U.S. patent application number 15/948944 was filed with the patent office on 2018-08-16 for method and system for determining the concentration of an analyte in a fluid sample.
The applicant listed for this patent is Labcyte Inc.. Invention is credited to Richard N. Ellson, Joseph D. Olechno, Ian Sinclair, Richard G. Stearns, Jonathan Wingfield.
Application Number | 20180231442 15/948944 |
Document ID | / |
Family ID | 55747494 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180231442 |
Kind Code |
A1 |
Ellson; Richard N. ; et
al. |
August 16, 2018 |
METHOD AND SYSTEM FOR DETERMINING THE CONCENTRATION OF AN ANALYTE
IN A FLUID SAMPLE
Abstract
A method and system are provided for detecting the concentration
of an analyte in a fluid sample. The method and system involve
analysis of a volatilized, ionized fluid sample using a mass
spectrometer or other ionic analyte detection device that provides
a signal proportional in intensity to the quantity of ionized
analyte detected. The improvement involves replacement of a
necessary non-analyte component in the fluid sample with a
substitute component that serves the same purpose as the original
component but is either more volatile than the original component
and/or the analyte or undergoes a reaction to provide lower
molecular weight reaction products, and results in an increased
intensity in signal and signal-to-noise ratio. Acoustic fluid
ejection is a preferred method of generating nanoliter-sized
droplets of fluid sample that are then volatilized, ionized, and
analyzed. Also provided are zwitterionic compounds suitable as the
substitute components that when ionized and heated decompose to
provide carbonic dioxide, a nitrogenous species such as ammonia, an
amine, or nitrogen gas, and a volatile aromatic compound.
Inventors: |
Ellson; Richard N.; (San
Jose, CA) ; Stearns; Richard G.; (San Jose, CA)
; Olechno; Joseph D.; (San Jose, CA) ; Sinclair;
Ian; (Warrington, GB) ; Wingfield; Jonathan;
(Macclesfield, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Labcyte Inc. |
San Jose |
CA |
US |
|
|
Family ID: |
55747494 |
Appl. No.: |
15/948944 |
Filed: |
April 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14887320 |
Oct 19, 2015 |
9939352 |
|
|
15948944 |
|
|
|
|
62065600 |
Oct 17, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6848 20130101;
H01J 49/0431 20130101; G01N 33/6851 20130101; C12Q 1/68 20130101;
H01J 49/04 20130101; G01N 1/28 20130101; Y10T 436/24 20150115; Y10T
436/25875 20150115; H01J 49/0422 20130101 |
International
Class: |
G01N 1/28 20060101
G01N001/28; H01J 49/04 20060101 H01J049/04 |
Claims
1. An improved method for determining the concentration of an
analyte in a fluid sample that additionally comprises a necessary
non-analyte original component, the method comprising volatilizing
and ionizing the sample, and introducing the ionized, volatilized
sample into an ionic analyte detection device that provides a
signal proportional in intensity to the quantity of ionized analyte
detected, wherein the improvement comprises: acoustically
generating nanoliter-sized droplets of the fluid sample prior to
volatilization and ionization, such that the fluid sample is
introduced into the ionic analyte detection device in the form of
nanoliter-sized droplets; and substituting for the necessary
non-analyte original component a substitute component that: (a)
functions as the original component in the fluid sample; (b) (i) is
more volatile than the necessary original component or (ii) upon
volatilizing the fluid sample, undergoes a reaction to yield at
least one reaction product that is more volatile than the necessary
original component; and (c) results in an increase in the intensity
of the signal and/or a greater signal-to-noise ratio than either
the signal intensity or signal to noise ratio obtained using the
original component.
2. (canceled)
3. The method of claim 1, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 5 nl.
4. The method of claim 3, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 2.5 nl.
5. The method of claim 4, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 50 pl.
6. The method of claim 5, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 1 pl.
7. The method of claim 1, wherein the improvement further includes
using focused acoustic ejection to generate the nanoliter-sized
droplets of the fluid sample.
8. The method of claim 7, wherein the acoustic ejection is carried
out using an acoustic ejector that directs focused acoustic energy
into a reservoir containing the fluid sample in a manner that
results in the rapid ejection of consistently sized fluid droplets
from the surface of the fluid sample.
9. The method of claim 1, wherein the ionic analyte detection
device comprises a mass spectrometer.
10. The method of claim 9, wherein the ionizing comprises chemical
ionization, field desorption ionization, electrospray ionization,
atmospheric pressure chemical ionization, matrix-assisted laser
desorption ionization, or inductively coupled plasma
ionization.
11. The method of claim 1, wherein the analyte comprises a drug, a
metabolite, an inhibitor, a ligand, a receptor, a catalyst, a
synthetic polymer, or an allosteric effector.
12. The method of claim 1, wherein the analyte is a
biomolecule.
13. The method of claim 12, wherein the biomolecule comprises a
nucleotide analyte, a peptidic analyte, or a saccharidic
analyte.
14. The method of claim 1, wherein the necessary non-analyte
original component comprises an original salt and the substitute
component comprises a substitute salt.
15. The method of claim 14, wherein the original salt and the
substitute salt function as buffer salts for the fluid sample, such
that volatilization of the fluid sample results in gas phase
extraction of the substitute buffer salt.
16. The method of claim 15, wherein the substitute salt comprises
singly charged ions formed from weak acids or weak bases.
17. The method of claim 16, wherein the substitute salt comprises
ammonium bicarbonate, ammonium formate, ammonium acetate,
pyridinium acetate, pyridinium formate, ethylmorpholinium acetate,
trimethylamino acetate, or trimethylamino formate.
18. The method of claim 17, wherein the substitute salt comprises
ammonium bicarbonate, ammonium formate, or ammonium acetate.
19-40. (canceled)
41. The method of claim 1, where the increase in analyte signal
intensity and/or signal-to-noise ratio is at least 10%.
42. The improved method of claim 41, where the increase in analyte
signal intensity and/or signal-to-noise ratio is at least 25%.
43. The method of claim 1, wherein the droplets introduced into the
ionic analyte detection device comprise the analyte and the
substitute component.
44. An improved method for determining the concentration of an
analyte in each of a plurality of fluid samples that additionally
comprises a necessary non-analyte original component, the method
comprising volatilizing and ionizing the samples and introducing
each ionized, volatilized sample into an ionic analyte detection
device that provides a signal proportional in intensity to the
quantity of ionized analyte detected, wherein the improvement
comprises substituting for the necessary non-analyte original
component a substitute component that functions as the original
component in the fluid sample, is more volatile than the necessary
original component, and results in an increase in the intensity of
the signal and/or a greater signal-to-noise ratio than either the
signal intensity or signal to noise ratio obtained using the
original component, and additionally comprises (a) providing the
fluid samples in each of a plurality of fluid reservoirs; (b)
acoustically coupling an acoustic droplet ejector to a first of the
fluid reservoirs; (c) activating the ejector to generate focused
acoustic radiation toward the first reservoir and into the fluid
sample therein, in a manner effective to eject nanoliter-sized
droplets of the fluid sample into the ionic analyte detection
device; (d) positioning another of the fluid reservoirs and the
acoustic droplet ejector in acoustic coupling relationship; (e)
repeating step (c); and (f) repeating steps (d) and (e) with
additional fluid reservoirs in the plurality of fluid reservoirs at
a rate of greater than 5 reservoirs per second.
45. The method of claim 44, wherein the droplets comprise both the
analyte and the substitute component.
46. The method of claim 44, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 5 nl.
47. The method of claim 46, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 2.5 nl.
48. The method of claim 47, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 50 pl.
49. The method of claim 48, wherein the nanoliter-sized droplets of
fluid sample have a mean droplet size of less than about
approximately 1 pl.
50. The method of claim 44, wherein the rate is greater than 10
reservoirs per second.
51. The method of claim 50, wherein the rate is greater than 25
reservoirs per second.
52. The method of claim 44, wherein the ionic analyte detection
device comprises a mass spectrometer.
53. The method of claim 52, wherein the ionizing comprises chemical
ionization, field desorption ionization, electrospray ionization,
atmospheric pressure chemical ionization, matrix-assisted laser
desorption ionization, or inductively coupled plasma
ionization.
54. The method of claim 44, wherein the analyte comprises a drug, a
metabolite, an inhibitor, a ligand, a receptor, a catalyst, a
synthetic polymer, or an allosteric effector.
55. The method of claim 44, wherein the analyte is a
biomolecule.
56. The method of claim 44, wherein the biomolecule comprises a
nucleotide analyte, a peptidic analyte, or a saccharidic
analyte.
57. The method of claim 44, wherein the necessary non-analyte
original component comprises an original salt and the substitute
component comprises a substitute salt.
58. The method of claim 57, wherein the original salt and the
substitute salt function as buffer salts for the fluid sample, such
that volatilization of the fluid sample results in gas phase
extraction of the substitute buffer salt.
59. The method of claim 58, wherein the substitute salt comprises
singly charged ions formed from weak acids or weak bases.
60. The method of claim 59, wherein the substitute salt comprises
ammonium bicarbonate, ammonium formate, ammonium acetate,
pyridinium acetate, pyridinium formate, ethylmorpholinium acetate,
trimethylamino acetate, or trimethylamino formate.
61. The method of claim 60, wherein the substitute salt comprises
ammonium bicarbonate, ammonium formate, or ammonium acetate.
62. The method of claim 44, wherein the fluid reservoirs are
arranged in an array.
63. The method of claim 62, wherein the fluid reservoirs are
contained within a substrate comprising an integrated multiple
reservoir unit.
64. The method of claim 63, wherein the integrated multiple
reservoir unit is a microwell plate and the fluid reservoirs are
wells therein.
65. The method of claim 62, wherein the fluid reservoirs are tubes
in a tube rack.
66. An improved method for determining the concentration of an
analyte in each of a plurality of fluid samples that additionally
comprises a necessary non-analyte original component, the method
comprising volatilizing and ionizing the samples and introducing
each ionized, volatilized sample into an ionic analyte detection
device that provides a signal proportional in intensity to the
quantity of ionized analyte detected, wherein the improvement
comprises substituting for the necessary non-analyte original
component a substitute component that functions as the original
component in the fluid sample, is more volatile than the necessary
original component, and results in an increase in the intensity of
the signal and/or a greater signal-to-noise ratio than either the
signal intensity or signal to noise ratio obtained using the
original component, and additionally comprises (a) providing the
fluid samples in each of a plurality of fluid reservoirs; (b)
acoustically coupling an acoustic droplet ejector to a first of the
fluid reservoirs; (c) activating the ejector to generate focused
acoustic radiation toward the first reservoir and into the fluid
sample therein, in a manner effective to eject nanoliter-sized
droplets of the fluid sample into the ionic analyte detection
device, wherein the droplets have a mean droplet size of less than
approximately 50 pl; (d) positioning another of the fluid
reservoirs and the acoustic droplet ejector in acoustic coupling
relationship; (e) repeating step (c); and (f) repeating steps (d)
and (e) with additional fluid reservoirs in the plurality of fluid
reservoirs at a rate of greater than 10 reservoirs per second.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 14/887,320,
filed Oct. 19, 2015, which claims priority under 35 U.S.C. .sctn.
119(e)(1) to provisional U.S. Patent Application Ser. No.
62/065,600, filed Oct. 17, 2014, the disclosures of which are
incorporated by reference herein.
TECHNICAL FIELD
[0002] The present invention relates generally to improved methods
for detecting the presence or quantity of an analyte in a fluid
sample, and more particularly relates to improvements in such
methods where nanoliter-sized fluid droplets are generated and the
sample accurately analyzed. The invention finds utility in the
fields of analytical chemistry, biological research,
pharmaceuticals, and medicine.
BACKGROUND
[0003] Accurate determination of the presence, identity,
concentration, and/or quantity of an ionized species in a sample is
critically important in many fields. Most techniques used in such
analyses involve ionization of species in a fluid sample prior to
introduction into the analytical equipment employed. The choice of
ionization method will depend on the nature of the sample and the
analytical technique used, and many ionization methods are
available, including, without limitation, chemical ionization,
electron impact ionization, desorption chemical ionization, and
atmospheric pressure ionization, including electrospray ionization
and atmospheric pressure chemical ionization.
[0004] The presence of contaminants in biological samples
undergoing analysis is obviously problematic for a number of
reasons. Contaminants might cause interference with the analytical
procedure, chemically or physically altering the sample or the
analyte itself. Contaminants may also be mistaken for analyte or
vice versa, such that the concentration measured concentration of
analyte may be significantly higher or lower than the actual
concentration. The same problem can be caused by necessary
components of the fluid sample, such as buffer salts, surfactants,
and other species that may be essential for biochemical steps
preceding the analysis.
[0005] Mass spectrometry is a well-established technique that
involves the detection of an analyte in ionized form. In this
technique, sample molecules are ionized and the resulting ions are
sorted by mass-to-charge ratio. For analytes contained in a fluid
sample, the sample is typically converted to an aerosol that
undergoes desolvation, vaporization, and ionization in order to
form fluid ions.
[0006] The presence of non-analyte species can be particularly
problematic in mass spectrometry, where analyte concentration may
be very low and the number and concentration of contaminants and
interfering components may be relatively high. One study has
documented over 650 contaminants and interfering components
frequently found in biological samples undergoing mass
spectrometric analysis. B. O. Keller et al. (2008), "Interferences
and contaminants encountered in modern mass spectrometry," Anal.
Chim. Acta 627(1):71-81. These include salts, buffering agents,
endogenous compounds, surfactants, drugs, metabolites, and
proteins. When these species reduce detection sensitivity by
decreasing the signal-to-noise ratio and give rise to a flawed
analysis, the problem has been characterized as "ion suppression."
See Weaver et al. (2006) Rapid Communications in Mass Spectrometry
20:2559-64.
[0007] It has been postulated that "the main cause of ion
suppression is a change in the spray droplet solution properties
caused by the presence of nonvolatile or less volatile solutes,"
i.e., solutes that are nonvolatile or less volatile than the
analyte; see Annesley (2003) "Ion Suppression in Mass
Spectrometry," in Clinical Chemistry 49(7):1041-1044, citing King
et al. (2000) J. Am. Soc. Mass Spectrom. 11:942-50. The reference
explains that the nonvolatile or less volatile contaminants and
components change the efficiency of droplet formation or droplet
volatilization, which in turn affects the quantity of charged
analyte in the gas phase that ultimately reaches the detector.
Annesley cites studies showing that molecules of higher mass tend
to suppress the signal of smaller molecules and that more polar
analytes are susceptible to ion suppression. Annesley at 1042,
citing Sterner et al. (2000) J. Mass Spectrom. 35:385-91 and
Bonfiglio et al. (1999) Rapid Commun. Mass Spectrom. 13:1175-85.
Weaver et al. cites several possible mechanisms underlying ion
suppression: (1) competition for charge between analyte and
ion-suppressing agent, leading to reduced ionization of analyte;
(2) large concentrations of ion-suppressing agents causing an
increase in surface tension as well as an increase in droplet
viscosity, in turn resulting in decreased evaporation efficiency;
and (3) gas phase reactions between the ionized analyte and other
species in the sample, resulting in an overall loss of charge from
the analyte ions. Weaver et al. at 2562.
[0008] As electrospray ionization (ESI) has a relatively complex
ionization mechanism, relying heavily on droplet charge excess,
there are additional factors to consider when exploring the cause
of ion suppression and potential solutions. It has been widely
observed that for many analytes, at high concentrations, ESI
exhibits a loss of detector response linearity, perhaps due to
reduced charge excess caused by analyte saturation at the droplet
surface, inhibiting subsequent ejection of gas phase ions from
further inside the droplet. Thus, competition for space and/or
charge may be considered as a source of ion suppression in ESI.
Both physical and chemical properties of analytes (e.g. basicity
and surface activity) determine their inherent ionization
efficiency. Biological sample matrices naturally tend to contain
many endogenous species with high basicity and surface activity,
and the total concentration of these species in the sample will
thus quickly reach levels at which ion suppression can be
expected.
[0009] Another explanation of ion suppression in ESI considers the
physical properties of the droplet itself rather than the species
present. As noted above, high concentrations of interfering
components give rise to increased surface tension and viscosity
that in turn reduce evaporation efficiency, and this is known to
have a marked effect on ionization efficiency.
[0010] An additional theory to explain ion suppression in ESI
relates to the presence of non-volatile species that can either
cause co-precipitation of analyte in the droplet (thus preventing
ionization) or prevent the contraction of droplet size to the
critical radius required for ion evaporation and/or charge residue
mechanisms to form gas phase ions efficiently. It should also be
pointed out that the degree of ion suppression may be dependent on
the concentration of the analyte being monitored, and with the
ever-increasing demand to lower detection threshold, ion
suppression may become a more and more serious problem.
[0011] Ion suppression has primarily been addressed by de-salting
the fluid sample using dialysis, liquid chromatography, solid-phase
extraction, or ion exchange. These processes require time,
materials, and equipment, and can reduce the available quantity of
an already small sample. In addition, certain ionic or ionizable
species may be essential to maintain in the sample, such as buffer
systems.
[0012] An ideal method to address ion suppression would:
[0013] Eliminate the need for additional process steps and
materials, including clean-up and de-salting;
[0014] Eliminate the need for additional processing time;
[0015] Be adaptable to use with very small sample sizes, consume a
small portion of the small sample size, allow for detection of low
analyte concentrations, and be composed of very small droplets;
and
[0016] Be capable of implementation in a high speed analytical
system such as high throughput mass spectrometry, optimally
enabling analysis of up to at least 50,000 samples per day or more;
and
[0017] Eliminate the need for pre-analysis "clean-up" of the sample
to remove contaminants and interfering components.
SUMMARY OF THE INVENTION
[0018] Accordingly, the present invention addresses the
aforementioned need in the art by providing an improved method for
accurately determining the concentration of an analyte in a fluid
sample.
[0019] In one embodiment, an improved method is provided for
determining the concentration of an analyte in a fluid sample that
also contains a necessary non-analyte component, where the method
comprises volatilizing and ionizing the fluid sample, and
introducing the ionized, volatilized sample into an ionic analyte
detection device that provides an analyte signal proportional in
intensity to the quantity of analyte detected, e.g., a mass
spectrometer, wherein the improvement comprises replacing the
necessary non-analyte original component with a substitute
component that (a) functions as the necessary non-analyte component
in the fluid sample, i.e., serves the same purpose with respect to
the fluid sample, (b) is more volatile than the original component
and/or the analyte, and/or undergoes a chemical reaction to yield
at least one reaction product that is more volatile than the
original component and/or the analyte, and (c) results in an
increase in the intensity of the analyte signal and/or a greater
signal to noise ratio than either the signal intensity or signal to
noise ratio obtained using the original component.
[0020] In another embodiment, acoustic ejection is used to generate
nanoliter-sized droplets that are then volatilized, ionized, and
analyzed, wherein "nanoliter-sized" droplets are defined herein as
droplets of 5 nl or less. In acoustic ejection, an acoustic ejector
directs focused acoustic energy into a reservoir containing the
fluid sample in a manner that results in the ejection of fluid
droplets from the surface of the fluid sample. Acoustic ejection
provides many advantages over other droplet generation
methodologies; for instance, acoustic fluid ejection devices are
not subject to clogging, misdirected fluid or improperly sized
droplets, and acoustic technology does not require the use of
tubing or any invasive mechanical action. Acoustic ejection
technology as described, for example, in U.S. Pat. No. 6,802,593 to
Ellson et al., enables rapid sample processing and generation of
droplets in the nanoliter or even picoliter range. In addition,
acoustic ejection enables control over droplet size as well as
repeated generation of consistently sized droplets. See U.S. Pat.
No. 6,416,164 B1 to Stearns et al., incorporated by reference
herein. As explained in that patent, the size of acoustically
ejected droplets from a fluid surface can be carefully controlled
by varying the acoustic power, the acoustic frequency, the
toneburst duration, and/or the F-number of the focusing lens.
[0021] In a further embodiment, a zwitterionic compound is used as
the substitute non-analyte component in the method of the
invention. Upon volatilization, the zwitterionic compound undergoes
a chemical reaction to yield at least one reaction product that is
more volatile than the original non-analyte component and/or the
analyte.
[0022] In another embodiment, a method is provided an improved
method is provided for determining the concentration of an analyte
in a fluid sample that also contains a necessary non-analyte
component, where the method comprises volatilizing and ionizing the
fluid sample, and introducing the ionized, volatilized sample into
an ionic analyte detection device that provides an analyte signal
proportional in intensity to the quantity of analyte detected,
where a substitute component is selected to replace the necessary
non-analyte component and, upon volatilizing the fluid sample,
undergoes a reaction to yield at least one reaction product that is
more volatile than the necessary original component and/or the
analyte.
[0023] In another embodiment, a method is provided as above wherein
the aforementioned reaction is a decomposition reaction.
[0024] In a further embodiment, a method is provided as above
wherein the reaction involves chemical, photolytic, or thermal
cleavage of a linkage in the substitute component that provides
lower molecular weight reaction products that do not cause any
significant ion suppression and/or are more volatile than the
original component.
[0025] In still a further embodiment, a system is provided for
determining the concentration of an analyte in a fluid sample that
comprises a mass spectrometer, an acoustic ejector to generate
droplets of fluid sample, and a means for volatilizing and ionizing
the droplets prior to introduction into the mass spectrometer, the
improvement which comprises replacing at least one necessary
component in the fluid sample with a substitute component that
serves the same function as the original component but results in
an increase in intensity of analyte signal and/or an increase in
signal-to-noise ratio relative to the intensity of the analyte
signal and/or signal-to-noise ratio, respectively, obtained using
the original component.
[0026] In another embodiment, the substitute component of the
system contains a linkage that can be chemically, thermally, or
photolytically cleaved to provide lower molecular weight reaction
products that do not cause any significant ion suppression and/or
are more volatile than the original component. When the substitute
component contains a photolytically cleavable linkage, the system
further includes a source of radiation effective to cleave the
linkage.
[0027] The method and system of the invention generally provide for
an increase in the intensity of analyte signal and/or a greater
signal-to-noise ratio that is at least 10% and preferably at least
25% relative to the intensity of the analyte signal and the
signal-to-noise ratio obtained without the substitute
component.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a plot of normalized signal at m/z=195 versus
ammonium acetate (.diamond-solid.) or sodium chloride (.box-solid.)
concentration (mM), as described in connection with the mass
spectrometric detection of caffeine described in Example 1.
[0029] FIG. 2 is a plot of signal intensity for detected caffeine
analyte versus salt concentration for six different buffer salts,
as described in Example 2.
[0030] FIG. 3 is a plot of luminescence versus time for the kinase
assay carried out in Example 3, conducted using a volatile
magnesium salt (magnesium acetate) and, for purposes of comparison,
a nonvolatile magnesium salt (magnesium chloride).
[0031] FIG. 4 schematically illustrates the synthesis of cleavable
buffer compound (30).
[0032] FIG. 5 schematically illustrates the synthesis of cleavable
buffer compound (31), using two alternate routes.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by one of ordinary
skill in the art to which the invention pertains. Specific
terminology of particular importance to the description of the
present invention is defined below.
[0034] In this specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, "an analyte"
refers not only to a single analyte but also to a combination of
two or more different analytes, "a substitute component" refers to
a single such component or to a plurality (e.g., a mixture) of
components, and the like.
[0035] The term "ionizable" as used herein refers to a species that
is capable of undergoing ionization. As such, an "ionizable"
species herein may be in electronically neutral form or in ionized
(or "ionic") form, as an individual ion or as a component of a
salt. The ionizable species may also be present in both
electrically neutral and ionized forms, such as will be the case in
a buffer system. As an example, acetic acid (CH.sub.3COOH) is an
ionizable compound that may be present in electronically neutral
form, with a protonated carboxyl group, or it may be present in
ionized form, with the proton removed to give the acetate ion
(CH.sub.3COO.sup.-), or it may be present as a combination of the
electronically neutral and ionized forms. As another example, a
zwitterion containing a carboxylic acid group and an amino group
may be in electrically neutral form or in ionized form in which the
carboxylic acid group is ionized to carboxylate and the amino group
is protonated to give a cationic nitrogen-containing
substituent.
[0036] The term "volatile" is used herein to refer to the relative
tendency of an ion, salt, or compound to leave the surface of a
fluid droplet and enter the vapor phase under the vaporization
conditions and using the vaporization methods discussed herein. The
term is used in a comparative sense herein, such that the
substitute species is "volatile" insofar as it is more likely than
either the original non-analyte component or the analyte to
volatilize under the volatilization conditions employed in
conjunction with described method.
[0037] The terms "contaminant" and "component" are used to refer to
species in the fluid sample that cause ion suppression. The term
"contaminant," however, refers to a species that is unintentionally
or accidentally introduced into the sample and may have been
present in a solvent, reagent, surfactant, or the like, while the
term "component" refers to a species that serves a necessary and
intended purpose, such as species that are required for biochemical
processing preceding the analysis and/or species that are necessary
to maintain a chemical or physical parameter of the fluid sample,
e.g., buffer systems to maintain pH.
[0038] The terms "acoustic radiation" and "acoustic energy" are
used interchangeably herein and refer to the emission and
propagation of energy in the form of sound waves. As with other
waveforms, acoustic radiation may be focused using a focusing
means, as discussed below.
[0039] The terms "focusing means" and "acoustic focusing means"
refer to a means for causing acoustic waves to converge at a focal
point, either by a device separate from the acoustic energy source
that acts like a lens, or by the spatial arrangement of acoustic
energy sources to effect convergence of acoustic energy at a focal
point by constructive and destructive interference. A focusing
means may be as simple as a solid member having a curved surface,
or it may include complex structures such as those found in Fresnel
lenses, which employ diffraction in order to direct acoustic
radiation. Suitable focusing means also include phased array
methods as are known in the art and described, for example, in U.S.
Pat. No. 5,798,779 to Nakayasu et al. and Amemiya et al. (1997)
Proceedings of the 1997 IS&T NIP 13 International Conference on
Digital Printing Technologies, pp. 698-702.
[0040] The terms "acoustic coupling" and "acoustically coupled"
used herein refer to a state wherein an object is placed in direct
or indirect contact with another object so as to allow acoustic
radiation to be transferred between the objects without substantial
loss of acoustic energy. When two items are indirectly acoustically
coupled, an "acoustic coupling medium" is needed to provide an
intermediary through which acoustic radiation may be transmitted.
Thus, an ejector may be acoustically coupled to a fluid, e.g., by
immersing the ejector in the fluid or by interposing an acoustic
coupling medium between the ejector and the fluid to transfer
acoustic radiation generated by the ejector through the acoustic
coupling medium and into the fluid.
[0041] The term "reservoir" as used herein refers to a receptacle
or chamber for holding or containing a fluid. Thus, a fluid in a
reservoir necessarily has a free surface, i.e., a surface that
allows a droplet to be ejected therefrom. In its one of its
simplest forms, a reservoir consists of a solid surface having
sufficient wetting properties to hold a fluid merely due to contact
between the fluid and the surface.
[0042] The term "fluid" as used herein refers to matter that is
nonsolid or at least partially gaseous and/or liquid. A fluid may
contain a solid that is minimally, partially or fully solvated,
dispersed or suspended. Examples of fluids include, without
limitation, aqueous liquids (including water per se and salt water)
and nonaqueous liquids such as organic solvents and the like.
[0043] The invention accordingly provides an improved method for
determining the concentration of an analyte in a fluid sample,
where the fluid sample contains, in addition to the analyte, a
necessary non-analyte component, for instance a buffer system
including a buffer salt for maintaining pH or a salt for
maintaining ionic strength, and wherein the method involves
volatilizing and ionizing the fluid sample and introducing the
ionized, volatilized sample into an ionic analyte detection device,
such as a mass spectrometer, that generates an analyte signal
proportional in intensity to the quantity of ionized analyte
detected. The improvement provided by the invention involves
employing a substitute component for the original non-analyte
component that will serve the same purpose as the original
component but is more volatile than the original component and/or
the analyte or undergoes a chemical reaction upon volatilization to
yield a reaction product that is more volatile than the original
component and/or the analyte. The substitute component results in a
stronger analyte signal and/or an increased signal-to-noise ratio
relative to the analyte signal and signal-to-noise ratio obtained
with the original non-analyte component. Preferred substitute
components, at least in part because of volatility considerations,
increase the signal-to-noise ratio by at least 20%, and
particularly preferred such components increase the signal-to-noise
ratio by 50% or more. The purpose of the necessary non-analyte
component may be, as noted, maintaining a pre-determined pH or a
required ionic strength.
[0044] The analyte in the fluid sample may be any analyte of
interest. Examples of analytes include, without limitation, drugs,
metabolites, inhibitors, ligands, receptors, catalysts, synthetic
polymers, and allosteric effectors. Often, the analyte is a
"biomolecule," i.e., any organic molecule, whether naturally
occurring, recombinantly produced, chemically synthesized in whole
or in part, or chemically or biologically modified, that is, was or
can be a part of a living organism. The term encompasses, for
example, nucleotidic analytes, peptidic analytes, and saccharidic
analytes.
[0045] Nucleotidic analytes may be nucleosides or nucleotides per
se, but may also comprise nucleosides and nucleotides containing
not only the conventional purine and pyrimidine bases, i.e.,
adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U),
but also protected forms thereof, e.g., wherein the base is
protected with a protecting group such as acetyl, difluoroacetyl,
trifluoroacetyl, isobutyryl or benzoyl, and purine and pyrimidine
analogs. Suitable analogs will be known to those skilled in the art
and are described in the pertinent texts and literature. Common
analogs include, but are not limited to, 1-methyladenine,
2-methyladenine, N.sup.6-methyladenine, N.sup.6-isopentyl-adenine,
2-methylthio-N.sup.6-isopentyladenine, N,N-dimethyladenine,
8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine,
5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,
2-methylguanine, 7-methylguanine, 2,2-dimethylguanine,
8-bromo-guanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine,
8-thioguanine, 5-fluoro-uracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil,
5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,
5-(methyl-aminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,
2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,
uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,
pseudouracil, 1-methylpseudouracil, queosine, inosine,
1-methylinosine, hypoxanthine, xanthine, 2-aminopurine,
6-hydroxyaminopurine, 6-thiopurine and 2,6-diaminopurine. In
addition, the terms "nucleoside" and "nucleotide" include those
moieties that contain not only conventional ribose and deoxyribose
sugars, but other sugars as well. Modified nucleosides or
nucleotides also include modifications on the sugar moiety, e.g.,
wherein one or more of the hydroxyl groups are replaced with
halogen atoms or aliphatic groups, or are functionalized as ethers,
amines, or the like.
[0046] Nucleotidic analytes also include oligonucleotides, wherein
the term "oligonucleotide," for purposes of the present invention,
is generic to polydeoxyribo-nucleotides (containing
2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to
any other type of polynucleotide which is an N-glycoside of a
purine or pyrimidine base, and to other polymers containing
nonnucleotidic backbones. Thus, an oligonucleotide analyte herein
may include oligonucleotide modifications, for example,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoramidates, carbamates, etc.), with
negatively charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), and with positively charged linkages
(e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters),
those containing pendant moieties, such as, for example, proteins
(including nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.). There is no
intended distinction in length between the terms "polynucleotide"
and "oligonucleotide," and these terms are used interchangeably.
These terms refer only to the primary structure of the molecule. As
used herein the symbols for nucleotides and polynucleotides are
according to the IUPAC-IUB Commission of Biochemical Nomenclature
recommendations (Biochemistry 9:4022, 1970).
[0047] "Peptidic" analytes are intended to include any structure
comprised of one or more amino acids, and thus include peptides,
dipeptides, oligopeptides, polypeptides, and proteins. The amino
acids forming all or a part of a peptidic analyte may be any of the
twenty conventional, naturally occurring amino acids, i.e., alanine
(A), cysteine (C), aspartic acid (D), glutamic acid (E),
phenylalanine (F), glycine (G), histidine (H), isoleucine (I),
lysine (K), leucine (L), methionine (M), asparagine (N), proline
(P), glutamine (Q), arginine (R), serine (S), threonine (T), valine
(V), tryptophan (W), and tyrosine (Y), as well as non-conventional
amino acids such as isomers and modifications of the conventional
amino acids, e.g., D-amino acids, non-protein amino acids,
post-translationally modified amino acids, enzymatically modified
amino acids, .beta.-amino acids, constructs or structures designed
to mimic amino acids (e.g., .alpha.,.alpha.-disubstituted amino
acids, N-alkyl amino acids, lactic acid, .beta.-alanine,
naphthylalanine, 3-pyridylalanine, 4-hydroxyproline,
0-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, and nor-leucine), and other
non-conventional amino acids, as described, for example, in U.S.
Pat. No. 5,679,782 to Rosenberg et al. Peptidic analytes may also
contain nonpeptidic backbone linkages, wherein the naturally
occurring amide --CONH-- linkage is replaced at one or more sites
within the peptide backbone with a non-conventional linkage such as
N-substituted amide, ester, thioamide, retropeptide (--NHCO--),
retrothioamide (--NHCS--), sulfonamido (--SO.sub.2NH--), and/or
peptoid (N-substituted glycine) linkages. Accordingly, peptide
analytes can include pseudopeptides and peptidomimetics. Peptide
analytes can be (a) naturally occurring, (b) produced by chemical
synthesis, (c) produced by recombinant DNA technology, (d) produced
by biochemical or enzymatic fragmentation of larger molecules, (e)
produced by methods resulting from a combination of methods (a)
through (d) listed above, or (f) produced by any other means for
producing peptides.
[0048] Saccharidic analytes include, without limitation,
monosaccharides, disaccharides, oligosaccharides, polysaccharides,
mucopolysaccharides or peptidoglycans (peptido-polysaccharides) and
the like.
[0049] An exemplary embodiment of the invention involves
replacement of a standard, relatively nonvolatile buffer salt
associated with ion suppression by a substitute buffer salt that is
more volatile than the standard buffer salt and provides for
reduced ion suppression as evidenced by an increased analyte signal
and/or an increased signal-to-noise ratio. It is established that
relatively nonvolatile salts in fluid samples are frequent
contaminants that can cause ion suppression and thus imprecise or
incorrect results.
[0050] In this embodiment, the necessary non-analyte component is
the salt component of a standard buffer system, and the substitute
component is a more volatile salt that serves the same purpose,
i.e., maintains the same pH, and that reduces or eliminates the ion
suppression seen with the standard buffer salt. In general, salts
of strong acids or bases are not sufficiently volatile for the
present purpose. For instance, sodium cations, potassium cations,
calcium cations, and tetrabutylammonium cations should be avoided,
as should anions such as sulfates and nitrates. A notable exception
is hydrochloric acid, which although a strong acid in water, is a
relatively weak acid in the gas phase. Further, ions bearing
multiple charges, such as sulfates, citrates and phosphates, are
not likely to be volatile. Finally, higher molecular weight salts
such as fatty acid salts are not likely to be volatile except at
very high temperatures.
[0051] Examples of relatively volatile buffer salts that may
replace the conventional buffer salts, or original non-analyte
component, include, without limitation, the following:
[0052] Ammonium bicarbonate
[0053] Ammonium formate
[0054] Ammonium acetate
[0055] Ammonium propionate
[0056] Ammonium butyrate
[0057] Pyridinium formate
[0058] Pyridinium acetate
[0059] Pyridinium propionate
[0060] Pyridinium butyrate
[0061] Ethylmorpholinium acetate
[0062] Dimethylamino formate
[0063] Dimethylamino acetate
[0064] Dimethylamino propionate
[0065] Dimethylamino butyrate
[0066] Methylethylamino formate
[0067] Methylethylamino acetate
[0068] Methylethylamino propionate
[0069] Methylethylamino butyrate
[0070] Diethylamino formate
[0071] Diethylamino acetate
[0072] Diethylamino propionate
[0073] Diethylamino butyrate
[0074] Within the aforementioned group, preferred volatile buffer
salts include the following:
[0075] Ammonium bicarbonate
[0076] Ammonium formate
[0077] Ammonium acetate
[0078] Pyridinium acetate
[0079] Pyridinium formate
[0080] Ethylmorpholinium acetate
[0081] Trimethylamino acetate
[0082] Trimethylamino formate
[0083] It is to be understood that the aforementioned salts are
merely representative, and that other salts may also be used,
providing that they serve the same purpose as the salt they are
replacing and that they meet the volatility and enhanced
signal-to-noise criteria set forth herein.
[0084] Candidate salts may be readily tested for volatility using
methods well known to those of ordinary skill in the art. Such
methods include, for example, a dry residue analysis, in which the
candidate salt or buffer composition is placed in a volatile
solvent and then heated to dryness. The presence of any dry residue
suggests that the salt is not sufficiently volatile for use in the
present purpose. Those candidate salts established as sufficiently
volatile are then tested for their capability to reduce ion
suppression, by conducting a comparison of the candidate salt with
the necessary non-analyte component the candidate salt is intended
to replace. Volatility may also be evaluated using butyl acetate
number, a measure of relative evaporation rates, as will be
appreciated by those in the field.
[0085] Generally, although not necessarily, the ionic detection
device is a mass spectrometer. It will be appreciated that various
volatilization techniques are available in connection with mass
spectrometry, including thermal methods and electrospray, and any
effective volatilization technique may be used in conjunction with
the present method. Any of a number of known ionization means may
also be used, including chemical ionization, field desorption
ionization, electrospray ionization, atmospheric pressure chemical
ionization, matrix-assisted laser desorption ionization, and
inductively coupled plasma ionization, and, again, any effective
ionization technique may be advantageously employed herein.
Depending on the nature of the analyte, mass spectrometric
measurements can be performed in negative or positive mode, with
acidic analytes preferentially ionizing in the negative mode and
basic analytes preferentially ionizing in the positive mode.
[0086] In a preferred embodiment, the improved method of the
invention employs acoustic ejection to produce very small droplets
that are then volatilized, ionized, and analyzed. These small
droplets are nanoliter-sized droplets, defined herein as a droplets
containing at most about 5 nl of fluid sample, preferably not more
than about 2.5 nl, more preferably less than 1 nl, most preferably
smaller than about 50 pl, and optimally less than about 1 pl.
Acoustic ejection of droplets from the surface of a fluid sample is
effected using an acoustic ejector as will be described in detail
below. Acoustic ejection technology is particularly suited to
high-throughput mass spectrometry (HTMS), insofar as HTMS has been
hampered by the lack of easily automated sample preparation and
loading, the need to conserve sample, the need to eliminate cross
contamination, the inability to go directly from a fluid reservoir
into the analytical device, and the inability to generate droplets
of the appropriate size.
[0087] The present method has proved to be unexpectedly effective
with nanoliter-sized droplets, as seen in the Examples herein. It
is well understood that fundamental fluid physics changes as the
size scale is decreased, i.e., as fluid droplets become smaller and
smaller. Otherwise applicable principles of diffusion and mixing
tend not to apply to nanoliter-sized droplets, nor are conventional
analyses workable when attempting to predict the flow dynamics of
an element, ion, or compound moving from the interior of a
nanoliter-sized droplet to the droplet surface, e.g., for purposes
of volatilization.
[0088] Acoustic ejection of droplets that are then volatilized
provides numerous advantages. In volatilizing droplets that are
nanoliter-sized, e.g., using a thermal volatilization technique,
the large surface area of these small droplets facilitates the gas
phase extraction of the relatively volatile substitute component,
leaving behind the charged analyte in the evaporating droplet.
Thus, by enabling gas phase extraction of the substitute component,
e.g., the substitute buffer salt or the like, acoustic ejection
eliminates the need for liquid phase or solid phase "clean up" of a
fluid sample to remove interfering components that cause ion
suppression. This significantly increases the number of samples
that can be analyzed using mass spectrometry or the like in a given
period of time. With current commercially available methodology,
the need for an additional step to remove buffer, other salts,
detergents, and any other non-analyte species results in a
processing time in the range of 7-20 seconds per sample, while the
present invention enables processing time of well under 1 second
per sample, typically on the order of about 0.3 seconds per sample.
Combining this feature with the fact that the present process can
be carried out using far smaller sample sizes and with the fact
that the analyte signal obtained is increased by virtue of the gas
phase extraction step means that the invention enables a far more
rapid, economical, and accurate method for analyzing fluid samples
using mass spectrometry or other ionic detection devices.
[0089] Acoustic ejection, as noted above, enables rapid sample
processing as well as generation of nanoliter-sized droplets of
predetermined and consistent size; see U.S. Pat. No. 6,416,164 to
Stearns et al., cited and incorporated by reference earlier herein.
The aforementioned patent describes how the size of acoustically
ejected droplets from a fluid surface can be carefully controlled
by varying the acoustic power, the acoustic frequency, the
toneburst duration, and/or the F-number of the focusing lens. An
additional advantage of using acoustic ejection in conjunction with
the present invention is that droplets can be ejected from a very
small sample size, on the order of 5 .mu.l or less. This is
particularly advantageous when sample availability is limited and a
small fluid sample must be analyzed out of necessity. In terms of
processing capability, U.S. Pat. No. 6,938,995 to Mutz et al.
explains that acoustic ejection technology, used in conjunction
with acoustic assessment of fluid samples in a plurality of
reservoirs, can achieve analysis of over 5, 10, or even 25
reservoirs per second, translating to well in excess of 50,000
fluid samples per day.
[0090] In one embodiment, then, the improved method of the
invention makes use of an acoustic ejector as a fluid sample
droplet generation device, the device including at least one
reservoir to contain the fluid sample, an acoustic ejector, and a
means for positioning the acoustic ejector in acoustic coupling
relationship with the reservoir. Typically, a single ejector is
used that is composed of an acoustic radiation generator and a
focusing means for focusing the acoustic radiation generated by the
acoustic radiation generator. However, a plurality of ejectors may
be advantageously used as well. Likewise, although a single
reservoir may be used, the device typically includes a plurality of
reservoirs.
[0091] Examples of acoustic ejection devices useful in conjunction
with the present invention are described in detail in U.S. Pat. No.
6,802,593 to Ellson et al., U.S. Pat. No. 7,270,986 to Mutz et al.,
U.S. Pat. No. 7,439,048 to Mutz et al., and U.S. Pat. No. 6,603,118
to Ellson et al., incorporated by reference herein. As described
therein, an acoustic ejection device may be constructed to include
multiple reservoirs as an integrated or permanently attached
component of the device. However, to provide modularity and
interchangeability of components, it is preferred that device be
constructed with removable reservoirs. Generally, the reservoirs
are arranged in a pattern or an array to provide each reservoir
with individual systematic addressability. In addition, while each
of the reservoirs may be provided as a discrete or stand-alone
item, in circumstances that require a large number of reservoirs,
it is preferred that the reservoirs be attached to each other or
represent integrated portions of a single reservoir unit. For
example, the reservoirs may represent individual wells in a well
plate. Many well plates suitable for use with the device are
commercially available and may contain, for example, 96, 384, 1536,
or 3456 wells per well plate, having a full skirt, half skirt, or
no skirt. Well plates or microtiter plates have become commonly
used laboratory items. The Society for Laboratory Automation and
Screening (SLAS) has established and maintains standards for
microtiter plates in conjunction with the American National
Standards Institute, including the footprint and dimension
standards ANSI/SLAS 1-2004. The wells of such well plates typically
form rectilinear arrays.
[0092] However, the availability of such commercially available
well plates does not preclude the manufacture and use of
custom-made well plates in other geometrical configurations
containing at least about 10,000 wells, or as many as 100,000 to
500,000 wells, or more. Furthermore, the material used in the
construction of reservoirs must be compatible with the fluid
samples contained therein. Thus, if it is intended that the
reservoirs or wells contain an organic solvent such as
acetonitrile, polymers that dissolve or swell in acetonitrile would
be unsuitable for use in forming the reservoirs or well plates.
Similarly, reservoirs or wells intended to contain DMSO must be
compatible with DMSO. For water-based fluids, a number of materials
are suitable for the construction of reservoirs and include, but
are not limited to, ceramics such as silicon oxide and aluminum
oxide, metals such as stainless steel and platinum, and polymers
such as polyester, polypropylene, cyclic olefin copolymers (e.g.,
those available commercially as Zeonex.RTM. from Nippon Zeon and
Topas.RTM. from Ticona), polystyrene, and polytetrafluoroethylene.
For fluids that are photosensitive, the reservoirs may be
constructed from an optically opaque material that has sufficient
acoustic transparency for substantially unimpaired functioning of
the device.
[0093] In addition, to reduce the amount of movement and time
needed to align the acoustic radiation generator with each
reservoir or reservoir well during operation, it is preferable that
the center of each reservoir be located not more than about 1
centimeter, more preferably not more than about 1.5 millimeters,
still more preferably not more than about 1 millimeter and
optimally not more than about 0.5 millimeter, from a neighboring
reservoir center. These dimensions tend to limit the size of the
reservoirs to a maximum volume. The reservoirs are constructed to
contain typically no more than about 1 mL, preferably no more than
about 1 .mu.L, and optimally no more than about 1 nL, of fluid. To
facilitate handling of multiple reservoirs, it is also preferred
that the reservoirs be substantially acoustically
indistinguishable.
[0094] A vibrational element or transducer is used to generate
acoustic radiation. In some instances, the acoustic radiation
generator is comprised of a single transducer. In addition, the
transducer may use a piezoelectric element to convert electrical
energy into mechanical energy associated with acoustic radiation.
Alternatively, multiple element acoustic radiation generators such
as transducer assemblies may be used. For example, linear acoustic
arrays, curvilinear acoustic arrays or phased acoustic arrays may
be advantageously used to generate acoustic radiation that is
transmitted simultaneous to a plurality of reservoirs.
[0095] An added element in the form of a gas phase extraction
device thermally volatilizes droplets ejected using an acoustic
radiation generator as just described, and extracts unwanted
species from the droplets prior to analysis using, e.g., a mass
spectrometer. Replacement of conventionally used components with
more volatile substitutes (e.g., buffer salts) or substitutes that
undergo a chemical reaction to provide volatile species,
facilitates ready gas phase extraction of these components, thus
eliminating or at least substantially reducing ion suppression or
other types of interference seen with the conventional
compounds.
[0096] As noted earlier herein, the method of the invention employs
a substitute component for the original non-analyte component that
serves the same purpose as the original component but is either (1)
more volatile than the original component and/or the analyte or (2)
undergoes a chemical reaction upon volatilization to yield a
reaction product that is more volatile than the original component
and/or the analyte. Embodiment (1) is discussed above. Now turning
to embodiment (2), the substitute component is in this case not
necessarily more volatile than the original component and/or the
analyte, but is rather selected to undergo a chemical reaction that
yields at least one volatile reaction product. The substitute
component may be a zwitterionic compound that undergoes an
intramolecular conversion, as will be discussed in detail below, or
the substitute component may be a nonzwitterionic compound that can
be chemically cleaved to yield a volatile reaction product.
Alternatively, the substitute component may be a compound that
undergoes a thermally induced reaction to give rise to at least one
volatile reaction product. The compound may also be selected to
undergo a photocatalytic reaction that results in at least one
volatile reaction product, where, it is to be understood, the term
"volatile reaction product" refers to a reaction product that is
more volatile than the original component and/or the analyte.
[0097] Zwitterionic Compound as the Substitute Component:
[0098] When a zwitterionic compound is used as the substitute
component, e.g., as a buffer salt in a fluid sample requiring a
buffer system, the zwitterionic compound is selected so as to
undergo a chemical reaction at elevated temperature and/or under
reduced pressure during the volatilization process, to provide at
least one reaction product that is more volatile than the original
non-analyte component and/or the analyte. The zwitterionic
compounds described infra are useful in other methods as well,
e.g., in any method that involves ionization and volatilization of
a buffered fluid sample followed by detection of the analyte in the
ionized and volatilized sample.
[0099] By "zwitterion" or a "zwitterionic" compound as those terms
are used herein is meant a compound that contains a pair of
ionizable groups, one that ionizes to form a positively charged
species and the other that ionizes to form a negatively charged
species. The former is typically a nitrogen atom-containing group
such as a substituted or unsubstituted amine or a diazo
substituent, and the latter is generally although not necessarily a
carboxyl (COOH) substituent. It will be appreciated that at higher
pH values only the nitrogen atom-containing group will bear a
charge, such that the compound is cationic, while at lower pH
values only the carboxyl-containing group will be ionized, such
that the compound is anionic. At intermediate pH values, generally
in the range of about 5 to about 8, both entities bear a charge and
it is in this form that the reactions described below proceed in an
optimal manner.
[0100] In one embodiment, the zwitterionic compound is a partially
unsaturated compound, i.e., a compound that contains at least one
unsaturated bond, such as a double bond, and is "pre-aromatic" in
the sense that the intramolecular decomposition results in the
generation of a volatile aromatic compound. For instance, the
partially unsaturated, pre-aromatic zwitterionic compound may
comprise a partially unsaturated, pre-aromatic core to which a
carboxylic acid group (--COOH) and a nitrogen-containing
substituent are covalently bound, wherein the nitrogen-containing
substituent may be selected from amino, primary amino, secondary
amino, tertiary amino, and diazo. In this example, the carboxylic
acid group and the nitrogen-containing substituent are positioned
with respect to each other so that the intramolecular decomposition
yields a volatile aromatic compound (i.e., an aromatic compound
that is more volatile than the original component and/or the
analyte), releases carbon dioxide, and generates a
nitrogen-containing compound such as ammonia, a substituted or
unsubstituted amine (generally gaseous), or nitrogen gas. The
pre-aromatic core is generally cyclic, although an acyclic core is
suitable provided that the aforementioned intramolecular
decomposition reaction results in a volatile compound, preferably a
volatile aromatic compound, in addition to release of carbon
dioxide and a nitrogen-containing compound. This pre-aromatic
zwitterionic compound containing pre-aromatic core Q and the
nitrogen-containing substituent N* may be represented by structure
(1)
##STR00001##
When the core Q is cyclic, transformation from the pre-aromatic
compound can occur according to the following scheme (I) when the
carboxylate group and the N* substituent are bound to adjacent
carbon atoms in a ring:
##STR00002##
In Scheme (I), Ar represents the volatile aromatic reaction
product, and each N** reaction product corresponds to each N*
substituent. This reaction may be illustrated using a more specific
example in which, solely for purposes of illustration, the core Q
comprises a cyclohexa-1,3-diene ring, such that, as shown in Scheme
(II), the volatile aromatic reaction product is benzene:
##STR00003##
[0101] The N* substituent, in cationic form, may be, for instance,
--(NH.sub.3).sup.+--(NHR.sup.1).sup.+, (NR.sup.2R.sup.3).sup.+,
(NR.sup.4R.sup.5R.sup.6).sup.+, or diazo, such that the N** will
be, respectively, ammonia (NH.sub.3), NH.sub.2R.sup.1,
NHR.sup.2R.sup.3, NR.sup.4R.sup.5R.sup.6, or nitrogen gas
(N.sub.2). R.sup.1 through R.sup.6 are non-hydrogen substituents
such as lower alkyl, i.e., a C.sub.1-C.sub.6 alkyl group,
preferably a C.sub.1-C.sub.3 alkyl group, that may or may not be
substituted with substituents that will not interfere in the
detection process or interact with any of the components of the
sample in a deleterious manner. The decomposition reaction thus
results in two products that will not be detected, i.e., the
volatile aromatic compound and carbon dioxide, and to the
nitrogenous species N** which, is unlikely to cause ion
suppression.
[0102] In Scheme (II), the cyclohexa-1,3-diene reactant may or may
not be substituted with one or more ring substituents that do not
interfere with the chemical reaction shown or with any other
compounds that are present, and are generally selected from the
same group of substituents as R.sup.1 through R.sup.6, and the
carboxylate and cationic nitrogen-containing substituent that are
shown as ortho to each other may be in either cis or trans
relationship (compound (2))
##STR00004##
but are preferably in the trans configuration (compound (3)):
##STR00005##
The optionally present substituents are illustrated in structure
(4)
##STR00006##
in which, as shown, there are i substituents where i is in the
range of zero to 6 inclusive, wherein the substituents, represented
as R, may be the same or different. In one particular case, wherein
N* is diazo, it is preferred that the carboxylate and diazo
substituent are in the trans position with a substituent attached
to the same carbon as the diazo group, where that substituent,
shown as R.sup.7 in compound (5), is C.sub.1-C.sub.6 alkyl,
preferably C.sub.1-C.sub.3 alkyl (omission of R.sup.7 results in a
compound that is too unstable for the present purpose):
##STR00007##
[0103] In a related embodiment, the zwitterionic compound is an
aromatic compound substituted with a carboxylate group and a diazo
group, such as ortho-diazo benzoic acid, in which case the
intramolecular reaction results in the N** reaction product benzyne
(Scheme (III):
##STR00008##
Benzyne is a very reactive compound that will serve as an
intermediate to further reaction. In this case, a second reactant,
e.g., water, or a diene, is introduced into the sample to react
with benzyne. Water will result in reaction to give phenol, while
proper selection of the diene, as will be understood by a skilled
practitioner, will yield a volatile reaction product. As one
example, furan may serve as the diene, in which case reaction with
benzyne gives the volatile product
1,2,3,4-tetrahydro-1,4-epoxynaphthalene. As another example,
anthracene may serve as the diene, resulting in the volatile
reaction product triptycene.
[0104] The zwitterionic compound may be cyclic or acyclic, and, if
cyclic, it may be monocyclic, bicyclic, or polycyclic, and may
contain aromatic rings as well as a molecular segment that is only
partially unsaturated. When the chemical reaction of the
zwitterionic compound results in a volatile aromatic compound as a
reaction product, this is generally, although not necessarily, by
addition of a double bond to a ring structure to generate 4n+2
aromaticity. Examples include addition of a double bond to a
substituted or unsubstituted cyclohexyl-1,3-dienyl ring to generate
a benzene ring, or addition of a double bond to a substituted or
unsubstituted dihydrofuran ring to generate furan, an aromatic.
Thus, the zwitterionic compound may, in one instance, comprise a
cyclohexyl-1,3-dienyl core that is substituted with the --COOH and
--N* moieties at adjacent carbon atoms, such that the compound
comprises 5-carboxyl-6-N*-cyclohexa-1,3-diene, e.g.,
5-carboxyl-6-amino-cyclohexa-1,3-diene, which may be further
substituted as indicated above, and which converts to a substituted
or unsubstituted benzene ring following reaction. A further
zwitterionic compound may comprise a cyclohexa-1,4-diene core
substituted with --COOH and --N* in the para configuration, such
that the compound is or contains
3-carboxyl-6-N*-cyclohexa-1,4-diene, either unsubstituted or
substituted as above, which converts via the decomposition reaction
to a substituted or unsubstituted benzene ring. Another
zwitterionic compound may comprise, for instance, dihydrofuran
substituted at the 2-position and 3-position with the --COOH and
--N* moieties, such that the compound comprises
2-carboxyl-3-N*-2,3-dihydrofuran or
2-N*-3-carboxyl-2,3-dihydrofuran, either unsubstituted or
unsubstituted as described earlier herein, which, upon reaction,
converts to substituted or unsubstituted furan, an aromatic
molecule.
[0105] Specific examples of these zwitterionic compounds include,
without limitation, the following (for simplicity, the compounds
are shown in uncharged form; it will be understood, however, that
at intermediate pH values each compound will contain both an
anionic species and a cationic species, i.e., a carboxylate group
--COO and a positively charged nitrogen atom):
6-Amino-3,4-dimethylcyclohexa-2,4-diene-1-carboxylic Acid
##STR00009##
[0106] 1,6-Diamino-2,4-diene-1-carboxylic Acid
##STR00010##
[0107] 6-Amino-1,4-dimethylcyclohexa-2,4-diene-1-carboxylic
Acid
##STR00011##
[0108] 6-Aminocyclohexa-2,4-diene-1-carboxylic Acid
##STR00012##
[0109] 6-(Dimethylamino)cyclohexa-2,4-diene-1-carboxylic Acid
##STR00013##
[0110] 6-(Ethylamino)cyclohexa-2,4-diene-1-carboxylic Acid
##STR00014##
[0111] 3-Amino-2,3-dihydrofuran-2-carboxylic Acid
##STR00015##
[0112] 4-Aminocyclohexa-2,5-dienecarboxylic Acid
##STR00016##
[0113] 10-Amino-9,10-dihydroanthracene-9-carboxylic Acid
##STR00017##
[0114] 4-Amino-1,4-dihydronaphthalene-1-carboxylic Acid
##STR00018##
[0115] 2-Amino-1,2-dihydronaphthalene-1-carboxylic Acid
##STR00019##
[0116] 2-Amino-1,2-dihydronaphthalene-1-carboxylic Acid
##STR00020##
[0117] 4-(Ethylamino)-1,4-dihydronaphthalene-1-carboxylic Acid
##STR00021##
[0119] These zwitterionic compounds may be obtained commercially or
synthesized using methods known in the art and described in the
pertinent literature. Carboxylate-containing zwitterions can be
synthesized using any of a variety of techniques for combining a
carboxyl-containing compound with an amine or other nitrogenous
compound. Zwitterionic sulfonic acid-containing compounds such as
zwitterionic detergents and buffers can generally be synthesized
from substituted or unsubstituted 1,2-oxathiolane-2,2-dioxide and a
substituted or unsubstituted amine, according to Scheme (IV):
##STR00022##
[0120] Other Chemically Cleavable Substitute Components:
[0121] Alternatively, a chemically cleavable nonzwitterionic
compound can be selected to serve as the substitute component,
wherein the chemically cleavable compound contains at least one
linkage that can be cleaved with an acid, base, or other reagent.
Representative linkages hydrolytically cleavable in the presence of
acids or bases include the following: carboxylate ester
(--(CO)--O--); enol ether (--CH.dbd.CH--O--); acetal
(--O--CR.sub.2--O--); hemiacetal (--CH(OH)--O--); anhydride
(--(CO)--O--(CO)--); carbonate (--O--(CO)--O--); amide
((--(CO)--NH--); N-substituted amide (--(CO)--NR--); urethane
(--O--(CO)--NH--); N-substituted urethane (--O--(CO)--NR--); imido
(--CH.dbd.N--); N,N-disubstituted hydrazo (--NR--NR--); thioester
(--(CO)--S--); phosphonic ester (--P(O)(OR)--O--); sulfonic ester
(--SO.sub.2--OR--); ortho ester (--C(OR).sub.2--O--); and betaine
ester (R--O--(CO)--N(W).sub.4.sup.+X.sup.- where the R' may be the
same or different non-hydrogen substituents and X.sup.- is the
associated counterion). Other chemically cleavable linkages
include, without limitation: the hydroxylamine-cleavable linkage
--(CO)--O--CH.sub.2--CH.sub.2--O--(CO)--; the thiol-cleavable
linkage --S--S, also cleavable upon treatment with trisubstituted
phosphines such as triphenylphoephine; periodate cleavable
cis-diols --CH(OH)--CH)OH--; and the fluoride-cleavable linkage
--(O)--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--(N-
H)--(CO)--. Surfactants containing chemically cleavable linkages
have been described, along with synthesis thereof. these include
ProteaseMAX (V2071) (Promega); RapiGest SF (Waters); PPS Silent
Surfactant (Agilent); MaSDeS (see Chang et al. (January 2015) J.
Proteome Res. 14(3)); Invitrosol (Life Technologies, Inc.);
Progenta AALS I (sodium 2,2-dihexoxypropyl sulfate, from Protea
Biosciences); Progenta AALS II (sodium 2,2-diheptoxypropyl sulfate,
also from Protea Biosciences); Progenta CALS I (2,2-dihexoxypropyl
ammonium bromide, also from Protea Biosciences); and Progenta CALS
II (2,2-diheptoxypropyl ammonium bromide, also from Protea
Biosciences). These surfactants and analogs thereof can be used
advantageously in the present compositions, as can buffers and
other compounds containing these and other chemically cleavable
linkages.
[0122] Thermally Cleavable Substitute Components:
[0123] Substitute components can also contain linkages that are
cleavable with heat, such that they serve the same purpose as the
original component in the fluid sample but cleave into smaller
compounds upon volatilization of the sample, where those smaller
compounds are either volatile or unlikely to cause ion suppression.
Thermally cleavable linkages include ester linkages, carbamate
linkages, carbonate linkages, urethane-type linkages
(--O--(CO)--NH--) and N-substituted urethane linkages
(--O--(CO)--NR--) in which the nitrogen atom of the linkage is
substituted with a non-hydrogen substituent such as lower alkyl.
Other thermally cleavable linkages include furan-maleimide
Diels-Alder adducts (see Szalal et al. (2007) Macromolecules 40(4):
818-823), oxirane and thiirane-based linkages, and
ester-substituted sulfones that thermally decompose to an ester and
gaseous SO.sub.2 according to the following scheme
##STR00023##
wherein R is a nonhydrogen substituent, generally an alkyl group
(e.g., piperylene, as described by Eckert and Liotta, "Designing
Smart Surfactants" printed from
www.chbe.gatech.edu/eckert/pdf/-surfactant.pdf, Internet site
accessed on Oct. 19, 2015).
[0124] Photolytically Cleavable Substitute Components:
[0125] Incorporation of one or more photolytically cleavable sites
into the substitute component allows for irradiation-induced
cleavage prior to introduction of the sample into the mass
spectrometer or other analytical device. The sample fluid or fluids
can be irradiated in the gas phase, i.e., after fluid droplet
ejection but prior to entry into the mass spectrometer, or they may
be irradiated in the liquid phase, e.g., in a well plate or other
container or group of containers. Irradiation in the gas phase
enables real-time conversion to the cleavage products, while
irradiation in the liquid phase, where sample fluid is present in a
multiplicity of containers or wells, does not.
[0126] Photolytically cleavable sites can be readily incorporated
into the substitute component, e.g., the buffer, surfactant, or the
like, using synthetic organic techniques known to those of ordinary
skill in the art and/or described in the pertinent texts and
literature. One type of photolytically cleavable linkage is
composed of an ortho-nitrobenzyl group as in the following
representative structure
##STR00024##
where R* is generally a nitrogen atom or oxygen atom bound to the
rest of the molecule. Such structures thus include
##STR00025##
and
##STR00026##
as well as the N-substituted analog
##STR00027##
in which R, again, is a non-hydrogen substituent such as lower
alkyl, and the symbol represents attachment to the remainder of the
molecule. Another photolytically cleavable linkage includes, by way
of example, the cinnamic acid-type linkage
##STR00028##
described, for example, by Sakai et al. (15 Jun. 2012) J. Colloid
and Interface Sci. 376(1):160-164, with respect to the
photocleavable surfactant
##STR00029##
Other photolytically cleavable linkages include benzyl ethers
(which cleave to form alcohols), carbamate linkages (which cleave
to form amines), 1,3-dithiane linkages (which cleave to form
carbonyl groups); ortho-nitroanilide linkages (which cleave to form
carboxyl groups), benzoin-type linkages (which cleave to form
phosphate groups), and the like. See, e.g., Pelliccioli et al.
(2002) Photochem. Photobiol. Sci. 1:441-458, and Greene et al.,
Protective Groups in Organic Synthesis, 3.sup.rd edition, John
Wiley & Sons (New York, N.Y.: 1999).
[0127] Those of ordinary skill in the art will be able to use known
methods of organic synthesis and/or methods described in the
literature to synthesize suitable zwitterionic and/or cleavable
compounds that can be used as the substitute component herein.
Additionally, known buffers (e.g., the Good's buffers; see Good et
al. (1966) Biochemistry 5(2):467-477), surfactants, and the like
may be modified to incorporate such cleavable linkers.
[0128] One representative buffer of interest is the
acetal-containing compound (25)
##STR00030##
which decomposes photolytically and/or in the presence of acid as
follows to give 2-aminoethanol and 2-formylbenzoic acid. Another
buffer of interest is compound (26)
##STR00031##
which can be photolytically cleaved to give methamine
(CH.sub.3--NH.sub.2) and 4-formyl-3-nitrosobenzoic acid.
[0129] Compounds having the general structure
##STR00032##
are suitable substitute components herein, particularly as an
acid-cleavable buffer. In (27), L.sup.1 and L.sup.2 are
C.sub.1-C.sub.6 hydrocarbyl linkages, generally C.sub.2-C.sub.4
hydrocarbyl, R.sup.8, R.sup.9, R.sup.10, and R.sup.11 are
independently selected from H and C.sub.1-C.sub.16 hydrocarbyl
(e.g., alkyl, cycloalkyl, alkenyl, etc., particularly lower alkyl),
R.sup.12 is lower alkyl, and R.sup.13 is either --COOH or
--CH.sub.2OSO.sub.3H. When R.sup.13 is --COOH, the compound may be
represented as (28), while when R.sup.13 is --CH.sub.2OSO.sub.3H,
the compound may be represented as (29):
##STR00033##
##STR00034##
In a preferred embodiment, R.sup.8, R.sup.9, R.sup.10, and R.sup.11
are H, L.sup.1 and L.sup.2 are --CH.sub.2CH.sub.2--, and R.sup.12
is methyl, such that generic compounds (28) and (29) have the
structures (30) and (31), respectively:
##STR00035##
Both compounds can be synthesized from methyl pyruvate. Compound
(30) has a pI of approximately 9.44 and is optimally employed as a
buffer at a pH in the range of approximately 7.5 to 10.5. Compound
(31) has a pI of approximately 9.44 and is optimally employed as a
buffer at a pH in the range of approximately 8.0 to 11.0. FIG. 4
schematically illustrates the synthesis of compound (30), while
FIG. 5 schematically illustrates the synthesis of compound (31).
Implementation of the individual reaction steps shown therein will
be within the purview of those skilled in the art and/or will
become apparent upon reference to an analogous reaction in the
texts or literature.
[0130] It is to be understood that while the invention has been
described in conjunction with a number of specific embodiments, the
foregoing description as well as the examples that follow are
intended to illustrate and not limit the scope of the invention.
Other aspects, advantages and modifications will be apparent to
those skilled in the art. All patents, patent applications, and
publications mentioned here are hereby incorporated by reference in
their entireties.
Example 1
[0131] In this example, the impact of buffer volatility on the mass
spectrometric determination of caffeine was evaluated using
ammonium acetate as a volatile buffer salt and sodium chloride as a
nonvolatile buffer salt. Fluid samples were prepared with varying
concentrations of either ammonium acetate or sodium chloride, at 1
mM, 2 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, and 500 mM.
Sample droplets were generated using a modified Echo.RTM. 555
liquid handler (Labcyte Inc., Sunnyvale, Calif.). The instrument
was modified to move the transducer assembly to the exterior of the
instrument underneath a nozzle to allow the droplet stream to be
exposed to 195.degree. C. heat, thus enabling gas phase extraction
of volatile salt, and pulled into a beta version of the SQ Detector
II mass spectrometer (Waters Micromass).
[0132] FIG. 1 is a plot of normalized signal at m/z=195 versus
ammonium acetate (.diamond-solid.) or sodium chloride (.box-solid.)
concentration (mM). As may be seen, the concentration of caffeine
detected decreases rapidly at even low sodium chloride
concentrations, a phenomenon not seen with ammonium acetate. This
result indicates that the more volatile buffer salt allows for
analyte detection, even at higher buffer salt concentrations, while
the less volatile buffer salt does not.
Experimental for Examples 2 and 3
[0133] All samples were analysed on a Waters SQ Detector 2 mass
spectrometer fitted with a standard electrospray source running a
source block temperature of 80.degree. C., a desolvation
temperature of 250.degree. C. and a gas flow rate of 400 liters/hr.
The electrospray probe voltage was set to 3500 V and the sample
cone to 25 V. All samples were introduced using a Harvard 22
syringe pump fitted with a Hamilton 250 .mu.l syringe operating at
6 .mu.l/min. All reagents were purchased from Sigma Aldrich as dry
powders except HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid, a zwitterionic buffer), which was supplied as a 1M solution.
1M stock solutions were prepared for each of the salts using HPLC
grade water and a caffeine stock solution of 2 mg/ml, again in HPLC
grade water. Serial dilutions were performed on each of the salt
stock solutions to generate 1 mM, 5 mM, 25 mM and 50 mM working
solutions. To 1 ml of each of these working solutions 50 pl of the
caffeine stock solution was added to produce a 10 .mu.g/ml caffeine
solution for analysis. For each sample a syringe infusion was
performed and the absolute ion signal heights for the M+H and M+Na
peaks of caffeine (at 195 Da and 217 Da) were summed and plotted.
Between each sample, the syringe and probe were flushed with water
to remove any salt residues.
Example 2
[0134] This example describes an additional evaluation of the
impact of buffer volatility on the mass spectrometric determination
of caffeine, with magnesium acetate used as a volatile buffer salt
and magnesium chloride as a nonvolatile buffer salt. The signal
intensity of caffeine and its sodium adduct was evaluated in four
concentrations of six salt systems. The data, plotted in FIG. 2,
shows that standard ESI system sensitivity is heavily impacted by
non-volatile salts and less impacted by a volatile salt, at similar
concentrations. That is, the uppermost data points in the plot
correspond to the signal obtained with fluid samples containing
caffeine and ammonium acetate as a buffer salt. The experiment may
be repeated to carry out mass spectrometric determinations of other
analytes with substantially the same results.
Example 3
[0135] In this example, a kinase assay was conducted in which
magnesium is required by the enzyme used, and the assay measures
the increase in concentration of a phosphorylated peptide substrate
with time. One set of samples was formulated with magnesium
chloride, a relatively nonvolatile compound, and a second set of
samples was formulated with magnesium acetate, a more volatile
compound. The assay results using each type of magnesium salt are
illustrated in FIG. 3, in which time, in minutes, is represented on
the X-axis, and luminescence is represented on the Y-axis. The
graph comparing results obtained for the assay employing magnesium
chloride (.diamond-solid.) with those obtained for the assay
employing magnesium acetate (.box-solid.) show that the assay was
not hindered or biased by the source of magnesium ions.
[0136] In many assay systems, certain non-volatile and/or highly
electronegative buffer components are used; however, these
components are not unique creating the biological or chemical
outcome of the desired assay. The switch does not show any
significant change in results for the range of up to 60 as this is
within the noise of the measurement. From the previous examples for
measurement of analyte with the absence of the non-volatile
analyte, this assay system would provide improved results with gas
phase extraction prior to MS loading due to reduce ion suppression
in the analyte.
* * * * *
References