Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SAMPLE PREPARATION FOR MASS SPECTROMETRY ANALYSIS SYSTEMS
FIELD OF THE INVENTION
The present invention refers to the field of chemical analysis and more
particularly
concerns the preparation of samples, such as, but not limited to, blood
samples and
other biological substances, for analysis through mass spectroscopy or similar
techniques.
BACKGROUND
to Dry blood spot techniques (DBS) are gathering a lot of attention.
Through traditional
DBS, a small drop of blood, obtained by pricking the finger, heel or toe of
the subject,
is gathered on a market spot of on special paper. The blood spot is later
punched out,
mixed with a solvent containing an internal standard or not through processes
such
as shaking, vortexing, centrifugation and/or sonication. These steps are time-
consuming which limits the analytical throughput.
DBS is very advantageous over liquid blood or plasma sample collection as it
is much
less intrusive for the subject, and because DBS paper or card can easily be
stored
and transported without requiring special care or refrigeration. A drawback of
DBS is
however the lengthy extraction process including the punching step, which
reduces
the analysis throughput and increases the analysis time usually performed in
mass
spectroscopy systems. There is therefore the need for new sampling support as
well
as new sample preparation methods procedures which conserve the advantages of
standard DBS whiles alleviating at least some of its drawbacks.
SUMMARY OF THE INVENTION
In accordance with a first aspect of the invention, there is provided a method
for
preparing a sample for analysis. The method includes the steps of:
a) Collecting said sample, said collecting including the substeps of:
2
i. providing a sampling tip including a hollow body having opposite open
extremities respectively defining an input end and an output end, the body
defining a cavity between said open extremities, the sampling tip further
including a filling material provided within the cavity;
ii. depositing the sample on the filling material from the input end of the
body;
and
iii. allowing the sample to dry on the filling material for a sufficient
drying
period; and
b) extracting the sample from the cavity, said extracting including the
substeps of:
i. inserting a solvent within the cavity from the output end of the hollow
body
and allowing the solvent to contact the sample for a contact period; and
ii. expelling the sample and solvent mix out of the cavity from the output end
of the hollow body.
is In
accordance with another aspect of the invention, there is further provided a
sampling tip for receiving a sample, which can be used in a method such as
described above. The sampling tip includes a hollow body, preferably conical
or
pyramidal-shaped, having opposite open extremities respectively defining an
input
end and an output end, the body defining a cavity between said open
extremities. A
filling material is provided within the cavity for receiving the sample from
the input end
of the body. The output end of the body is designed to allow an extraction of
the
sample therefrom. Preferably, the input end of the body is adapted for
connection to
an automated system.
In accordance with yet another aspect of the invention, there is provided a
method for
collecting an analyte sample for analysis. The collecting method includes the
steps of:
i. providing a sampling tip including a hollow body having opposite
open
extremities respectively defining a sample input end and a sample output
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end, the body defining a cavity between said open extremities, the sampling
tip further including a filling material provided within the cavity;
ii. depositing the sample on the filling material from the input end
of the body;
and
iii. allowing the sample to dry on the filling material for a sufficient
drying
period.
Advantageously, the collected sample can be easily stored and transported
without
requiring complex conservation measures.
In accordance with another aspect of the invention, there is also provided a
method
for extracting a sample from the cavity of a sampling tip in which it has been
collected
according to the method above. The extracting method includes the steps of:
i. inserting a solvent within the cavity from the output end of the hollow
body
and allowing the solvent to contact the sample for a contact period; and
ii. expelling the sample and solvent mix out of the cavity from the output
end
of the hollow body of the sampling tip.
The samples are preferably received after extraction in the wells of an
analysis
systems. An optional step of drying the samples in these wells may also be
provided.
Preferably, the collecting method, extracting method or both can be performed
by an
automated system. Further preferably, a plurality of samples can be processed
by a
single automated system, either serially or in parallel, through the use of a
plurality of
corresponding sampling tips.
It will be readily understood that the samples prepared according to
embodiments of
the invention described herein can be analysed through any one of a number of
techniques.
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Other features and advantages of the invention will be better understood upon
a
reading of preferred embodiments thereof with reference to the appended
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGs. 1A through 1C illustrate the steps of a sample collecting method
according to
an embodiment of the invention; FIGs. 1D and 1F illustrate the steps of a
sample
extracting method according to an embodiment of the invention.
FIGs. 2A to 2C are cross-sectional views of a sampling tip at various stages
of the
collecting and extracting methods shown in FIGs. 1A though 1E.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
The present invention relates to the preparation of samples for subsequent
analysis,
in particular for mass spectrometry analysis. It will be readily understood
that
embodiments of the present invention may be used in conjunction with a
plurality of
sample analysis processes, such as, but not limited to, Laser Diode Thermal
Desorption (LDTD), direct analysis techniques such as, but not limited to,
MALDI as
well as chromatographic system combined or not to mass spectrometry or
spectrometry.
The sample to be analysed may be any appropriate substance, preferably in the
liquid
state, and further preferably biological substances such as blood, plasma,
saliva,
urine, liver cells or biological tissues, It may also be embodied by buffer
solutions and
aqueous samples such but not limited to water from river, also to plant or
plant
extract, food or food extract and forensic samples etc. It will be readily
appreciated
that embodiments of the present invention may for example replace traditional
dried
blood spot (DBS) or dried plasma spot (DPS) techniques with highly
advantageous
results.
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Embodiments of the present invention generally involve collecting the sample
in a
sampling tip and extracting the sample from this sampling tip, for example
through
injection into wells of the analytical system through which the sample is to
be
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CA 02766635 2012-01-25
analysed. While preferred embodiments of a sampling tip, collecting method,
extracting method and overall sample preparation process are presented below,
one
skilled in the art will understand that various aspects of the disclosed
embodiments
may be used individually and that numerous variants thereof could be used
without
5 departing from the scope of the present invention.
Sampling tip
Referring to FIG. 2A, there is shown a sampling tip 20 for receiving a sample
according to one embodiment of the invention. The sampling tip 20 includes a
hollow
to body 22. Preferably, the hollow body 22 is conical or pyramidal-shaped,
but it could
alternatively have a different profile, such as tubular or irregular-shaped.
The hollow
body 22 has opposite open extremities respectively defining an input end 24
and an
output end 26, the body 22 defining a cavity 28 between its open extremities.
In the
illustrated embodiment, the input end 24 is embodied by the wider extremity of
the
conical-shaped body 22, whereas the output end 26 is embodied by the thinner
extremity. Preferably, the hollow body is made of plastic, inert polymer,glass
or inert
metal, with different cavity volumes and body length. Markings, a bar code or
other
forms of identification could be provided on the hollow body for
identification and
traceability purposes, or on a separate label attached to the hollow body.
A filling material 30 is provided within the cavity 28 of the hollow body 22.
The filling
material is preferably fibrous, and can for example be made of cellulose
fiber,
polymeric material, glass fiber, and the material may optionally be treated
with
chemical or by a physical treatment such as heat for example. In the preferred
embodiment, the filling material forms a three dimensional mass substantially
blocking the cavity along a portion of its length. It may be introduced in the
cavity in
any appropriate manner, form example by forming one or more balls of fiber
introduced into the cavity from one of its extremities. The filling material
preferably
defined a sample-receiving surface 32 facing the input end 24 of the hollow
body 22,
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for receiving the sample thereon. Although the sample-receiving is shown as
flat in
the enclosed drawings, it may have a different shape, such as curved or
irregular.
The output end 26 of the body 22 is designed to allow an extraction of the
sample
therefrom, as will be further explained below with reference to an extraction
method
according to one embodiment of the invention. Preferably, the input end 24 of
the
body 22 is adapted for connection to an automated system, as will also be
further
explained below.
lo Sample collecting method
Referring to FIGs. 1A to 1C, the steps of a sample collecting method according
to an
embodiment of the invention are illustrated. Although the illustrated
embodiment
involves an automated system for processing a plurality of samples, one
skilled in the
art will readily understand that the collecting method according to the
invention could
.. be embodied through a manual process and/or on a single sample without
departing
from the scope of the present invention.
The method first involves (FIG. 1A) providing a number of sampling tips 20,
one for
each sample to be collected. In the illustrated embodiment, the sampling tips
20 are
mounted on a tip holder 36 which can be embodied a supporting structure having
a
plurality of openings 38 therein shaped to receive and hold the sampling tips.
As
mentioned above, the sampling tips 20 include a hollow body having opposite
input
and output ends, and they are preferably mounted in the tip holder so that the
input is
easily accessible.
?5
In the illustrated embodiment, the collecting method is robotized through the
use of
one or more sampler robots 42. Optionally, the sampler robot 42 may be used in
a
preliminary step of collecting samples 40 from a sample holder 46 having a
plurality of
wells 48, each well containing one such sample 40 (see FIG. 1A). The sampler
robot
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preferably includes a plurality of sampling containers spaced apart in the
same
configuration as the wells 48 of the sample holder 46. The sampling containers
are
adapted to extract the samples 40 from the wells 48, for example through an
appropriate pump mechanism. Additionally or alternatively, the same or a
different
sampler robot 42 may be used to insert the sampling tips 20 into the openings
38 of
the tip holder 36.
The collecting method next involves depositing a sample 40 on the filling
material 30
of each sampling tip 20, from the input end 24 of the corresponding hollow
body 22.
io For this purpose, the sampler robot 42 is preferably moved over the tip
holder 36 with
the sampling containers 44 aligned with the input ends 24 of the sampling tips
20, and
the samples 40 ejected out of the containers 44 into the sampling tips 20 (see
FIG.
1B). As shown in FIG. 2B, the sample 40 in each sampling tip 20 is retained on
the
sample-receiving surface 32 of the filling material 30, therefore facing the
input end
is .. 24 of the sampling tip 20.
The collecting method next involves allowing the sample 40 to dry on the
filling
material 30 for a sufficient drying period 50. The length of the drying period
50 may
vary depending on the application, amount of sample on the filling material,
nature of
20 the sample, etc. It may range from a few seconds to a few hours or days. By
"sufficient", it is understood that the drying period is long enough so that
the liquid
within the sample has completely evaporated, leaving a dry residual onto
and/or into
the filling material. For example, purely for illustrative purposes and for
the sake of
comparison, in the case of blood samples, it is usually recommended en the
field of
25 DBS to leave the sample to dry for at least 2 hours, although.
Once the sample is sufficiently dried the sampling tips 20 may be removed from
the
tip holder, manually, through the sampler robot 42 (see FIG. 1C) or using a
different
device.
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As those skilled in the art will readily understand, dried samples can be
stored,
packaged and transported without the need for special measures or equipment.
As
such, once collected in the sampling tips, the samples can be easily
transferred to
another location and/or stored for a long period of time, to be analysed in a
different
context.
Sample extracting method
Referring to FIGs. 1D and 1E, there is also provided a method for extracting a
sample
from the cavity of a sampling tip in which it has been collected, for example
according
to the method above.
The extracting method according to the illustrated embodiment of the invention
is
preferably performed on an automated system, and further preferably on the
same
system used for the sample collection, including sampler robot 42. It will be
readily
understood that this is in no way [imitative of the present invention, and
that the
sample collection and extraction can be performed at completely different
locations
through completely different systems without departing from the scope of the
present
invention.
The extracting method first includes the step of inserting a solvent 34 within
the cavity
of each sampling tip, and allowing the solvent to be in contact with the
sample for a
contact period 56.
The solvent may be any substance appropriate for use in the context of the
analytical
system through which the sample is destined to be processed. As such it
preferably
includes an internal standard or any organic or inorganic chemicals or a
mixture of all
the above, which might improve the extraction efficiency. For example, the
solvent
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may be embodied by Methanol, Acetonitrile, Water or Ethyl acetate, to name
only a
few.
Preferably, the solvent is inserted into the cavity 28 through the output end
26 of the
hollow body. In the illustrated embodiment, with particular reference to FIG.
1D, each
sampling tip is adapted for connection to a pump mechanism part of the sampler
robot which is positioned over a solvent plate 52 provided with wells 54 each
containing a sufficient quantity of solvent. The pump mechanism is activated
in order
to draw solvent in each sampling tip 20 from the corresponding well 54. A
quantity of
solvent sufficient to completely soak the filling material 30 and cover the
sample 40
should preferably be used.
Advantageously, it is believed that for some embodiments of the invention, the
contact period 56 may be very short, for example of the order of few seconds.
Of
course, the actual length of the contact period may vary with a number of
factors from
one embodiment to the next. Preferably, the contact period is between a few
seconds
and a few hours. The contact period should be determined in order for the
sample to
contact sufficiently well with the solvent to allow its subsequent extraction,
as shown
in FIG. 2C.
The extraction solvent should be in contact with the filling material as well
as with the
dry samples. In some embodiments of the invention, it may be advtageous to
aspirate
a volume of air following the extraction solvent aspiration in order for the
extraction
solvent to reach the region of the cavity where the filling material and the
dry sample
are provided.
The extracting method finally includes expelling the sample and solvent mix
out of the
cavity from the output end of the body of each sampling tip. The sample and
solvent
mix is transferred to a location appropriate for the subsequent analysis of
the sample.
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In the illustrated embodiment (FIG. 1E), this is accomplished by positioning
the
sampler robot 42 over an analysis support 58 provided with an appropriate
number of
wells 60, and reversing the pump mechanism to push out the liquid contents of
the
sampling tip into the wells.
5
One skilled in the art will readily understand that a wide number of systems
and
configurations could be used to accomplish the collecting and the extracting
methods
in an automated manner, using for example a system similar to the Liquid
Handling
Workstation from Hamilton (corporate name) or the Direct Drive Robot from
Agilent
10 (corporate name).
Experimental results
Human blood was spiked with Dextrorphan at concentrations from 1 to 500 ng/mL.
15pL of blood were spotted onto a FTA DMPK-C card and 3 pl were deposited onto
is the paper fibers filled into a tip. Blood was dried at room temperature
for 2 hours.
The 3mm punches were extracted with 50pL of methanol ¨water (75/25) containing
deuterated IS (vortex and centrifuged for 2 minutes). 4 pl of the extract were
deposited into the well. The DBS in tips were extracted by aspiration of 10 pl
of the
methanol/water solution with a contact time of 10 seconds and by expulsing the
extract directly into Lazwell. This process was performed on a Hamilton
Nimbus.
After drying, plates were analyzed in LDTD-MS/MS.
The traditional DBS card extracts analyzed in LDTD-MS/MS gave an excellent
linearity over the calibration range (r2 = 0.99). With the criteria of 5 times
the analyte
signal over the blank, we got a limit of quantification (LLOQ) of 5 ng/mL. The
accuracy at the LLOQ was higher than 90 % and varies between 5 % for the
other
calibration points. The precision was evaluated by running the same batch
within the
same day (intra-day) and on different days within the same week (inter-batch).
The
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average precision at the LLOQ was below 15% while it's below 10% at the other
calibration points.
Analyzing the extracts from the DBS in tips gives similar results as compared
to
traditional DBS cards in term of LLOQ, linearity and accuracy. The
reproducibility
(%CVs) of 12 extractions of the same blood set as QC at 100 ng/mL gives 8.5%
in
ratio and 13.7% in total area count. Variation of the internal standard area
over all
samples extracted in the experiment showed 15.3 %. Sample volume deposition on
the tip was optimized in conjunction with the extraction solvent volume. The
optimal
sampling volume is 3 pl of blood and the optimal extraction volume is 10p1 of
methanol-water (75-25). The extraction time reach a maximum at 10 seconds
contact
time and the fiber amount of 2 mg filled a volume of 6 pl. The volume expulsed
from
the tip is 8pL and this volume is directly deposited into the well. Using a
commercial
robot to performed the extraction-spotting process in a 96 tips configuration
lead to 21
seconds effective extraction which represents 0.2 second per sample.
Of course, numerous modifications could be made to the embodiments above
without
departing from the scope of the present invention.
