U.S. patent application number 10/505837 was filed with the patent office on 2006-07-13 for method and system for high-throughput quantitation of small molecules using laser desorption and multiple-reaction-monitoring.
This patent application is currently assigned to MDS SCIEX. Invention is credited to Tung Chau, John J. Corr, Thomas R. Covey, William H. Fisher.
Application Number | 20060151691 10/505837 |
Document ID | / |
Family ID | 28675456 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060151691 |
Kind Code |
A1 |
Covey; Thomas R. ; et
al. |
July 13, 2006 |
Method and system for high-throughput quantitation of small
molecules using laser desorption and
multiple-reaction-monitoring
Abstract
A mass spectrometry quantitation technique enables
high-throughput quantitation of small molecules using a
laser-desorption (e.g., MALDI) ion source coupled to a
triplequadrupole mass analyzer. The ions generated from the ion
source are collisionally damped/cooled, and then quantitatively
analyzed using the triple-quadrupole analyzer operated in the
multiple-reaction-monitoring (MRM) mode. Significantly improved
measurement sensitivity is obtained by applying laser pulses to the
ion source at a high pulse rate of about SOOHz or higher. This
allows the data acquisition to be performed rapidly, and the speed
of about one second for each sample point on the ion source target
has been achieved.
Inventors: |
Covey; Thomas R.; (Ontario,
CA) ; Corr; John J.; (Ontario, CA) ; Fisher;
William H.; (Ontario, CA) ; Chau; Tung;
(Berkeley, CA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
MDS SCIEX
CONCORD
ON
L4K 4M1
|
Family ID: |
28675456 |
Appl. No.: |
10/505837 |
Filed: |
March 27, 2003 |
PCT Filed: |
March 27, 2003 |
PCT NO: |
PCT/IB03/01915 |
371 Date: |
July 5, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60368195 |
Mar 28, 2002 |
|
|
|
Current U.S.
Class: |
250/290 ;
250/282 |
Current CPC
Class: |
H01J 49/0481 20130101;
H01J 49/0031 20130101; H01J 49/4215 20130101; H01J 49/164 20130101;
H01J 49/004 20130101 |
Class at
Publication: |
250/290 ;
250/282 |
International
Class: |
B01D 59/44 20060101
B01D059/44 |
Claims
1. A method of quantitatively detecting small molecules,
comprising: providing an ion source having a target surface
carrying a sample material containing a type of small molecules to
be detected; operating a laser to apply a plurality of laser pulses
to a selected area on the target source, wherein each laser pulse
generates a plume of analyte ions from the sample material on the
target surface; collisionally damping the analyte ions in the
plumes with a damping gas; passing the collisionally damped analyte
ions into a triple-quadrupole mass analyzer operated in a
multiple-reaction monitoring mode to select ions of a precursor
type derived from small molecules of the type to be detected and
ions of a product type created by fragmenting ions of the precursor
type; counting ions of the product type selected by the
triple-quadrupole mass analyzer.
2. A method as in claim 1, wherein the step of operating operates
the laser at a pulse rate of about 500 Hz or higher.
3. A method as in claim 2, where in the pulse rate of the laser is
between about 500 Hz and 1500 Hz.
4. A method as in claim 3, wherein the pulse rate of the laser is
between about 1000 Hz and 1500 Hz.
5. A method as in claim 1, further including the step of generating
a calibration curve for measurements in the
multiple-reaction-monitoring mode.
6. A method as in claim 1, wherein the damping gas is provided in a
radio-frequency ion guide operated to provide confinement to the
analyte ions.
7. A method as in claim 1, wherein the step of operating operates
the laser at a pulse rate selected to deplete the sample material
in the selected area of the target surface within about one
second.
8. A method of quantitatively analyzing a sample material,
comprising: providing an ion source having a target surface
carrying the sample material; operating a laser at a pulse rate of
about 500 Hz or higher to apply a plurality of laser pulses to a
selected area on the target source, wherein each laser pulse
generates a plume of analyte ions from the sample material on the
target surface; collisionally damping analyte ions in the plumes
with a damping gas; passing the collisionally damped analyte ions
into a triple-quadrupole mass analyzer operated in a
multiple-reaction monitoring mode to select ions of a precursor
type and ions of a product type created by fragmenting ions of the
precursor type; counting ions of the product type selected by the
triple-quadrupole mass analyzer.
9. A method as in claim 8, where in the pulse rate of the laser is
between about 500 Hz and 1500 Hz.
10. A method as in claim 8, wherein the pulse rate of the laser is
between about 1000 Hz and 1500 Hz.
11. A method as in claim 8, further including the step of
generating a calibration curve for measurements in the
multiple-reaction-monitoring mode.
12. A method as in claim 8, wherein the damping gas is provided in
a radio-frequency ion guide operated to provide confinement to the
analyte ions.
13. A method as in claim 8, wherein the pulse rate is selected to
deplete the sample material in the selected area of the target
surface within about one second.
14. A system for quantitative analyses of a sample material,
comprising: a target surface carrying the sample material; a laser
for generating laser pulses directed to the target surface, the
laser being controlled to fire at a pulse rate of about 500 Hz or
higher, wherein each laser pulse generates a plume of analyte ions
from the sample material on the target surface; a damping gas
provided in an ion path of the plumes of analyte ions for
collisionally damping the analyte ions in the plumes; a
triple-quadrupole mass analyzer disposed in the ion path after the
damping gas and operated in a multiple-reaction monitoring mode to
select from the analyte ions of a precursor type and ions of a
product type created by fragmenting ions of the precursor type; and
means for counting ions of the product type selected by the
triple-quadrupole mass analyzer.
15. A system as in claim 14, wherein the laser is operated at a
pulse rate between about 500 Hz and 1500 Hz.
16. A system as in claim 15, wherein the pulse rate of the laser is
between about 1000 Hz and 1500 Hz.
17. A system as in claim 14, further includes a radio-frequency ion
guide in which the damping gas is provided, the RF ion guide being
operated to provide confinement of the analyte ions.
18. A system as in claim 14, wherein the sample material is of a
type of small molecules.
Description
RELATED APPLICATION
[0001] This application claims the priority of U.S. Provisional
Application 60/368,195, filed Mar. 28, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates generally to mass
spectrometry, and more particularly to a way to perform
high-throughput quantitation of small molecules.
BACKGROUND OF THE INVENTION
[0003] Quantitative analyses of pharmaceutically and biologically
important compounds, such as drugs and metabolites, are important
applications of mass spectroscopy. Traditionally, ion sources based
on electrospray (ESI) ionization and atmospheric pressure chemical
ionization (APCI) are used in combination with triple-quadrupole
mass spectrometers to provide quantitative analysis. The
combination provides both high sensitivity and high specificity.
ESI and APCI both generate ions from flowing liquid streams, and
are therefore used by pumping organic and aqueous solvent streams
containing the compounds to be analyzed through the source. Liquid
chromatography is commonly used as an on-line separation technique
prior to the mass spectrometer. Thus, samples can be introduced by
injecting a known volume containing the sample into the liquid
flow, and using the mass spectrometer to monitor specific
combinations of ion mass/charge values that correspond to known
precursor and product fragment ions using the scan mode known as
multiple-reaction-monitoring (MRM) mode. During the scan, samples
are injected sequentially, at a rate in the order of 1 per 10
second, due to limitations in autosamplers, as well as limitation
imposed by the natural width of the eluting peak Once the sample
has passed through the ion source, it is ionized and dissipated in
the source, with only a small fraction of the ions generated from
the sample actually being sampled into the mass spectrometer
system.
[0004] Matrix assisted laser desorption/time-of-flight (MALDI/TOF)
is a different type of mass spectrometer technique, in which
samples are mixed with a UV-adsorbing compound (the matrix),
deposited on a surface, and then ionized with a fast laser pulse. A
short burst or plume of ions is created in the ion source of the
mass spectrometer by the laser, and this plume of ions is analyzed
by a time-of-flight mass spectrometer, by measuring the flight time
over a fixed distance (starting with the ion creating pulse). This
technique is inherently a pulsed ionization technique (required for
the time-of-flight mass spectrometer) as well as a batch-processing
technique, since samples are introduced into the ion source in a
batch (of samples located in small spots on a plate) rather than in
a continuous flowing liquid steam. MALDI/TOF has been almost
exclusively used for the analysis of biopolymers such as peptides
and proteins. The technique is sensitive and works well for fragile
molecules such as those mentioned, and the TOF method is
particularly suitable for the analysis of high-mass compounds.
However, until recently, there has been no viable method of doing
true MS/MS with this type of instrument. Instead, the method of
post-source decay (PSD) is used to provide some fragmentation
information. In this technique, precursor ions are selected in the
flight tube with an ion gate, and then those ions that fragment
before the ion mirror (due to excess energy carried away from the
source) can be mass resolved. This technique provides relatively
poor sensitivity and mass accuracy, and is not considered to be a
high performance MS/MS technique. The MALDI technique also suffers
from the fact that while the mass accuracy and resolution can be
very high (up to 30,000 resolution at low mass, and accuracy of a
few parts-per-million), these important features are difficult to
achieve because they depend on the microstructure of the sample
surface (roughness), the laser fluence, and other instrumental
characteristics which can be hard to control. Good mass accuracy
typically requires that calibration compounds be placed on the
sample surface close to the actual sample itself. The MALDI/TOF
technique has mainly been used for spectral analyses. Some previous
attempts have been made to use MALDI for quantitative analysis, but
they have met with limited success because of the poor precision
obtained with MALDI/TOF.
[0005] Recently, the method of combining MALDI with orthogonal TOF
has been introduced by a group at the University of Manitoba. This
technique, called Orthogonal MALDI, or "oMALDI.TM." (trademark of
Applied Biosystems/MDS SCIEX Instruments, Concord, Ontario, Canada)
as described in U.S. Pat. No. 6,331,702 (assigned to the University
of Manitoba), is an apparatus and method enabling a pulse source,
such as a MALDI source, to be coupled to a variety of spectrometer
instruments, in a manner which more completely decouples the
spectrometer form the source and provides a more continuous ion
beam with smaller angular and velocity spreads. In this technique,
ions generated from a MALDI source as plumes (typically at the rate
of less than 20 Hz, with pulse widths of a few nanoseconds from the
laser pulse) are collisionally cooled in a relatively high pressure
region containing a damping gas within an RF ion guide. Collisions
with the damping gas convert the plumes into a quasi-continuous
beam. This quasi-continuous beam is then analyzed with orthogonal
time-of-flight, in which the ions enter orthogonally to the axis of
the TOF and are pulsed sideways.
[0006] There are several advantages to this combination that are
not available from conventional MALDI/TOF. The TOF resolution and
mass accuracy are decoupled from the source conditions such as
laser fluence and sample morphology. The ions are slowed to near
thermal energies from which they can conveniently be re-accelerated
to tens of electron volts for collisionally activated decomposition
(CAD) in a collision cell. The flux of ions in the beam is low
enough (through having beam stretched out in time) that a
time-to-digital converter (TDC) can be used for ion detection. The
result is that high mass accuracy and resolution can be achieved
under a wide range of operating conditions. In addition, a mass
resolving quadrupole and collision cell can be placed before the
TOF analyzer to provide an MS/MS configuration. Precursor ions from
the MALDI source are collisionally cooled, then selected by the
quadrupole mass filter, fragmented in the collision cell, and the
fragments mass analyzed by the TOF. This provides high mass
resolution and sensitivity for MS/MS of MALDI ions, which has not
been previously available. This MS/MS configuration is referred to
as QqTOF, where Q refers to the mass filter quadrupole and q refers
to the RF-only collision cell.
[0007] The Manitoba group recognized that the oMALDF.TM. technique
allows a MALDI source to be efficiently coupled to a quadrupole
mass spectrometer system, because of the near-continuous nature of
the ion beam. However, there is no recognition that this might
offer improved ability to measure sample concentrations
quantitatively.
SUMMARY OF THE INVENTION
[0008] In view of the foregoing, the present invention provides a
mass spectrometry quantitation technique that enables
high-throughput quantitation of small molecules using a
laser-desorption (e.g., MALDI) ion source coupled to a
triple-quadrupole mass analyzer. As used herein, the term "small
molecules" means compounds that are not inherently polymeric in
nature and, as such, are not composed of repeating subunit classes
of compounds. Small molecules fall outside the realm of biological
macromolecules or polymers, which are composed of repeating subunit
entities such as proteins and peptides (composed of amino acid
subunits), DNA and RNA (composed of nucleic acid subunits), or
cellulose (composed of sugar subunits).
[0009] In accordance with the invention, the ions generated by
laser-desorption of a sample material of a small molecule are
collisionally damped/cooled, and then quantitatively analyzed using
the triple-quad operating in the multiple-reaction-monitoring (MRM)
mode. In according with a feature of the invention, significantly
improved measurement sensitivity is obtained by applying laser
pulses to the ion source at a high pulse rate, preferably about 500
Hz or higher. This allows the data acquisition to be performed
rapidly, and the speed of one second or so for each sample point on
the ion source target has been achieved.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic view of an embodiment of a mass
spectrometer system in accordance with the invention that includes
a MALDI ion source and a triple-quadrupole mass analyzer operated
in the MRM mode for high-throughput quantitation of small
molecules;
[0011] FIG. 2 is a schematic close-up view of the MALDI ion source
of the mass spectrometer system of FIG. 1;
[0012] FIG. 3 is a schematic view of an alternative arrangement in
which the MALDI ion source is in a differentially pumped vacuum
chamber;
[0013] FIG. 4 is a schematic view of another alternative embodiment
in which the MALDI ion source is at atmospheric pressure;
[0014] FIG. 5 is a chart showing exemplary MRM data taken using the
high-throughput quantitation technique of the invention;
[0015] FIG. 6 is a chart showing an exemplary calibration
curve;
[0016] FIG. 7 is a chart showing an exemplary calibration curve
similar to that of FIG. 6 but for a lower concentration range;
[0017] FIG. 8 is a chart showing exemplary data taken using a low
laser pulse rate typically used in conventional MALDI/TOF mass
spectroscopy;
[0018] FIG. 9 is a chart showing the effect of laser pulse rate on
the width of the MRM peaks;
[0019] FIG. 10 is a chart showing a close-up view of a portion of
the chart of FIG. 9;
[0020] FIG. 11 is a chart showing an example of the ratio of the
fragment ion intensity to the M+H intensity for Prazosin; and
[0021] FIG. 12 is a chart showing examples of MRM peak areas as a
function of laser pulse rate.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Referring now to the drawings, wherein like reference
numerals refer to like elements, FIG. 1 shows an embodiment of a
mass spectrometer system that includes an ion source and a mass
analyzer. In accordance with the invention, the ion source is a
matrix-assisted-laser-desorption ion (MALDI) source 20 coupled to a
collision-damping setup 22, and the mass analyzer is a
triple-quadrupole device 30 that is operated in the
multiple-reaction-monitoring (MRM) mode. To activate the MALDI ion
source, laser pulses generated by a laser 40 are directed onto a
sample target 36 of the MALDI ion source 20. A described in greater
detail below, the laser is of a type capable of firing at a pulse
rate of a relatively high rate, such as about 500 Hz or higher.
[0023] The mass spectrometer is connected to a data acquisition
system 50, which includes data acquisition electronics 52 for data
collection, and a computer 56 programmed to control the operations
of the system to perform mass spectrometry studies. Particularly,
the computer 56 controls the pulse rate of the laser 40, and
controls, via interface to the data acquisition electronics 52, the
operation of the triple-quadrupole mass analyzer ("triple-quad") 30
to carry out the MRM study.
[0024] As shown in FIG. 2, in a preferred embodiment, the ions to
be analyzed are generated from the target 36 of the MALDI source
inside a vacuum chamber 60. The ultraviolet (UV) light 62 generated
by the laser 40 is transmitted though a UV lens 66 into the vacuum
chamber 60 and directed onto the surface of the MALDI sample target
36. Each laser pulse generates a plume 70 of ions from the sample
target 36. This plume 70 is collisionally cooled by the gas in the
vacuum chamber and confined by the quadrupole set Q0 disposed
adjacent the sample target 36.
[0025] FIG. 3 shows an alternative embodiment in which the sample
target 36 is disposed in a vacuum region 72 that is separated by
partition 76 from the vacuum region 60 in which the quadrupole set
Q0 sits. This arrangement allows the plume 70 coming off the sample
target 36 to be exposed to a collision-damping gas at a pressure
higher than the pressure in the second vacuum region 60.
[0026] FIG. 4 shows another alternative embodiment in which the
sample target 36 is positioned in the atmosphere outside the vacuum
region 72. As a result, the plume 70 of ions is created in
atmospheric pressure. The plume 70 of ions then passes through the
differentially pumped vacuum region 72 and enters the vacuum region
60 of the quadrupole set Q0.
[0027] Returning to FIG. 1, in the illustrated embodiment, the
triple-quad 30 includes three sets of quadrupole rods designated
Q1, Q2, and Q3. When the triple-quad 30 is operated in the MRM
mode, the first quadrupole rod set Q1 is operated to select a
"precursor" ion from the plume 70 of ions generated by the MALDI
source 20. The second quadrupole rod set Q2 is operated to cause
fragmentation of the precursor ion selected by the first quadrupole
set Q1 by means of collisions with the gas in the space confined by
the rods Q2. The third quadrupole rod set Q3 is then operated to
select a particular `product` ion from the ions generated by
fragmenting the precursor ion. The product ion selected by the
quadruple rods Q3 passes through and an aperture 80 and is
collected by an electrical pulse generation device 82, such as a
CHANNELTRON.RTM. electron multiplier device known to those skilled
in the art. The pulses generated by the pulse generation device 82
are detected by the data acquisition electronics 52, which
typically includes pulse detection devices and counters, etc. The
data collected by the data acquisition electronics 52 are sent to
the computer 56 for storage, display, and analyses. For purposes of
the MRM mode detection, the pulses generated by the pulse
generation device 82 are collected and counted as a function of the
duration of time the sample target is ablated by laser pulses.
[0028] The present invention is based on the unexpected result that
high throughput quantitation of small molecules can be achieved by
combining a triple quad mass analyzer operating in the MRM mode
with a MALDI source activated with laser pulses at a high
repetition rate, such as about 500 Hz or higher, preferably between
about 500 Hz and 1500 Hz, and collisionally damping the ion plumes
generated by the laser pulses. The result was unexpected because
prior to the discovery it was unknown whether the use of a MALDI
source would allow quantitative analyses for small molecules, or
what the sensitivity would be, of if there would be sufficient
speed of analysis to accept a sensitivity compromise, if any. The
inventors have discovered that the use of a high laser pulse rate
provides enhanced sensitivity, the ability to make very high
throughput quantitative measurements on certain compounds that
could not be adequately detected under high throughput conditions
using laser pulse rates typical in traditional MALDI, and much
better reproducibility of the signal. The ability to use relatively
high laser fluence without degrading the mass spectrometer signal
is believed to be due to the presence of a damping gas in the ion
path, which cools the ions through collisions. The collisional
cooling also converts the pulsed ion beam into a quasi-continuous
ion beam, which can be efficiently analyzed with a triple
quadrupole mass spectrometer using the MRM mode of operation. The
higher the laser pulse rate, the more continuous the ion beam
becomes.
[0029] Due to the high sensitivity and throughput of the
quantitation technique of the invention, measurements can be
performed at a high speed. It has been shown that a laser pulse
rate of about 1000-1500 Hz allows throughput rates well under one
sample per second. Since high throughput quantitation is the goal,
it is not desired to "hunt and peck" around on a sample spot, it is
desired to aim "at" the sample spot and start taking
quantitation-quality data. Choice of matrix, and hence sample spot
formation may be influenced by this requirement. Many matrix
materials have been tried, and the matrix material that provides
the best mix of sensitivity and spot-to-spot and day-to-day
reproducibility is .alpha.-cyano (.alpha.-Cyano-4-hydroxycinnamic
acid)(a.k.a. HCCA). HCCA is also typically used for MALDI/TOF
analysis of peptides and proteins.
[0030] In operation, samples to be analyzed are deposited on a
sample target plate that typically may contain from 96 to 384, or
more, sample spot positions. One of the main application areas of
this quantitation technique is the quantitation of pharmaceutical
compounds and their metabolites or reaction products. Solutions
containing the material of interest are typically extracted from a
biological sample such as blood or urine or plasma, or from a
buffer solution containing enzymes that have been used to react
with the samples. Some simple clean-up procedure maybe used in
order to remove most of the unwanted salts or proteins. A small
volume, usually less than 1 microliter, is then mixed with a matrix
solution. The matrix solution is selected in order to efficiently
adsorb ultraviolet light at the wavelength of the laser, which is,
for example, 335 nanometers. The mixture of sample solution and
matrix is deposited on the sample plate, and allowed to dry on the
plate, forming a spot of crystalized material that contains the
sample of interest. The plate is inserted into the ion source of
the mass spectrometer. In one configuration, the plate is inserted
into a holder that is moved by stepper motors such that the sample
spot of interest is in front of the ion optics of the mass
spectrometer. An O-ring around the sample plate provides a vacuum
seal. The laser is fired repetitively at the sample spot in order
to desorb and ionize the sample. The ions of interest (both those
of the internal standard and those of the analyte) are monitored by
the mass spectrometer, using dwell times in the range of a few
milliseconds to several hundred milliseconds, depending on the
laser pulse rate. As described in greater detail below, in
accordance with the invention, the laser is fired at a high rate,
from about 500 Hz up to, for example, about 1500 Hz. In one method,
the plate remains stationary while the laser is fired for a fixed
period of time (e.g. 1 second), and the ion signal intensity is
integrated for this time period in order to provide a measure of
the amount of sample consumed. In another method, the laser is
fired until the ion signal is reduced to a low level, indicating
that the sample is fully depleted in this region. In another
method, the sample plate is moved in a small pattern in order to
bring new regions of sample into the path of the laser light as the
ion signal is being measured. This can provide a more
representative signal if the sample is inhomogeneously dispersed,
but more time is required to process each sample. The second method
is described in more detail by the following example.
[0031] Samples for analysis are mixed in a predetermined ratio with
the HCCA MALDI matrix solution, such as 1:1 ratio that reduces the
analyte concentration to half of the original concentration.
Samples are deposited onto the target plate using a manual pipette
or any other liquid handling device capable of accurately
delivering volumes in the 0.1 to 2 ul range. The liquid drops on
the target plate are allowed to fully dry and crystallize before
the target plate is placed into the MALDI source.
[0032] An example of the high-throughput quantitation process in
accordance with the invention is described below. A fresh part of
the sample spot is presented in front of the laser for the duration
of the data acquisition. For quantitative MRM analysis an internal
standard is include in the sample, and is therefore present in the
sample spot. The chromatographic (signal as a function of time)
data acquisition is started (for both the analyte and the internal
standard), with the laser light not striking the sample spot. The
laser light is permitted to strike the sample spot and ablate the
sample from the same location on the sample spot (i.e. the sample
is not moved during ablation). This causes the ion signal to
increase significantly from the background level, reach a peak, and
then decrease back to the background level as the sample is
completely desorbed. The laser light is stopped from striking the
sample spot once ion signal has returned to the background level.
The laser is then moved on to the next location on the sample
target from which data will be taken. The next location may be
another location in the same sample spot or a completely different
sample spot.
[0033] To provide a reference, data are taken for the same ion
pairs for a "matrix blank" from a sample spot containing only the
matrix and the sample solvent in a predetermined ratio, such as
1:1. From the data that present ion signals as a function of time,
which look much like LC/MS flow injection peaks, the peak areas for
the analyte and internal standard peaks are calculated, and the
ratio of analyte area to internal standard area for each peak is
taken, and results are plotted accordingly.
[0034] FIG. 5 gives an example of the type of MRM data acquired
using this technique. In this case the laser was fired at two
discrete locations on each of five sample spots. The analyte was 25
pg/ul Haloperidol (a commercially available compound). Data was
acquired using a 20 ms dwell time to monitor the 376.0/165.1 m/z
ion pair. The laser was operated at 1400 Hz and .about.6 uJ per
pulse. For such MRM quantitative analyses samples of 0.2 to 1 ul
are deposited onto the target plate (above data was from 0.2 ul
spots). There are at least 10 data points per peak in all cases.
The average peak width is given by a Full Width at Half Maximum (FW
of 130 msec, which offers the possibility of routine analytical
throughput at speeds not attainable from typical atmospheric
pressure ionization sources used on mass spectrometers, such as the
previously mentioned ESI and APCI sources.
[0035] Using this method, calibration curves can be generated, such
as the one shown in FIG. 6 for Lidoflazine, a commercially
available compound. A concentration of 5 pg/ul Prazosin was
included in the sample preparation, and was used as the internal
standard. All MRM concentration data points were acquired in
triplicate with a 10 msec dwell time for the analyte ion pair and a
10 msec dwell time for the internal standard. The ion pairs
monitored were 386.2/122.0 for Lidoflazine, and 384.2/247.0 for
Prazosin, the internal standard. The calibration curve used peak
areas, and the analyte peak areas were ratioed to the internal
standard peak areas, and a linear fit with no weighting, was used.
The calibration curve covers the wide range 0.5 pg/ul to 2000
pg/ul, and includes blanks. The curve is very linear, with
r=0.9979. FIG. 7 shows the same data as FIG. 6, but this time it is
only analyzed over the range 0.5 pg/ul to 100 pg/ul, which is of
much greater analytical interest. Over this smaller concentration
range, the data has been re-analyzed and the calibration curve is,
again, very linear, with r=0.9957.
[0036] As mentioned above, the laser pulse rate has a very
significant influence on the possible speed of analysis, and hence
on sample throughput. To provide a contrast, FIG. 8 shows MRM data
taken with a Nitrogen laser operating at 40 Hz and a pulse energy
of .about.18 uJ per pulse. Even though this pulse rate is much
lower than the laser pulse rate used in the technique of the
invention, it is actually "high" for conventional MALDI use. In
this case, the laser was fired at two discrete locations on each of
five sample spots. The analyte was 25 pg/ul Diltiazem (a
commercially available compound), and 0.2 ul sample spots were
used. Data was acquired using a 500 ms dwell time to monitor the
414.9/178.1 m/z ion pair. The average peak with is given by a Full
Width at Half Maximum (FWHM) of 4.51 sec. This FWHM is much greater
than the value of 130 msec for the 1400 Hz data in FIG. 5
(approximately 34 times as much). In general, for lower frequencies
the use of higher pulse energies causes the sample to be ablated
more rapidly, yielding narrow peaks and hence higher throughput
possibilities than for low pulse energies at the same lower
frequencies. However, higher laser pulse energies can cause
increased molecular fragmentation in the ion source region and a
resulting decrease in MS/MS sensitivity. The much narrower peaks
provided by higher pulse rates offer the ability to acquire data in
a much more high throughput manner.
[0037] FIG. 9 shows the effect of the laser pulse rate on the width
of MRM peaks for Haloperidol. The laser pulse energy was kept fixed
while the laser pulse rate was varied, and the FWHM was measured
for each frequency. FIG. 10 is an expansion of the data shown in
FIG. 7. The pulse width decreased from .about.17 sec. at a laser
pulse rate 10 Hz to .about.0.1 sec. at a laser pulse rate of 1400
Hz. This is a decrease of .about.155 times, permitting much higher
sample throughput.
[0038] Higher laser pulse rates provide other benefits as well.
Higher pulse rates at lower energy cause less molecular
fragmentation in the ion source region that results in more
precursor ions on which to perform MS/MS. Experiments were
performed in which single MS Q1 spectra were taken as the laser
pulse rate was varied. The intensity of the molecular ion (M+H) was
measured as well as the intensity of the major fragment ion
corresponding to M+H. FIG. 11 shows the ratio of the fragment ion
intensity to the M+H intensity for Prazosin.
[0039] As the laser pulse frequency was varied the MS scan speeds
were adjusted so that the same number of laser shots occurred for
data taken at different frequencies. Molecular fragmentation was
reduced by about a factor of two as the laser pulse rate was
increased from 40 Hz to 1400 Hz. Since higher laser pulse rates
cause less molecular fragmentation in the ion source, there is more
molecular ion left intact on which to perform MS/MS experiments,
such as MRM. FIG. 12 shows MRM peak area as a function of laser
pulse rate, for Haloperidol and Prazosin. It is seen that there is
a 60% to 100% increase in MRM peak area as the laser pulse rate was
increased from 10 Hz to 1400 Hz.
[0040] The quantitation technique of the present invention offers
several advantages over both conventional MALDI/TOF and orthogonal
MALDI/TOF (or MALDI QqTOF). First, the sensitivity is significantly
improved over MALDI QqTOF because of the high sensitivity of the
triple quadrupole in an MRM mode, compared to that of a QqTOF. In
the QqTOF, significant ion losses are encountered due to duty cycle
limitations of the orthogonal TOF method, which only samples a
portion of the ion beam (with the efficiency being lower at low
mass than at high mass). Experience has shown that the absolute
sensitivity or efficiency is 10 to 50 times better with MRM in a
triple quadrupole than with the equivalent experiment on a
QqTOF.
[0041] A second advantage is provided by the fact that MS/MS is a
very specific detection technique, in which chemical noise
background is usually very low. This is because only specific
precursor/product ion combinations are monitored. In MALDI/TOF
(where there is no efficient MS/MS capability), the chemical noise
is usually high, especially at low mass. This chemical noise is due
to matrix-related ions that are present in high abundance, and can
obscure the signal from low-mass analyte ions. Therefore, the MS/MS
capability of the triple quadrupole can allow the sensitive
detection of even low mass ions that are present at much lower
intensity than the matrix-related ions. Furthermore, MALDI/TOF has
such a large ion flux that a transient recorder detection system
must be used. This has the disadvantage of being somewhat noisy, so
that single-ion events may not be detected. With the technique of
the invention, the pulses are stretched out in time so that the ion
flux is much lower, even if the same number of ions per pulse are
received, so that a time-to digital converter can be used for pulse
counting. This benefits MS/MS, since the noise levels are very
low.
[0042] Thirdly, the fact that the mass spectrometer performance (in
this case, the triple quadrupole) is independent of the laser
fluence and sample morphology, allows the possibility of rapidly
desorbing the sample from the surface, in order to improve the rate
at which samples can be analyzed. For example, in MALDI/MS, the
laser fluence must be kept low, near the ionization threshold, in
order that the mass resolution and mass accuracy are not
significantly affected. However, because of collisional cooling of
the ion beam, the laser energy can be increased to the point just
below that at which the sample will be thermally degraded occurs.
This can allow more rapid desorption of the sample, and therefore
allow more samples to be processed in a short period of time.
Furthermore, the fact that the mass spectrometer analytical
performance is independent of the sample morphology means that a
larger region of the sample can be ionized at one time, by using a
larger diameter laser beam. Inhomogeneities in the sample will have
no effect on the mass spectrometer performance (mass resolution or
mass position), in contrast to the situation with MALDI/TOF.
Furthermore, the quasi-continuous nature of the ion beam allows the
use of pulse counting methods (since the ion flux is still rather
weak). Pulse-counting is inherently the most noise-free detection
method for MS/MS, allowing the best signal-to-noise ratio.
[0043] The combination of a collisionally cooled MALDI ion source
with a triple quadrupole in MRM mode and with high laser pulse
rates therefore provides a very sensitive and rapid technique for
the quantitative analysis of biological and pharmaceutical samples
of small molecules. The ability to prepare samples off-line, and
deposit them on sample plates means that methods of parallel sample
processing can be used to extract and clean-up multiple samples
off-line. Since generally the mass spectrometer is the most
expensive part of the analytical system, the ability to prepare the
samples for analysis in a batch mode, significantly improves the
efficiency of the process.
[0044] In view of the many possible embodiments to which the
principles of this invention may be applied, it should be
recognized that the embodiments described herein with respect to
the drawing figures are meant to be illustrative only and should
not be taken as limiting the scope of the invention. Therefore, the
invention as described herein contemplates all such embodiments as
may come within the scope of the following claims and equivalents
thereof.
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