 by a gas flow.
Calibration/lockmass/lock mobility compounds may be used in the various
techniques described herein for calibrating the ion analyser or providing a
reference mass
to the ion analyser. The calibration, lockmass or lock mobility compound may
be
introduced via the matrix introduction conduit 203, via the sample transfer
tube 202, or in
another location.
Fig. 9A and Fig. 9B show two mass spectra obtained by analysing a sample of
porcine muscle according to an embodiment. The spectrum of Fig. 9A was
obtained whilst
introducing a lockmass compound (Leu-enk) into the analyser 207 through the
matrix
.. conduit 203. The peaks for the lockmass compound can be observed as the
first peaks in
the mass spectrum. The mass to charge ratios of the lockmass ions are known in
advance
and can be used to calibrate the mass analyser 207 such that the mass to
charge ratios of
the other ions detected can be determined more accurately. The mass spectrum
shown in
Fig. 9B was obtained using the same method as the spectrum in Fig. 9A, except
that no
.. lockmass compound was used in the analysis. It can be seen that the two
mass spectra
are substantially identical, except for the detection of the lockmass ions in
the mass
spectrum of Fig. 9A. It is therefore apparent that the introduction of a
lockmass compound
in the technique does not affect the mass spectra measured by the ion analyser
207.
Fig. 10 shows a plot resulting from a principle component analysis of porcine
brain
over four days. The data for Day 1 and Day 4 was obtained without the use of a
lockmass
compound, whereas the data for Day 2 and Day 3 was obtained with the use of a
lockmass
compound. The analysis was performed over the range of 600-900 mass units and
so the
lockmass ions are not shown in the plot, as the lockmass ions are outside of
this range
(see Fig. 9A). The principle component analysis shows that the data obtained
with use of
.. the lockmass compound is not separable from the data obtained without use
of the
lockmass compound, and that the variance due to the data being from different
days is
significantly greater than any variance due to the inclusion of the lockmass
compound.
Fig. 11 shows the results of a principle component analysis for the analysis
of data
obtained from porcine kidney cortex, porcine liver, porcine brain, porcine
heart muscle and
other porcine muscle. Some of the data was obtained using a lockmass compound
and
some of the data was obtained without the use of a lockmass compound. However,
the
data for each of the types of tissue is well clustered in a particular region
of the plot,
demonstrating that the use of a lockmass compound does not affect the analysis
and
tissue classification.
It has been determined that more than one different know lock mass compounds
may be used without adversely affecting analysis of the sample. Exact lock
mass
compound(s) may be used and/or external lock mass compound(s) may be used.
Date Recue/Date Received 2024-04-10
- 50 -
Figs. 12A and 11B show schematics of example configurations of the collision
surface that may be used in the present invention. Fig. 12A corresponds to the
collision
surface 209 shown in Figs. 2 and 3. For example, the collision surface 209 may
be a
spherical, stainless-steel collision surface 209a and may be mounted
approximately 6 mm
from the end of the inlet capillary 206 into the analyser 207. Fig. 12B shows
an alternative
collision surface 209 that may be used, in the form of a coil-shaped collision
surface 209b.
Ions may be transferred by the ion optics 212 to an analysis region (not
shown) of the ion
analyser 207. As discussed above, the ion optics 212 may comprise a Stepwave
(RTM)
ion guide.
It is contemplated that the collision surface may be other shapes, such as
substantially cylindrical, tubular, rod-shaped, hemispherical, teardrop-
shaped, plate-
shaped, concave, dish-shaped or conical. It is also contemplated that the
collision surface
may be formed by the inner surface of a hollow collision assembly having an
inlet and an
outlet. The aerosol may enter through the inlet and then impact on the inner
surface of the
collision assembly so as to form or release analyte ions. The analyte ions may
then
emerge from said collision assembly via said outlet. The inner cross-sectional
area of the
collision assembly may be either substantially constant or reduce in a
direction from the
inlet to said outlet, i.e. the collision assembly may be funnel-shaped,
tubular or cylindrical.
The embodiments relating to a hollow funnel-shaped collision assembly or a
hollow
cylindrical collision assembly have also been found to result in a high ion
yield (or improved
ionisation efficiency) coupled with a significant improvement in signal to
noise ratio.
Furthermore, these embodiments have also been found to result in less
contamination of
the collision assembly and downstream ion optics by background clusters which
are not of
analytical interest.
It has been recognised that the REIMS mechanism may lead to substantially
equal
generation of positively and negatively charged ions, which may subsequently
form
relatively large molecular clusters of neutral electrical charge. These
neutral clusters are
not manipulated well by electric fields within the analyser or spectrometer
and hence may
be eliminated, e.g., by the instrument ion optics 212. The collision surface
209 described
herein serves to break up the molecular clusters 205, releasing the ions so
that they may
be guided by the electrical fields within the analyser or spectrometer.
However, it has also
been recognised that the provision of the collision surface 209 may induce
cross-
contamination between measurements of different samples. For example, certain
bacterial metabolites were found to induce relatively strong memory effects
after only a
small number of repetitive measurements, e.g., certain sphingolipids produced
by
Bacteroides spp. or lipopolypeptides such as surfactin and lichenysin produced
by certain
Bacillus spp.. This cross-contamination could be mitigated by cleaning the
atmospheric
pressure interface before each analysis. However, this is undesirable,
particularly in
automated instruments. In order to avoid contamination of the collision
surface 209, the
surface may be heated, e.g., to several hundred degrees Celsius. For example,
heating the
collision surface 209 may cause carbonaceous deposits on the collision surface
209 to
react with oxygen introduced through the inlet capillary 206. The carbonaceous
deposits
Date Recue/Date Received 2024-04-10
- 51 -
will then be converted to CO2 gas, which can leave the collision surface 209
and hence not
contaminate the instrument during subsequent analyses. The coil-shaped
collision surface
209b of Fig. 12B provides a particularly reproducible heat distribution.
The collision element or surface 209 may be constructed from a material that
may
be heated by passing an electric current through it, e.g., by applying voltage
V in Fig. 12B,
enabling it to be easily heated during analysis. For example, the collision
surface 209 may
be manufactured out of a heat-resistant iron-chromium-aluminium (FeCrAl) alloy
such as
kanthal. Using such a heated collision surface 209 significantly reduces
memory effects
and thus the frequency of instrument cleaning may be greatly reduced. For
example,
thousands of database entries are able to be recorded without any memory
effects and
even prolonged exposure to lipopolypeptides did not result in any observed
carry-over.
The spectral profile obtained using the heated collision surface 209 may, in
some
cases, be different to the spectral profile obtained using the collision
surface 209 unheated,
for example, as shown in Figs. 13A and 13B.
Figs. 13A and 13B show the spectral profiles resulting from the analysis of
Bacteroides fagilis using a non-heated collision surface and a heated
collision surface,
respectively. This indicates that not all spectral constituents are thermally
stable enough to
be analysed using this type of heated surface technique. For example, the
effect of the
heated surface seems to be especially strong on phosphatidic acid (which is
common in,
e.g., fungi such as C. albicans) and sphingolipid species (which is common in
e.g.,
Bacteroidetes phylum), while it has generally little effect on the spectral
appearance
observed for phosphatidylglycerol and phosphatidylethanolamines (which are,
e.g., the
main phospholipid species in Proteus mirabilis).
As described above, the introduction of a matrix compound 204, such as
isopropyl
alcohol (IPA), upstream of the collision surface 209 has been found to improve
analyte
ionisation and sensitivity of the instrument. It has also been found that the
introduction of
the matrix compound 204 may restore spectral features that would otherwise be
missing by
using a heated collision surface rather than a non-heated collision surface.
For example,
Figs. 13A and 13B demonstrate that the use of a heated collision surface was
found to
eliminate spectral features such as ceramides in Bacteroides fragilis. The
introduction of
isopropanol into the sampled aerosol 201 before introduction into the mass
analyser 207 or
spectrometer was found to restore these spectral features and generate a mass
spectral
fingerprint similar to that of an atmospheric pressure interface with a non-
heated collision
surface. Furthermore, the addition of the matrix 204 (e.g., isopropanol) to
the sample
aerosol 201 led to similar or higher signal intensities as compared to direct
aerosol
introduction, and thus enables the use of a Venturi pump 213 for aerosol
transport.
Figs. 14A-14C show three mass spectra obtained by analysing a sample of
Candida albicans (yeast). The mass spectrum of Fig. 14A was obtained whilst
introducing
an isopropyl alcohol (IPA) matrix upstream of a heated collision surface
according to an
embodiment. The mass spectrum of Fig. 14B was obtained using the same method
as the
spectrum of Fig. 14A, except that no isopropyl alcohol (IPA) matrix was
introduced. The
mass spectrum of Fig. 14C was obtained using the same method as the spectrum
of Fig.
Date Recue/Date Received 2024-04-10
- 52 -
14B, except that the collision surface was not heated. Figs. 14A-14C show the
effects of
using a heated surface and an isopropanol matrix on the spectral appearance
for Candida
albicans (yeast). These examples show that using a heated collision surface
instead of a
non-heated collision surface results in significant changes of the spectral
appearance, with
many spectral features in Candida albicans being significantly reduced in
relative intensity
or disappearing altogether. The introduction of isopropanol into the system
having the
heated collision surface helps to circumvent this problem and creates a
spectrum that is
more similar to that obtained using a non-heated collision surface.
As described previously, lockmass compounds (such as Leu Enk) may be used. It
is contemplated herein that the intensities of the lockmass compound peaks may
be
monitored and used to determine if the matrix 204 is flowing at the desired
rate, e.g., by
introducing the lockmass compound along with the matrix 204. This may be used
to
determine that the matrix compound 204 is flowing consistently and is not a
variable flow.
In order to calibrate the instruments described herein a calibrant may be
introduced
into the instrument and analysed. For example, the instrument may be
calibrated before
analysis of the sample (e.g., tissue) and afterwards, to determine if there is
any mass shift.
The calibrant (e.g., sodium formate) may be injected into the instrument using
the matrix
(e.g., isopropanol) injection tube 203. However, it was discovered that the
optimum
distance between the outlet 232 of the matrix injection tube 230 and the inlet
240 of the
mass analyser 207 or spectrometer for calibration may be shorter than the
optimum
distance x for sample analysis, e.g., tissue analysis. In order to address
this, different IPA
capillary lengths may be used during calibration and tissue analysis. For
calibration, a
relatively long capillary may be used, whereas for tissue analysis a shorter
capillary may be
used.
As described above, the matrix introduction conduit may be arranged in various
different configurations. For example, the matrix introduction conduit may be
coaxial with
and arranged inside of the inlet tube to the mass spectrometer. The distance
from the exit
of the matrix introduction conduit to the downstream exit of the inlet tube to
the mass
spectrometer (i.e. to the entrance to the vacuum chamber) was found to be
important. It is
believed that larger distances allow better interaction between the analyte
and the matrix.
Fig. 15A shows the total ion current detected as a function of time for
several
different distances between the exit of the matrix introduction conduit and
the entrance to
the mass spectrometer vacuum chamber. Positive distances represent a distance
in the
direction downstream of the entrance to the mass spectrometer vacuum chamber,
whereas
negative distances represent the distance upstream of the entrance to the mass
spectrometer vacuum chamber. The sample analysed was porcine liver and the
matrix
was isopropyl alcohol. The matrix capillary was made from quartz glass, had an
outer
diameter of 360 pm and an inner diameter of 250 pm. A relatively strong
background noise
and strong IPA signal was observed when the exit of the matrix introduction
conduit was
arranged upstream of the entrance to the mass spectrometer vacuum chamber
(i.e. at
negative distances).
Date Recue/Date Received 2024-04-10
- 53 -
Figs. 15B-15F show the mass spectra obtained at the different distances of
Fig.
15A. Figs. 15B-15F show mass spectra obtained at distances of -10 mm, -20 mm, -
24
mm, 0 mm and +2 mm respectively. It can be seen that when the exit of the
matrix
introduction tube was arranged at or downstream of the entrance to the vacuum
chamber
(i.e. distances of 0 mm and +2 mm), the effect of the matrix was relatively
low. In contrast,
the further the exit of the matrix introduction tube was arranged upstream of
the entrance to
the vacuum chamber (i.e. more negative distances), the more influence the
matrix had. It
was confirmed that at a distance of 0 mm, increasing the matrix flow rate did
not improve
the total ion current observed.
Fig. 16A shows data corresponding to that of Fig. 15A, except wherein the data
was
obtained using a matrix introduction conduit having an inner diameter of 100
pm and
wherein different distances to Fig. 15A were used. As with Fig. 15A, the ion
signal
observed increased the more that the exit of the matrix introduction tube was
arranged
upstream of the entrance to the vacuum chamber (i.e. the more negative the
distance
was).
Fig. 16B shows the mass spectra obtained at the different distances of Fig.
16A.
Figs. 16B-16F show mass spectra obtained at distances of 0 mm, +2 mm, -1 mm, -
10 mm,
-20 mm, -30 mm, -40 mm and -50 mm respectively. It can be seen that when the
exit of
the matrix introduction tube was arranged at or downstream of the entrance to
the vacuum
chamber (i.e. distances of 0 mm and 2 mm), there the effect of the matrix was
relatively
low. In contrast, the further the exit of the matrix introduction tube was
arranged upstream
of the entrance to the vacuum chamber (i.e. more negative distances), the more
influence
the matrix had. The spectra are also less noisy than the spectra of Figs. 15B-
15F and the
matrix had a stronger effect. It was confirmed that at a distance of 0 mm,
increasing the
matrix flow rate did not improve the spectra.
Fig. 17A shows data corresponding to that of Fig. 15A, except wherein the data
was
obtained using a matrix introduction conduit having an inner diameter of 50 pm
and
wherein different distances to Fig. 15A were used. As with Fig. 15A, the ion
signal
observed in Fig. 17A increased the more that the exit of the matrix
introduction tube was
arranged upstream of the entrance to the vacuum chamber (i.e. the more
negative the
distance was).
Figs. 17B-17IB show the mass spectra obtained at the different distances of
Fig.
17A. Figs. 17B-17F show mass spectra obtained at distances of 0 mm, +2 mm, -2
mm, -10
mm, -20 mm, -30 mm, -40 mm and -50 mm respectively. It can be seen that when
the exit
of the matrix introduction tube was arranged at or downstream of the entrance
to the
vacuum chamber (i.e. distances of 0 mm and 2 mm), the effect of the matrix was
minimal.
In contrast, the further the exit of the matrix introduction tube was arranged
upstream of the
entrance to the vacuum chamber (i.e. more negative distances), the more
influence the
matrix had.
It has been found that the position of the matrix conduit affects the ion
signal more
than the flow rate of the matrix.
Date Recue/Date Received 2024-04-10
- 54 -
Figs. 18A-18C show three spectra obtained for matrix introduction conduits
having
internal diameters of 50 pm, 100 pm and 250 pm, respectively, when the exit of
each
conduit was arranged 20 mm upstream of the entrance to the vacuum chamber and
using
a matrix flow rate of 0.2 ml/min. The spectra show that the smaller the inner
diameter of
the matrix introduction conduit, the better and less noisy the spectra are.
It has also been found that tapering the exit end of the matrix introduction
conduit
improves the ion signal intensity detected.
As described above, e.g., in relation to Fig. 5B, a whistle arrangement may be
used
for sampling. In this arrangement the matrix introduction conduit may be
coaxial with the
inlet tube to the mass spectrometer. As described above, the distance x from
the exit of
the matrix introduction conduit to the entrance of the inlet tube to the mass
spectrometer
was found to be important.
Fig. 19A shows the total ion current detected as a function of time for
several
different distances between the exit of the matrix introduction conduit and
the entrance to
the mass spectrometer inlet tube, in the whistle arrangement. The sample
analysed was
porcine liver and the matrix was isopropyl alcohol. The matrix capillary was
made from
quartz glass, had an outer diameter of 360 pm and an inner diameter of 250 pm.
It can be
seen that the ion signal intensity was approximately the same for different
distances
between the matrix conduit outlet and the mass spectrometer tube inlet, until
a distance of
around 3-4 mm.
Figs. 19B to 19H show the mass spectra obtained at the different distances of
Fig.
19A. Figs. 19B-19F show mass spectra obtained at distances of 6 mm, 5 mm, 4
mm, 3
mm, 2 mm, 1 mm and 0 mm respectively. It can be seen that the spectra are very
similar
at distances up to around 3 mm.
Fig. 20A shows data corresponding to that of Fig. 19A, except wherein the data
was
obtained using a matrix introduction conduit having an inner diameter of 100
pm. It can be
seen that the ion signal intensity is approximately the same for different
distances between
the matrix conduit outlet and the mass spectrometer tube inlet, until a
distance of around 3
mm (although most intense at a distance of around 2 mm). At distances greater
than 3 mm
the ion signal intensity dropped.
Figs. 20B to 20G show the mass spectra obtained at the different distances of
Fig.
20A. Figs. 20B-20G show mass spectra obtained at distances of 5 mm, 4 mm, 3
mm, 2
mm, 1 mm and 0 mm respectively.
Fig. 21A shows data corresponding to that of Fig. 19A, except wherein the data
was
obtained using a matrix introduction conduit having an inner diameter of 50 pm
and
wherein data for additional distances are shown. It can be seen that the ion
signal intensity
is approximately the same for different distances between the matrix conduit
outlet and the
mass spectrometer tube inlet, until a distance of around 4 mm. At distances
greater than 4
mm the ion signal intensity dropped.
Figs. 21B to 211 show the mass spectra obtained at the different distances of
Fig.
21A. Figs. 21B-211 show mass spectra obtained at distances of 7 mm, 6 mm, 5
mm, 4
mm, 3 mm, 2 mm, 1 mm and 0 mm respectively.
Date Recue/Date Received 2024-04-10
- 55 -
Various biological samples were analysed using the methods and techniques
according to the various embodiments of the present invention. These analyses
demonstrated that the use of a collision surface and/or matrix improved the
ion signal
obtained from the analyte.
Figs. 22A-22C each show a mass spectrum obtained using a REIMS technique in
negative ion mode for the analysis of lamb liver. Each spectrum represents
data obtained
from five samples. The spectrum of Fig. 22A was obtained without the
introduction of a
matrix into the analyte stream and without the use of a collision surface. The
spectrum of
Fig. 22B was obtained with the introduction of a matrix (isopropyl alcohol at
a rate of 0.2
mUmin) into the analyte stream and without the use of a collision surface. The
spectrum of
Fig. 22C was obtained with the introduction of a matrix (isopropyl alcohol at
a rate of 0.2
mUmin) into the analyte stream and with the use of a collision surface. It can
be seen by
comparing these spectra that the use of a matrix increases analyte ion signal
intensities,
even without the use of a collision surface, and that the combined use of a
matrix and
collision surface significantly increases the intensity of analyte ion
signals.
Figs. 23A-23C each show a mass spectrum obtained using a REIMS technique in
positive ion mode for the analysis of lamb liver. Each spectrum represents
data obtained
from five samples. The spectrum of Fig. 23A was obtained without the
introduction of a
matrix into the analyte stream and without the use of a collision surface. The
spectrum of
Fig. 23B was obtained with the introduction of a matrix (isopropyl alcohol at
a rate of 0.2
mL/min) into the analyte stream and without the use of a collision surface.
The spectrum of
Fig. 23C was obtained with the introduction of a matrix (isopropyl alcohol at
a rate of 0.2
mUmin) into the analyte stream and with the use of a collision surface. As
with the
negative ion mode shown in Figs. 22A-22C, it can be seen by comparing the
positive ion
mode spectra of Figs. 23A-23C that the use of a matrix increases analyte ion
signal
intensities, even without the use of a collision surface, and that the
combined use of a
matrix and collision surface significantly increases the intensity of analyte
ion signals.
It was also discovered that the analysis of highly adipose tissues, such as
normal
breast tissue, may generate little of no ion signal without the use of a
matrix.
Fig. 24A shows a mass spectrum obtained from the analysis of normal breast
tissue
without the use of a matrix. Fig. 24B shows a mass spectrum obtained from the
analysis of
normal breast tissue with the use of isopropyl alcohol as a matrix. It can be
seen by
comparing these spectra that the use of a matrix significantly improves the
signal intensity
for analyte ions.
Analvsina sample spectra
A list of analysis techniques which are intended to fall within the scope of
the
present invention are given in the following table:
Analysis Techniques
Univariate Analysis
Date Recue/Date Received 2024-04-10
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Multivariate Analysis
Principal Component Analysis (PCA)
Linear Discriminant Analysis (LDA)
Maximum Margin Criteria (MMC)
Library Based Analysis
Soft Independent Modelling Of Class Analogy (SIMCA)
Factor Analysis (FA)
Recursive Partitioning (Decision Trees)
Random Forests
Independent Component Analysis (ICA)
Partial Least Squares Discriminant Analysis (PLS-DA)
Orthogonal (Partial Least Squares) Projections To Latent Structures (OPLS)
OPLS Discriminant Analysis (OPLS-DA)
Support Vector Machines (SVM)
(Artificial) Neural Networks
Multilayer Perceptron
Radial Basis Function (RBF) Networks
Bayesian Analysis
Cluster Analysis
Kernelized Methods
Subspace Discriminant Analysis
K-Nearest Neighbours (KNN)
Quadratic Discriminant Analysis (QDA)
Probabilistic Principal Component Analysis (PPCA)
Non negative matrix factorisation
K-means factorisation
Fuzzy c-means factorisation
Discriminant Analysis (DA)
Combinations of the foregoing analysis approaches can also be used, such as
PCA-LDA, PCA-MMC, PLS-LDA, etc.
Analysing the sample spectra can comprise unsupervised analysis for
dimensionality reduction followed by supervised analysis for classification.
By way of example, a number of different analysis techniques will now be
described
in more detail.
Multivariate analysis - Developing a Model for Classification
By way of example, a method of building a classification model using
multivariate
analysis of plural reference sample spectra will now be described.
Figure 25 shows a method 1500 of building a classification model using
multivariate
Date Recue/Date Received 2024-04-10
- 57 -
analysis. In this example, the method comprises a step 1502 of obtaining
plural sets of
intensity values for reference sample spectra. The method then comprises a
step 1504 of
unsupervised principal component analysis (PCA) followed by a step 1506 of
supervised
linear discriminant analysis (LDA). This approach may be referred to herein as
PCA-LDA.
Other multivariate analysis approaches may be used, such as PCA-MMC. The PCA-
LDA
model is then output, for example to storage, in step 1508.
The multivariate analysis such as this can provide a classification model that
allows
an aerosol, smoke or vapour sample to be classified using one or more sample
spectra
obtained from the aerosol, smoke or vapour sample. The multivariate analysis
will now be
described in more detail with reference to a simple example.
Figure 26 shows a set of reference sample spectra obtained from two classes of
known reference samples. The classes may be any one or more of the classes of
target
described herein. However, for simplicity, in this example the two classes
will be referred
as a left-hand class and a right-hand class.
Each of the reference sample spectra has been pre-processed in order to derive
a
set of three reference peak-intensity values for respective mass to charge
ratios in that
reference sample spectrum. Although only three reference peak-intensity values
are
shown, it will be appreciated that many more reference peak-intensity values
(e.g., - 100
reference peak-intensity values) may be derived for a corresponding number of
mass to
charge ratios in each of the reference sample spectra. In other embodiments,
the reference
peak-intensity values may correspond to: masses; mass to charge ratios; ion
mobilities
(drift times); and/or operational parameters.
Figure 27 shows a multivariate space having three dimensions defined by
intensity
axes. Each of the dimensions or intensity axes corresponds to the peak-
intensity at a
particular mass to charge ratio. Again, it will be appreciated that there may
be many more
dimensions or intensity axes (e.g., - 100 dimensions or intensity axes) in the
multivariate
space. The multivariate space comprises plural reference points, with each
reference point
corresponding to a reference sample spectrum, i.e., the peak-intensity values
of each
reference sample spectrum provide the co-ordinates for the reference points in
the
multivariate space.
The set of reference sample spectra may be represented by a reference matrix D
having rows associated with respective reference sample spectra, columns
associated with
respective mass to charge ratios, and the elements of the matrix being the
peak-intensity
values for the respective mass to charge ratios of the respective reference
sample spectra.
In many cases, the large number of dimensions in the multivariate space and
matrix
D can make it difficult to group the reference sample spectra into classes.
PCA may
accordingly be carried out on the matrix D in order to calculate a PCA model
that defines a
PCA space having a reduced number of one or more dimensions defined by
principal
component axes. The principal components may be selected to be those that
comprise or
"explain" the largest variance in the matrix D and that cumulatively explain a
threshold
amount of the variance in the matrix D.
Date Recue/Date Received 2024-04-10
- 58 -
Figure 28 shows how the cumulative variance may increase as a function of the
number n of principal components in the PCA model. The threshold amount of the
variance
may be selected as desired.
The PCA model may be calculated from the matrix D using a non-linear iterative
partial least squares (NIPALS) algorithm or singular value decomposition, the
details of
which are known to the skilled person and so will not be described herein in
detail. Other
methods of calculating the PCA model may be used.
The resultant PCA model may be defined by a PCA scores matrix S and a PCA
loadings matrix L. The PCA may also produce an error matrix E, which contains
the
variance not explained by the PCA model. The relationship between D, S, L and
E may be:
D = SLT + E
(1)
Figure 29 shows the resultant PCA space for the reference sample spectra of
Figs.
26 and 27. In this example, the PCA model has two principal components PC0 and
PC, and
the PCA space therefore has two dimensions defined by two principal component
axes.
However, a lesser or greater number of principal components may be included in
the PCA
model as desired. It is generally desired that the number of principal
components is at least
one less than the number of dimensions in the multivariate space.
The PCA space comprises plural transformed reference points or PCA scores,
with
each transformed reference point or PCA score corresponding to a reference
sample
spectrum of Figure 26 and therefore to a reference point of Figure 27.
As is shown in Figure 29, the reduced dimensionality of the PCA space makes it
easier to group the reference sample spectra into the two classes. Any
outliers may also be
identified and removed from the classification model at this stage.
Further supervised multivariate analysis, such as multi-class LDA or maximum
margin criteria (MMC), in the PCA space may then be performed so as to define
classes
and, optionally, further reduce the dimensionality.
As will be appreciated by the skilled person, multi-class LDA seeks to
maximise the
ratio of the variance between classes to the variance within classes (i.e., so
as to give the
largest possible distance between the most compact classes possible). The
details of LDA
are known to the skilled person and so will not be described herein in detail.
The resultant PCA-LDA model may be defined by a transformation matrix U, which
may be derived from the PCA scores matrix S and class assignments for each of
the
transformed spectra contained therein by solving a generalised eigenvalue
problem.
The transformation of the scores S from the original PCA space into the new
LDA
space may then be given by:
Z =SU
(2)
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where the matrix Z contains the scores transformed into the LDA space.
Figure 30 shows a PCA-LDA space having a single dimension or axis, wherein the
LDA is performed in the PCA space of Figure 29. As is shown in Figure 30, the
LDA space
comprises plural further transformed reference points or PCA-LDA scores, with
each
further transformed reference point corresponding to a transformed reference
point or PCA
score of Figure 29.
In this example, the further reduced dimensionality of the PCA-LDA space makes
it
even easier to group the reference sample spectra into the two classes. Each
class in the
PCA-LDA model may be defined by its transformed class average and covariance
matrix or
one or more hyperplanes (including points, lines, planes or higher order
hyperplanes) or
hypersurfaces or Voronoi cells in the PCA-LDA space.
The PCA loadings matrix L, the LDA matrix U and transformed class averages and
covariance matrices or hyperplanes or hypersurfaces or Voronoi cells may be
output to a
database for later use in classifying an aerosol, smoke or vapour sample.
The transformed covariance matrix in the LDA space V'o for class g may be
given
by
V'o=UT Vo U
(3)
where Vo are the class covariance matrices in the PCA space.
The transformed class average position zg for class g may be given by
soU = zo
(4)
where so is the class average position in the PCA space.
Multivariate Analysis - Using a Model for Classification
By way of example, a method of using a classification model to classify an
aerosol,
smoke or vapour sample will now be described.
Figure 31 shows a method 2100 of using a classification model. In this
example, the
method comprises a step 2102 of obtaining a set of intensity values for a
sample spectrum.
The method then comprises a step 2104 of projecting the set of intensity
values for the
sample spectrum into PCA-LDA model space. Other classification model spaces
may be
used, such as PCA-MMC. The sample spectrum is then classified at step 2106
based on
the project position and the classification is then output in step 2108.
Classification of an aerosol, smoke or vapour sample will now be described in
more
detail with reference to the simple PCA-LDA model described above.
Figure 32 shows a sample spectrum obtained from an unknown aerosol, smoke or
vapour sample. The sample spectrum has been pre-processed in order to derive a
set of
three sample peak-intensity values for respective mass to charge ratios. As
mentioned
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above, although only three sample peak-intensity values are shown, it will be
appreciated
that many more sample peak-intensity values (e.g., - 100 sample peak-intensity
values)
may be derived at many more corresponding mass to charge ratios for the sample
spectrum. Also, as mentioned above, in other embodiments, the sample peak-
intensity
values may correspond to: masses; mass to charge ratios; ion mobilities (drift
times);
and/or operational parameters.
The sample spectrum may be represented by a sample vector dx, with the
elements
of the vector being the peak-intensity values for the respective mass to
charge ratios. A
transformed PCA vector sx for the sample spectrum can be obtained as follows:
c1,1... = s,
(5)
Then, a transformed PCA-LDA vector zx for the sample spectrum can be obtained
as follows:
z,
(6)
Figure 33 again shows the PCA-LDA space of Figure 30. However, the PCA-LDA
space of Figure 33 further comprises the projected sample point, corresponding
to the
transformed PCA-LDA vector zx, derived from the peak intensity values of the
sample
spectrum of Figure 32.
In this example, the projected sample point is to one side of a hyperplane
between
the classes that relates to the right-hand class, and so the aerosol, smoke or
vapour
sample may be classified as belonging to the right-hand class.
Alternatively, the Mahalanobis distance from the class centres in the LDA
space
may be used, where the Mahalanobis distance of the point zõ from the centre of
class g
may be given by the square root of:
(zõ-zg)T (Vg)-1(z,-z9)
(8)
and the data vector d, may be assigned to the class for which this distance is
smallest.
In addition, treating each class as a multivariate Gaussian, a probability of
membership of the data vector to each class may be calculated.
Library Based Analysis - Developing a Library for Classification
By way of example, a method of building a classification library using plural
input
reference sample spectra will now be described.
Figure 34 shows a method 2400 of building a classification library. In this
example,
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the method comprises a step 2402 of obtaining plural input reference sample
spectra and a
step 2404 of deriving metadata from the plural input reference sample spectra
for each
class of sample. The method then comprises a step 2406 of storing the metadata
for each
class of sample as a separate library entry. The classification library is
then output, for
example to electronic storage, in step 2408.
A classification library such as this allows an aerosol, smoke or vapour
sample to
be classified using one or more sample spectra obtained from the aerosol,
smoke or
vapour sample. The library based analysis will now be described in more detail
with
reference to an example.
In this example, each entry in the classification library is created from
plural pre-
processed reference sample spectra that are representative of a class. In this
example, the
reference sample spectra for a class are pre-processed according to the
following
procedure:
First, a re-binning process is performed. In this embodiment, the data are
resannpled onto a logarithmic grid with abscissae:
= EN chant g ______________________________ / 10 gitimmaxi
¨.min ¨.min
where N chõ is a selected value and Irx] denotes the nearest integer below x.
In one
example, Nolan is 212 or 4096.
Then, a background subtraction process is performed. In this embodiment, a
cubic
spline with k knots is then constructed such that p% of the data between each
pair of knots
lies below the curve. This curve is then subtracted from the data. In one
example, k is 32.
In one example, p is 5. A constant value corresponding to the q% quantile of
the intensity
subtracted data is then subtracted from each intensity. Positive and negative
values are
retained. In one example, q is 45.
Then, a normalisation process is performed. In this embodiment, the data are
normalised to have mean yi. In one example, yi = 1.
An entry in the library then consists of metadata in the form of a median
spectrum
value Ai and a deviation value Di for each of the khan points in the spectrum.
The likelihood for the i'th channel is given by:
1 cc-112 r(c) 1
Pr(yiliii, Di) =
Di VirF(C ¨ 1/2) (
C ___________________________________________________ Dt? )
where 1/2 s C < co and where r(c) is the gamma function.
The above equation is a generalised Cauchy distribution which reduces to a
standard Cauchy distribution for C = 1 and becomes a Gaussian (normal)
distribution as
C ¨> 0. The parameter Di controls the width of the distribution (in the
Gaussian limit Di =
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is simply the standard deviation) while the global value C controls the size
of the tails.
In one example, C is 3/2, which lies between Cauchy and Gaussian, so that the
likelihood becomes:
, 31 1
=
4Di (3/2 (yi i)2/D1)3/2 it
For each library entry, the parameters Ili are set to the median of the list
of values
in the i'th channel of the input reference sample spectra while the deviation
Di is taken to
be the interquartile range of these values divided by N/2. This choice can
ensure that the
likelihood for the i'th channel has the same interquartile range as the input
data, with the
use of quantiles providing some protection against outlying data.
Library-Based Analysis - Using a Library for Classification
By way of example, a method of using a classification library to classify an
aerosol,
smoke or vapour sample will now be described.
Figure 35 shows a method 2500 of using a classification library. In this
example, the
method comprises a step 2502 of obtaining a set of plural sample spectra. The
method
then comprises a step 2504 of calculating a probability or classification
score for the set of
plural sample spectra for each class of sample using metadata for the class
entry in the
classification library. The sample spectra are then classified at step 2506
and the
classification is then output in step 2508.
Classification of an aerosol, smoke or vapour sample will now be described in
more
detail with reference to the classification library described above.
In this example, an unknown sample spectrum y is the median spectrum of a set
of
plural sample spectra. Taking the median spectrum y can protect against
outlying data on
a channel by channel basis.
The likelihood Ls for the input data given the library entry s is then given
by:
N chan
Ls = Pr(yly,D) = fl
where pi and Di are, respectively, the library median values and deviation
values
for channel i. The likelihoods Ls may be calculated as log likelihoods for
numerical safety.
The likelihoods Ls are then normalised over all candidate classes 's' to give
probabilities, assuming a uniform prior probability over the classes. The
resulting
probability for the class S- is given by:
Pr(lY) = ___________________________________ g
zs L(si/F)
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The exponent (1/F) can soften the probabilities which may otherwise be too
definitive. In one example, F = 100. These probabilities may be expressed as
percentages,
e.g., in a user interface.
Alternatively, RMS classification scores Rs may be calculated using the same
median sample values and derivation values from the library:
N chan
1102
Rs (y, D) = j.,1
"chan
L=1
Again, the scores Rs are normalised over all candidate classes 's'.
The aerosol, smoke or vapour sample may then be classified as belonging to the
class
having the highest probability and/or highest RMS classification score.
Methods of medical treatment, surgery and diagnosis and non-medical methods
Various different embodiments are contemplated. According to some embodiments
the methods disclosed above may be performed on in vivo, ex vivo or in vitro
tissue. The
tissue may comprise human or non-human animal tissue.
Various surgical, therapeutic, medical treatment and diagnostic methods are
contemplated.
However, other embodiments are contemplated which relate to non-surgical and
non-therapeutic methods of mass spectrometry which are not performed on in
vivo tissue.
Other related embodiments are contemplated which are performed in an
extracorporeal
manner such that they are performed outside of the human or animal body.
Further embodiments are contemplated wherein the methods are performed on a
non-living human or animal, for example, as part of an autopsy procedure.
The various embodiments described herein provide an apparatus and associated
method for the chemical analysis of aerosols and gaseous samples containing
analytes
using mass spectrometry or other gas-phase ion analysis modalities. The method
starts
with the introduction of an aerosol or other gaseous sample 201 containing the
analyte into
an enclosed space, where the sample 201 is mixed with a low molecular weight
matrix
compound 204. This homogeneous or heterogeneous mixture is then introduced
into the
atmospheric interface of a mass spectrometer 102 or ion mobility spectrometer
via inlet
206. On the introduction of the mixture into the low pressure regime of the
analytical
instrument, aerosol particles containing molecular constituents of the sample
and the
matrix compound are formed, which are accelerated by the free jet expansion.
The mixed
composition aerosol particles 205 are subsequently dissociated via collisions
with solid
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collision surfaces 209. The dissociation events produce neutral and charged
species,
including the molecular ions 210 of the chemical constituents of the sample.
The ions 210
may be separated from the neutral species by using electric fields, e.g., by
using an ion
guide 212, such as a Stepwave (RTM) ion guide so as to guide ions 210 on a
different path
to the neutral species. The molecular ions 210 are then subjected to mass or
mobility
analysis. This provides a simple solution for the analysis of molecular
constituents of
aerosols in an on-line fashion without the application of high voltages or
lasers.
The method and device provides a solution for the on-line mass spectrometric
and
ion mobility spectrometric analysis of gas phase or aerosol-type samples.
According to various embodiments the matrix compound 204 may be mixed into the
sample aerosol 201 as a vapour or as a liquid at any point prior to
introduction of the
sample into the ion analyser device 207.
Although the embodiments described above relate to a particular solid
collision
surface geometry for performing the surface induced dissociation of the
clusters, it will be
appreciated that other geometries can be implemented (provided that the
clusters impact
the collision surface 209 at sufficiently high velocity to induce
dissociation).
Although the embodiments described above result in the generation of gas phase
analyte ions due to the impact with the collision surface, it is contemplated
that additional
ionisation techniques may be used downstream of the collision surface in order
to generate
the analyte ions.
Although the embodiments described above impact the mixture of matrix and
analyte on a collision surface in order to atomise the mixture, it is
contemplated that
alternative atomisation techniques may be used.
Although the present invention has been described with reference to various
embodiments, it will be understood by those skilled in the art that various
changes in form
and detail may be made without departing from the scope of the invention as
set forth in
the accompanying claims.
Date Recue/Date Received 2024-04-10
