U.S. patent application number 11/811281 was filed with the patent office on 2008-01-24 for devices and methods for separating phospholipids from biological samples.
Invention is credited to Patrick Kevin Bennett, Kenneth Charles Van Horne.
Application Number | 20080020485 11/811281 |
Document ID | / |
Family ID | 34226192 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080020485 |
Kind Code |
A1 |
Bennett; Patrick Kevin ; et
al. |
January 24, 2008 |
Devices and methods for separating phospholipids from biological
samples
Abstract
Device and methods for the removal of phospholipids from
biological samples are disclosed and described. Removal of
phospholipids may be desirable for the analysis of the
phospholipids themselves, or to prevent the phospholipids from
conflicting with and effectively masking other analytes in the
sample for which identification or quantification is sought.
Inventors: |
Bennett; Patrick Kevin;
(Salt Lake City, UT) ; Van Horne; Kenneth Charles;
(Denver, CO) |
Correspondence
Address: |
THORPE NORTH & WESTERN, LLP.
8180 SOUTH 700 EAST, SUITE 350
SANDY
UT
84070
US
|
Family ID: |
34226192 |
Appl. No.: |
11/811281 |
Filed: |
June 8, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10655740 |
Sep 4, 2003 |
7256049 |
|
|
11811281 |
Jun 8, 2007 |
|
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|
Current U.S.
Class: |
436/177 ;
422/255; 436/17 |
Current CPC
Class: |
G01N 1/34 20130101; Y10T
436/107497 20150115; Y10T 436/255 20150115; Y10T 436/25375
20150115 |
Class at
Publication: |
436/177 ;
422/255; 436/017 |
International
Class: |
G01N 1/28 20060101
G01N001/28; B01J 19/00 20060101 B01J019/00; G01N 37/00 20060101
G01N037/00 |
Claims
1. A device for removing phospholipids from a biological sample,
comprising: a) a support; and b) at least one type of
phospholipotropic multivalent cation coupled to the support in a
concentration that is sufficient to capture and retain
phospholipids from the biological sample.
2. The device of claim 1, wherein the phospholipotropic multivalent
cation is a transition metal.
3. The device of claim 1, wherein the phospholipotropic multivalent
cation is a lanthanide.
4. The device of claim 1, wherein the phospholipotropic multivalent
cation is an actinide.
5. The device of claim 3, wherein the lanthanide is cerium.
6. The device of claim 1, wherein the phospholipotropic multivalent
cation is coupled to the support with an attachment selected from
the group consisting of an ionic bond, chelation, and combinations
thereof.
7. The device of claim 6, wherein the ionic bond utilizes an acid
active group.
8. The device of claim 7, wherein the acid active group is selected
from the group consisting of sulfonic acid, phosphoric acid,
carboxylic acid, acidic silanols acidic zirconia and combinations
thereof.
9. The device of claim 1, wherein the support is an inorganic salt
matrix.
10. The device of claim 1, wherein the support is a sorbent.
11. The device of claim 1, wherein the support includes a member
selected from the group consisting of alumina, silica, polymers,
carbon, zirconium, controlled-pore glass, diatomaceous earth, and
combinations thereof.
12. The device of claim 11, wherein the support includes a
functional group.
13. The device of claim 1, wherein the phospholipotropic
multivalent cation retains the phospholipid until the cation is
contacted with a solution that is sufficient to release the
phospholipid from the cation.
14. The device of claim 1, wherein the phospholipotropic
multivalent cation is coupled to the support until the cation is
contacted with an agent that is sufficient to release the cation
from the support.
15. A method of removing phospholipids from a biological sample,
comprising: a) contacting at least one type of phospholipotropic
multivalent cation with the biological sample; b) capturing
phospholipids in the sample with the cation; and c) separating the
cation and captured phospholipids from the sample.
16. The method of claim 15, further comprising the step of
separating the captured phospholipids from the cation.
17. The method of claim 16, further comprising the step of
collecting the phospholipids.
18. The method of claim 15, wherein the capturing includes
ionically associating the phospholipids with the phospholipotropic
multivalent cation.
19. The method of claim 15, wherein the phospholipotropic
multivalent cation is a transition metal.
20. The method of claim 15, wherein the phospholipotropic
multivalent cation is a lanthanide.
21. The method of claim 15, wherein the phospholipotropic
multivalent cation is an actinide.
22. The method of claim 20, wherein the lanthanide is cerium.
23. The method of claim 15, wherein the phospholipotropic
multivalent cation is coupled to a support.
24. The method of claim 23, wherein the support is an inorganic
salt matrix.
25. The method of claim 23, wherein the support includes a member
selected from the group consisting of alumina, silica, polymers,
carbon, zirconium, controlled-pore glass, diatomaceous earth, and
combinations thereof.
26. The method of claim 23, further comprising the step of
separating the phospholipotropic multivalent cation from the
support.
27. A method of making a device for removing phospholipids from a
biological sample, comprising: coupling at least one type of
phospholipotropic multivalent cation with a support in a manner
that preserves an affinity of the cation for phospholipids.
28. The method of claim 27, wherein the coupling is a mechanism
selected from the group consisting of ionic bonding, covalent
bonding, chelation, and combinations thereof.
29. The method of claim 27, wherein the cation is coupled to the
support using an acid active group.
30. The method of claim 27, wherein the acid active group is
selected from the group consisting of sulfonic acid, phosphoric
acid, carboxylic acid, acidic silanol, acidic zirconia, and
combinations thereof.
31. The method of claim 27, wherein the phospholipotropic
multivalent cation is a transition metal.
32. The method of claim 27, wherein the phospholipotropic
multivalent cation is a lanthanide.
33. The method of claim 27, wherein the phospholipotropic
multivalent cation is an actinide.
34. The method of claim 32, wherein the lanthanide is cerium.
35. The method of claim 27, wherein the support is an inorganic
salt matrix.
36. The method of claim 27, wherein the support includes a member
selected from the group consisting of alumina, silica, polymers,
carbon, zirconium, controlled-pore glass, diatomaceous earth, and
combinations thereof.
37. The method of claim 36, wherein the support includes a
functional group.
Description
PRIORITY CLAIM
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/655,740, filed Sep. 4, 2003, which is
hereby incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to devices and methods for
separating phospholipids from biological samples. Accordingly, the
present invention relates to the fields of chemistry, biology,
medicine, and pharmaceutics.
BACKGROUND
[0003] Biological sampling has long been known as a mechanism for
obtaining information concerning the physical condition of a
subject. A wide variety of sampling techniques and analyses are
known, the selection of which may depend on a number of factors,
including the specific sample medium, the information sought, and
the type of instrumentation to be used in performing the
analysis.
[0004] As a general matter, most analytical techniques are
customized to identify the presence and/or amount of a target
substance or constituent in the biological sample. However, in some
cases, the presence of certain substances within a sample may
interfere with the ability of the analytical technique to obtain
and provide such information. In cases where a conflict between
sample constituents is identified, attempts are often made to
design a sample preparation protocol that removes the problematic
substance from the sample to the extent that complete and reliable
identification or quantification of the target substance can be
achieved.
[0005] For example, phospholipids have recently been identified as
a potential cause of signal suppression or enhancement, during mass
spectroscopic analysis of biological samples for certain target
substances. Such suppression or enhancement impairs the accuracy
and precision of the resulting data. The chemical configuration of
phospholipids includes a hydrophilic polar head group and a
hydrophobic tail, which can allow the phospholipid to interact with
many different sample constituents. The ability of phospholipids to
interact with a variety of substances may be an aspect of their
potential to disrupt the assay. Thus, removing the phospholipids
from the biological sample prior to mass spectrometric analysis,
may allow greater accuracy and reliability of the analytical result
obtained.
[0006] In some instances, however, removal of phospholipids from a
biological sample may be desirable because the phospholipids are
themselves the target constituent of interest. In such cases,
isolation of the phospholipids from the sample may reduce or
eliminate conflicts with certain other constituents and thus
improve the reliability of the analysis for the phospholipids.
[0007] While removal or separation of phospholipids from a
biological sample may be desirable for the above-recited reasons,
it is to be noted that such removal should be selective in order to
prevent target substances from also being removed along with the
phospholipids. Further, when phospholipids are themselves the
target substance, the non-selective removal thereof may in some
aspects diminish the value of subsequent analytical results
obtained.
[0008] As a result, methods and devices for separating
phospholipids from biological samples, especially for selective
separation of phospholipids from biological samples have been
sought through research and development efforts.
SUMMARY OF THE INVENTION
[0009] Accordingly, the present invention provides methods and
devices for removing phospholipids from a biological sample. In one
aspect, such a device may include a support, and at least one type
of phospholipotropic multivalent cation coupled to the support in a
concentration that is sufficient to capture and retain
phospholipids from the biological sample.
[0010] A variety of phospholipotropic multivalent cations may be
used. However, in one aspect, the cation may be a transition metal.
In another aspect, the cation may be a lanthanide, for example
cerium. In yet another aspect, the cation may be an actinide.
[0011] A number of suitable attachment mechanisms may be used to
couple the multivalent cation to the support. Examples of such
mechanisms include without limitation, ionic bonding, and
chelation. However, in one aspect, the attachment may be via an
ionic bonding or association. Those of ordinary skill in the art
will recognize a wide range of possible chemical groups, or
moieties, that may be used to effect the ionic association between
the support and the multivalent cation. However, in one detailed
aspect of the present invention, the ionic bond may utilize an acid
active group. In a more detailed aspect, the acid active group may
be selected from the group consisting essentially of sulfonic acid,
phosphoric acid, carboxylic acid, acidic silanols, and combinations
thereof.
[0012] The particular configuration and materials selected for the
support may be determined in part by the manner in which the
particular device is to be used. For example, in some aspects, the
support may be configured as a flat membrane type structure. In
other aspects, it may be configured as a matrix suitable to fit
into a column, or other structure. Moreover, the materials for the
support may vary. For example, in one aspect, the support may
include a sorbent material. In another aspect, the support may
include an inorganic salt matrix. Examples of suitable sorbent
materials are known in the art and include without limitation,
alumina, silica, polymers, carbon, zirconium, controlled-pore
glass, diatomaceous earth, and combinations thereof. Additionally,
the support may include one or more types of functional groups.
[0013] The devices of the present invention may take a number of
shapes and sizes, and may optionally include various supporting
materials, such as external housings and fittings that allow the
device to interface with, and/or become attached to, other standard
laboratory equipment, such as a syringe body, or in-line column
fittings.
[0014] The present invention additionally encompasses methods for
the fabrication of the devices recited herein. In one aspect, such
a method may include the step of coupling at least one type of
phospholipotropic multivalent cation with a support in a manner
that preserves an affinity of the cation for phospholipids.
[0015] Methods of removing phospholipids from biological samples
using the devices disclosed herein are also encompassed by the
present invention. In one aspect, such a method may include the
steps of: a) contacting at least one type of phospholipotropic
multivalent cation with the biological sample; b) capturing
phospholipids in the sample with the cation; and c) separating the
cation and captured phospholipids from the sample. Optionally, such
methods may further include the steps of either separating the
captured phospholipids from the cation, or separating the cations
holding the phospholipids from a support to which the cations are
attached, and collecting the phospholipids.
[0016] As discussed more fully below, the capturing of the
phospholipids according to the methods of the present invention
primarily involves ionically associating the phospholipids with the
phospholipotropic multivalent cations as described herein.
Normally, the separation of the cation and captured phospholipids
from the sample will occur by effectively removing the sample from
the proximity of the cations, or visa versa. For example, as is
well known in the art of chromatography, a sample can be placed in
a fluid stream and passed through the cations, which are held
stationary, for example by coupling to a support as described
herein. In this case, as the fluid stream pushes the sample
through, or past the cations, the phospholipids in the sample
become captured by the cations while the sample moves on in the
fluid stream. Other alternative mechanisms for separating the
cations having the captured phospholipids from the biological
sample will be recognized by those of ordinary skill in the
art.
[0017] Desirably, the multivalent cation used will have a higher
affinity for phospholipids than for other sample constituents. In
some aspects of the invention such selectivity may be important in
maximizing the analytical results that can be obtained when
attempting to identify and measure analytes with which the
phospholipids compete or conflict during a particular type of
analysis.
[0018] There has thus been outlined, rather broadly, the more
important features of the invention so that the detailed
description thereof that follows may be better understood, and so
that the present contribution to the art may be better appreciated.
Other features of the present invention will become clearer from
the following detailed description of the invention, taken with the
claims, or may be learned by the practice of the invention.
DETAILED DESCRIPTION
[0019] Reference will now be made to the exemplary embodiments that
are described as follows, and specific language will be used herein
to describe the same. It will nevertheless be understood that no
limitation on the scope of the invention is thereby intended.
Alterations and further modifications of the inventive features
described herein, and additional applications of the principles of
the inventions as described herein, which would occur to one
skilled in the relevant art and having possession of this
disclosure, are to be considered within the scope of the
invention.
Definitions
[0020] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set forth below.
[0021] The singular forms "a," "an," and, "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a sample" includes reference to one or more
of such samples, and reference to "the target agent" includes
reference to one or more of such target agents.
[0022] As used herein, "biological sample" and "sample" may be used
interchangeably, and refer to a specimen collected from an organism
such as an animal, plant, bacteria, protozoa, fungus, virus, etc.
Also, the sample can also be prepared from excretions and wastes
produced by an organism, such as feces, urine, sweat, sap,
residues, etc. Those of ordinary skill in the art will recognize a
variety of ways for preparing such samples for analysis, such as by
dilution, or extraction in a liquid, among others.
[0023] As used herein, "analyte," "target analyte," "target agent,"
"target substance," and "constituent" may be used interchangeably,
and refer to an agent or substance whose presence, or quantity in a
biological sample is sought. Those of ordinary skill in the art are
familiar with such terms and their general meaning and use.
[0024] As used herein, "support" refers to a material that is
capable of having phospholipotropic cations coupled thereto. A
support may utilize a number of materials, or have a variety of
physical configurations to provide such a capacity. Moreover,
specific materials and configurations may be selected and combined
in order to provide a support having a desired function and
character. Examples of suitable configurations include without
limitation, substrates, matrices, membranes, semi-permeable
membranes, lattices, backings, bases, beds, molecular sieves,
powders, granulates, fibers, etc. Also, the support structure can
be rigid, malleable, hard, or soft. Additionally the support can be
pre-formed in a shape or free-flowing within a confined space. A
support can be in the form of solids, semi-solids, porous solids,
gels, hydrogels, fluids, creams, pastes, powders, particulates,
salt matrices, microspheres, nanospheres, nanoparticles, emulsions,
micelles, reverse micelles, colloids, bilayers, liposomes,
microemulsions, films, etc. The support can be heterogeneous and/or
homogeneous. Examples of materials that can be used include without
limitation, ceramics, hydrophilic substances, hydrophobic
substances, composites, crystalline structures, polymers, random
polymers, block co-polymers, multimer polymers, linear polymers,
branched polymers, segmented polymers, sorbents, inorganic salts,
aluminas, silicas, carbons, ziconiums, controlled-pore glass, and
diatomaceous earths, and various combinations and compounds
thereof. Furthermore, the support micro-structure can be fixed,
static, dynamic, mobile, fluid, and/or diffuse. The support can be
made of many sections and/or portions that aggregate, flocculate,
associate, and/or separate, which can also be segmented into
distinct portions with or without boundaries.
[0025] As used herein, "phospholipotropic" refers to an agent,
substance, or element that has an affinity for phospholipids. In
some aspects, the phospholipotropic agent may display a selectivity
for phospholipids over other analytes to which the agent may also
be attracted. The affinity of the phospholipotropic agent for the
phospholipid may be due to a variety of chemical or molecular
forces, such as van der Waals, London dispersion, ion-ion,
ion-dipole, dipole-dipole, and hydrogen bonding. Further, the
phospholipotropic substance and the phospholipid can associate by
ionic bonding or chelation.
[0026] As used herein, "lanthanide" refers to an element of the
periodic table, which can include any of the elements with atomic
numbers from 57 to 70. Examples of lanthanides include without
limitation lanthanum, cerium, praseodymium, neodymium, etc. In a
preferred aspect, the lanthanide may be cerium. Moreover, it is to
be understood that as used herein, "lanthanide" also includes
compounds and complexes containing a lanthanide element, such as
oxides, and other metal salts.
[0027] As used herein, "actinide" refers to an element of the
periodic table, which can include any of the elements with atomic
numbers from 89 to 103. Examples of actinides include without
limitation actinium, thorium, protactinium, etc. Moreover, it is to
be understood that as used herein, "actinide" also includes
compounds and complexes containing an actinide element, such as
oxides and other metal salts.
[0028] As used herein, "multivalent" refers to an atom or molecule
having a valence greater than one.
[0029] As used herein, "matrix" refers to a medium or surrounding
substance within which something else originates, develops, or is
contained, captured, or retained. Also, a matrix can be rigid,
malleable, hard, or soft. Additionally, a matrix can be pre-formed
in a fixed shape or configuration, or can be free-flowing within a
space.
[0030] As used herein, "sorbent" refers to composition and/or
material that can take up and hold another substance, as by
absorption or adsorption. Also, a sorbent can be rigid, malleable,
hard, or soft. Additionally a sorbent can be pre-formed in a fixed
shape or free-flowing within a space. A variety of sorbent
materials are known to those of ordinary skill in the art, which
can be used as a support material in the devices of the present
invention.
[0031] As used herein, "functional group" refers to an aspect of a
molecule or a combination of atoms in a molecule that gives the
molecule a characteristic chemical behavior. For example, organic
chemistry functional groups are typically submolecular structural
motifs, characterized by a specific elemental composition and
connectivity, which may confer reactivity upon the molecule that
contains them. In one aspect, a functional group can be an active
acid group. Also, a functional group may be substituted onto a
carbon in place of hydrogen on an organic molecule. Common
functional groups useful in the present invention include without
limitation, halides, alcohols, ethers, aldehydes, ketones, esters,
acids, including carboxylic acids, sulfuric acids, sulfonic acids,
and phosphoric acids, amines, amides, alkanes, alkenes, alkynes,
alkyl halides, aromatic hydrocarbons, nitrites, sulfides,
phosphates, azos, phosphodiesters, phenyls, pyridyls, isonitriles,
isocyanates, isothiocyanates, thioethers, etc.
[0032] Concentrations, amounts, solubilities, and other numerical
data may be presented herein in a range format. It is to be
understood that such range format is used merely for convenience
and brevity and should be interpreted flexibly to include not only
the numerical values explicitly recited as the limits of the range,
but also to include all the individual numerical values or
sub-ranges encompassed within that range as if each numerical value
and sub-range is explicitly recited. For example, a range of 0.01
to 6 should be interpreted to include not only the explicitly
recited limits of 0.01 and 6, but also to include individual
numbers such as 0.3, 0.6, 2.0, 2.3, 3.7, 5.4, and sub-ranges such
as 0.2-2.3, 1.3-3.9, 2.9-5.1, etc. This interpretation should apply
regardless of the breadth of the range or the characteristic being
described, and should apply to ranges having both upper and lower
numerical values, as well as open-ended ranges reciting only one
numerical value.
The Invention
[0033] The present invention encompasses methods and devices for
removing phospholipids from biological samples. Removal of
phospholipids from a biological sample has been discovered to be
advantageous for a number of reasons as alluded to above. For
example, in many cases, phospholipids may conflict with analytes
sought for identification or quantification in the biological
sample, or otherwise influence the analytical process. When this
happens, the phospholipids may effectively mask the desired
analytes and prevent accurate identification or quantification
thereof. Additionally, in some cases, the phospholipids may be
themselves desired for analysis. Accordingly, removal of the
phospholipids from the biological sample is desirable to improve
testing accuracy by reducing the possibility of interference from
other conflicting analytes.
[0034] As a general matter, the methods for removing phospholipids
from a biological sample in accordance with the present invention
include the steps of: a) contacting at least one type of
phospholipotropic multivalent cation with the biological sample; b)
capturing phospholipids in the sample with the cation; and c)
separating the cation and captured phospholipids from the sample. A
number of devices may be used to accomplish such a method. However,
such devices will generally include a support, and at least one
type of phospholipotropic multivalent cation coupled to the support
in a concentration that is sufficient to capture and retain
phospholipids from the biological sample.
[0035] Those of ordinary skill in the art will recognize a variety
of ways in which the phospholipotropic multivalent cation may be
brought into contact with the biological sample, and then separated
therefrom. Generally speaking, contact may be made by passing the
sample past the cations, or visa versa. For example, in one aspect,
the sample may be placed in a fluid stream which flows past
phospholipotropic multivalent cations that are held stationary on a
support. As the sample flows past the cations, the phospholipids
become captured by the cations and remain in place while the rest
of the sample is driven onward by the flow of the fluid stream.
[0036] In another aspect, the sample may be placed in a device that
is capable of expelling the sample with an amount of force, such as
a syringe. The cations are then coupled to a support that is placed
at an opening of the device, and when the sample is expelled from
the device it passes past the cations. The phospholipids become
captured by the cations, and the remaining portion of the sample
moves forward by the expulsion action of the device.
[0037] In yet another aspect, the cations may be placed in a
solution containing the sample. The sample may be agitated, or
otherwise manipulated so that the cations come into contact with,
and capture, the phospholipids. The cations with the attached
phospholipids are then removed from the solution, leaving the
remainder of the sample by itself. Those of ordinary skill in the
art will recognize a variety of mechanisms for removing the cations
from the solution.
[0038] Once the captured phospholipids and cations have been
separated from the biological sample, the phospholipids may be
separated from the cations and collected. A number of techniques
may be used for separating the phospholipids from the cations, such
as by contacting the phospholipids with a solution that is
sufficient to release the phospholipids from the cations. For
example, the phospholipids may be contacted with substances that
are capable of ionically out-competing the multivalent cations, or
otherwise forming a stronger bond, or association with the
phospholipids than the multivalent cations. Those of ordinary skill
in the art will recognize a number of possible chemistries that may
either have a higher affinity for the phospholipid than the
multivalent cation, or reduce the affinity of the cation for the
phospholipid. Examples of such competing substances include without
limitation, chelating agents, and solvents, such as phosphoric
acid, sulfuric acid, hydrochloric acid, hydrofluoric acid, oxalic
acid, and other strong organic and inorganic, or mineral acids.
Additionally, solvents having strong bases can remove the
phospholipids from the cations, such as sodium hydroxide, potassium
hydroxide, and ammonium hydroxide. Examples of chelating agents
include without limitation, EDTA, IMDA, and NTA, among other
chelating organics.
[0039] Alternatively, in some aspects of the invention, the
phospholipids may remain captured by the multivalent cations, and
the cations may be uncoupled from a support to which they are
attached. In this manner, it may be possible to further utilize the
multivalent nature of the cation, for example, by attaching other
chemical entities thereto which may help in quantitative or
qualitative analysis, such as fluorescent, phosphorescent, and
radioactive elements. A number of techniques may be used to remove
cations from a support to which they are coupled. Such techniques
will depend in large measure on the type of mechanism holding the
cation to the support, the materials of the support, etc. Those of
ordinary skill in the art will readily be able to determine various
ways of disconnecting a given cation from a given support. For
example, if a strong acid group, such as a sulfonic acid, is used
to couple the cation to the support, a solution of sulfuric or
another strong inorganic or organic acid may be brought into
contact with the cation, thus releasing it from the support.
[0040] The mechanism for capturing the phospholipids in the
biological sample will generally occur by an ionic association, or
bond that is formed between the phospholipids and the multivalent
cation. In one preferred aspect, the interaction may be an ionic
one between a highly oxophilic cation and the organo-phosphate
group on the phospholipids. However, in some aspects, additional
chemical or molecular forces, such as van der Waals, London
dispersion, ion-ion, ion-dipole, dipole-dipole, chelation, and
hydrogen bonding forces may provide an affinity for the
phospholipids, and aid in the capture thereof from the biological
sample. As will be appreciated by one of ordinary skill in the art,
the exact number and type of forces will be determined according to
the specific multivalent cation, or cation compounds that are used,
along with any additional phospholipotropic moieties present.
[0041] As noted above, the devices of the present invention that
may be used to carry out the methods recited herein will generally
include a support, and at least one type of phospholipotropic
multivalent cation coupled to the support in a concentration that
is sufficient to capture and retain phospholipids from the
biological sample. The specific support configuration and material
may be selected by one of ordinary skill in the art to provide a
device with specific characteristics or operational parameters.
[0042] For example, in some aspects, the support may be configured
as a flat membrane type structure. Such a flat configuration may be
desirable for the high surface area that it presents, and its
convenience in allowing a sample to pass along or through it.
Further, because of the structural integrity of such a membrane, it
may be used in an open or semi-open environment.
[0043] In other aspects, the support may be configured as a matrix
suitable to fit into a column, or other structure. Such
configurations may be especially desirable for use with an
apparatus that employs a continuous fluid stream into which the
sample is placed. In one aspect, such a matrix may be a single
connected structure. In another aspect, such a matrix may be
modular or segmented. In yet another aspect, such a matrix may be
particulate. For example, a powdered form of the below recited
materials may be packed into a column body, cylinder, or other
housing that is suitable for holding the particles of powder
together while a sample is passed therethrough. In another aspect,
the support may be configured in a bulk format suitable for direct
addition to the biological sample. Those of ordinary skill in the
art will readily recognize a wide variety of other possible
configurations that may be suitable for the support of the present
invention.
[0044] The support used in the devices of the present invention may
be made from a variety of materials. Those of ordinary skill in the
art will be familiar with many materials that are known for use in
somewhat similar devices, such as chromatography columns and
filters. One broad class of such materials is sorbents. Examples of
specific sorbents include without limitation, alumina, silica,
polymers, carbon, zirconium, controlled-pore glass, diatomaceous
earth, and combinations thereof. Examples of other classes of
materials that may be used include without limitation, fibers made
of glass, cellulose, organic polymers, quartz, silica, metals or
other materials without limitation. In addition, it is to be noted
that in some aspects, the support may be made from an inorganic
salt matrix. In such a case, the salt component, or accompanying
element of a phospholipotropic multivalent cation compound or
complex may act as the support for the cation. For example in the
compound cerium oxalate, the oxalate component may act as the
support for the cerium which is the multivalent cation.
[0045] The support used in the present invention may additionally
include one or more types of functional groups, or one or more
instances of a single functional group. Such functional groups may
be used for the purposes of coupling the multivalent cations to the
support, or for a variety of other reasons, such as providing
specific patterns of spacing, etc. so as to provide the support
with a specific anatomy. Examples of such functional groups may
include without limitation, ionic or ionizable groups, chelating
groups, hydrogen donating or accepting groups, pi-bonding groups,
etc. Optionally, the functional groups may include an active acid
group. Examples of useful acid groups include without limitation,
sulfonic, phosphoric, phosphonic, carboxylic and other organic
acids, dicarboxylic acids, acidic silanols and acidic zirconia
groups. In one aspect, the acid group may be a sulfonic acid. In
another aspect, the acid may be a phosphoric acid. In yet another
aspect, the acid may be an acidic silanols.
[0046] The phospholipotropic multivalent cation used in the present
invention may be coupled to the support using a variety of chemical
mechanisms that suitably retain the cations on the support.
Examples of such mechanisms include without limitation, molecular
forces such as van der Waals, London dispersion, ion-ion, and
ion-dipole interactions. Additional mechanisms include ionic
bonding, covalent bonding, and chelation. Further, the cation can
be directly coupled to the support, or indirectly through
functional groups, linking groups, and/or spacer groups. Such
functional groups, linkers and/or spacers are well known in the art
to one of ordinary skill and can include active acid groups as
recited above, various cross-linked and linear polymers, short and
long chain saturated or unsaturated aliphatics, etc. Accordingly,
the linker and/or spacer can be of nearly any length required in
order to allow the cation to retain its affinity for phospholipids.
In one aspect, the cation may be coupled to the support with an
ionic bond. In another aspect, the ionic bond may utilize an acid
active group, such as those used in connection with the functional
groups recited above.
[0047] For the purposes of allowing the cations to become detached
from the support after the phospholipids have been captured, as
recited above, the functional group, including linkers and spacers
can be configured to decouple or otherwise lose their integrity
upon application of the appropriate chemistry. Such decoupling can
be achieved by contacting the linker and/or spacer with a solvent
containing a substance that may induce the linker and/or spacer to
break-down, and allow the cation to disassociate from the support.
For example, in one aspect, the cation can be ionically coupled to
a linker that is hydrogen bonded with the support. In another
aspect, the cation can be chelated to a spacer that is covalently
bound to the support. In yet another aspect, the cation can be
ionically coupled to a spacer that is covalently bound to the
support. In an additional aspect, the cation can be ionically
coupled to a spacer that is ionically coupled with the support.
[0048] A wide variety of substances, many of which will be
recognized by those of ordinary skill in the art may be
phospholipotropic. However, in one aspect, the phospholipotropic
agent may be a multivalent cation. Transition metals may be one
source of acceptable cations. For example, copper, iron, nickel,
and cobalt all may be used. Other subclasses of transition metals
that are particularly suitable include actinides and lanthanides.
Examples of suitable actinides include without limitation,
actinium, thorium, protactinium, uranium, neptunium, plutonium,
americium, curium, berkelium, californium, einsteinium, fermium,
mendelevium, nobelium, and lawrencium. Additionally, examples of
suitable lanthanides include without limitation, cerium,
praseodymium, neodymium, promethium, samarium, europium,
gadolinium, terbium, dysprosium, holmium, erbium, thulium,
ytterbium, and lutetium. However, in a preferred embodiment of the
present invention, the multivalent cation is cerium.
[0049] It is to be noted that not only may such multivalent cations
be used, but also complexes thereof for the purpose of capturing
phospholipids from a biological sample. To this end, various salts,
such as lanthanide chlorides, fluorides, oxides, oxalates,
citrates, acetates, etc., and complexes such as organo-lanthanides
may be used. In one preferred aspect of the invention, the
multivalent cation-containing salt may be cerium oxalate.
[0050] In one aspect of the present invention, the multivalent
cation may have a higher affinity for phospholipids than for other
constituents of the biological sample. Such selectivity may be
important when analytes of interest exhibit different chemical or
physical properties than certain phospholipids. Examples of such
analytes include without limitation compounds lacking highly
oxygenated functional groups, for example many basic pharmaceutical
compounds. Without wishing to be bound by theory, it is speculated
that the selectivity shown by certain multivalent cations for
phospholipids may be due at least in part to the strongly oxophilic
character of the multivalent cations and their selective affinity
for the phosphate groups on the phospholipids. Further, as
mentioned above, the specific configuration of the support to which
the cation is bound may additionally enhance or induce, such
selectivity.
[0051] As previously noted, in some aspects, the devices of the
present invention may include a containment structure, such as an
external housing within which the support and phospholipotropic
multivalent cation are contained. Such a housing is especially
useful when the support is particulate, granular, or free-flowing
in nature. However, those of ordinary skill in the art will
recognize that such supporting structures may be used to improve
the functionality and ease of handling the support and cations
regardless of the particular form or configuration they utilize.
Specific materials and configurations for the housing or other
supporting structure which can be suitably used with the present
invention by allowing entry and exit of a biological samples will
be readily recognized by those of ordinary skill in the art, and
may be selected and employed as required by a number of variables,
including the type of samples being analyzed, the mode or mechanism
of analysis, the particular shape, form, or physical state of the
support and multivalent cations, etc.
[0052] The present invention additionally encompasses methods of
making a device for removing phospholipids from a biological
sample. In some aspects, such a method may include the step of
coupling at least one type of phospholipotropic multivalent cation
as recited herein, with a support as recited herein, in a manner
that preserves an affinity of the cation for phospholipids. Those
of ordinary skill in the art will recognize that a variety of
coupling mechanisms as recited herein may be suitable to allow the
cation to retain its affinity for phospholipids, and that such
mechanisms may be selected depending on a variety of factors,
including the specific cation to be used. Furthermore, those of
ordinary skill in the art will be able to determine optimal
coupling mechanisms for a given cation through routine
experimentation.
[0053] The following example sets are illustrative of particular
embodiments and devices and methods in accordance with the present
invention. Such examples are provided solely to convey a greater
understanding of the present invention to those of ordinary skill
in the art, and no limitations thereon are intended, or to be
inferred thereby.
EXAMPLES
[0054] The following Examples 1-10 are illustrative of different
sorbent chemistries utilizing a phospholipotropic multivalent
cation for phospholipid capture, and their preparation.
Example 1
[0055] Cerium on a phenylsulfonic acid-modified silica--a
commercial 50 mg column containing a silica-based phenylsulfonic
acid sorbent ("Cerex", Baldwin Park, Calif.) was treated with 2
milliliters of methanol, 2 milliliters of water, 2 milliliters of
saturated cerium acetate solution in water, 2 milliliters of water,
and 2 milliliters of methanol.
Example 2
[0056] Cerium on a phenylsulfonic acid-modified polymer--a
commercial 50 mg column containing a polymer-based phenylsulfonic
acid sorbent ("Cerex", Baldwin Park, Calif.) was treated with 2
milliliters of methanol, 2 milliliters of water, 2 milliliters of
saturated cerium acetate solution in water, 2 milliliters of water,
and 2 milliliters of methanol.
Example 3
[0057] Cerium on a propylsulfonic acid-modified silica--a
commercial 50 mg column containing a silica-based propylsulfonic
acid sorbent ("Cerex", Baldwin Park, Calif.) was treated with 2
milliliters of methanol, 2 milliliters of water, 2 milliliters of
saturated cerium acetate solution in water, 2 milliliters of water,
and 2 milliliters of methanol.
Example 4
[0058] Cerium on a propylcarboxylic acid-modified silica--a
commercial 50 mg column containing a silica-based propylcarboxylic
acid sorbent ("Cerex", Baldwin Park, Calif.) was treated with 2
milliliters of methanol, 2 milliliters of water, 2 milliliters of
saturated cerium acetate solution in water, 2 milliliters of water,
and 2 milliliters of methanol.
Example 5
[0059] Cerium on a propylsulfonic acid-modified silica--a
commercial 50 mg column containing a silica-based propylsulfonic
acid sorbent ("Cerex", Baldwin Park, Calif.) was treated with 2
milliliters of methanol, 2 milliliters of water, 2 milliliters of
saturated cerium chloride solution in water, 2 milliliters of
water, and 2 milliliters of methanol.
Example 6
[0060] Cerium on an ethyl-modified silica (active silanols on the
sorbent are the suspected acidic groups binding the cerium
cation)--a commercial 50 mg column containing a silica-based ethyl
sorbent ("Cerex", Baldwin Park, Calif.) was treated with 2
milliliters of methanol, 2 milliliters of water, 2 milliliters of
saturated cerium acetate solution in water, 2 milliliters of water,
and 2 milliliters of methanol.
Example 7
[0061] Cerium on a triamine tetraacetate-modified silica--a
disposable syringe-barrel column was packed with 50 mg of a
triamine tetraacetate-modified silica (Silicycle, Quebec, QC,
Canada)". This packed column was treated with 2 milliliters of
methanol, 2 milliliters of water, 2 milliliters of saturated cerium
acetate solution in water, 2 milliliters of water, and 2
milliliters of methanol.
Example 8
[0062] Cerium (III) oxalate hydrate, 99.9% (Aldrich Chemical Co.,
Milwaukee, Wis.) was used without further treatment or
preparation.
Example 9
[0063] Lanthanum carbonate hydrate, 99.9% (Aldrich Chemical Co.
Milwaukee, Wis.) was used without further treatment or
preparation.
Example 10
[0064] Cerium (III) citrate was prepared as follows--3.726 grams of
cerium (III) chloride heptahydrate in 30 milliliters of water was
added to 2.941 grams of sodium citrate dihydrate in 30 milliliters
of water. A white precipitate formed and was centrifuged out at
3500 RPM for 5 minutes. The supernatant was poured off. The
precipitate was then washed 5 times (to remove dissolved sodium
chloride) with 30 milliliters of water by (for each wash) a)
addition of water to the precipitate, b) vortex mixing for 2
minutes, and c) centrifugation at 3500 RPM for 5 minutes. The
precipitate was then washed in a similar manner 3 times (to remove
residual water) with 30 milliliters of methanol (for each
wash).
[0065] The following Examples 11-15 illustrative of different
device formats utilizing the sorbent materials from Examples 1-10
above.
Example 11
[0066] A measured amount of the sorbent was added in bulk to the
respective sample, vortexed, and the sorbent removed by
filtration.
Example 12
[0067] A measured amount of the sorbent was added in bulk to the
respective sample, vortexed, and the sorbent removed by
centrifugation followed by pouring off the supernatant.
Example 13
[0068] A disposable extraction column containing the
phospholipotropic multivalent cation sorbent was prepared as
described in Examples 1-6 above, by starting with a pre-packed
commercial column, and modifying the pre-packed column sorbent via
treatment with a protocol designed to capture the phospholipotropic
multivalent cation onto the pre-packed sorbent.
Example 14
[0069] A bulk sorbent was prepared as described in Examples 8-10
above, then the bulk sorbent was packed into a disposable syringe
barrel column, between two porous frits designed to contain the
sorbent yet allow liquid flow through the column.
Example 15
[0070] A column was prepared as described in Example 14 above, but
an additional layer of sodium sulfate and an additional frit was
placed above the sorbent bed to facilitate water removal from an
applied sample.
[0071] The following Examples 16-20 are illustrative of different
sample treatment methods utilizing the device formats from Examples
11-15 above, and utilizing the sorbents from Examples 1-10
above.
Example 16
[0072] Columns containing sorbents treated with a phospholipotropic
multivalent cation, and sorbents without such treatment were
prepared as follows:
[0073] a. A disposable column (see Example 13 above) was used,
containing 50 mg of the sorbent from Example 4 above ("CeCBA").
[0074] b. A second column was used, containing the same pre-packed
sorbent as in step "a." above, but this sorbent was not treated
with cerium acetate (referred to as "CBA"). Prior to sample
application (step "c." below), this column and the column from step
"a." above were treated with 2 milliliters of methanol.
[0075] c. A standard Bligh-Dyer extraction of lipids from EDTA
human plasma was performed. The lipid fraction from this extraction
(containing naturally-occurring phospholipids and
lysophospholipids, well-known by those experienced in the art) was
dried and reconstituted in an equal volume of methanol (sample
"STD"). 200 microliters of this methanol sample were further
processed separately through the "CeCBA" column (sample "CeCBA"),
and a separate 200 microliters was processed through the "CBA"
column (sample "CBA"). 10 microliters of each of these samples was
analyzed by positive ion ESI-LC/MS. Two parent ions were monitored,
representing two of the major lysophospholipids present in human
plasma: 496.0 and 524.0. Values under the parent ions in Table I
are raw area counts for the respective chromatographic peaks.
TABLE-US-00001 TABLE I Sample 496.0 % 524.0 % STD 2.4E+04 100%
1.9E+04 100% CBA 3.2E+04 133% 2.0E+04 105% CeCBA 1.5E+03 6% 7.0E+02
4%
[0076] As can be seen from the results in Table 1, the sample
processed through the untreated "CBA" column shows essentially no
phospholipid capture as compared to the sample "STD", while the
sample processed through the cerium acetate-treated "CeCBA" column
shows >90% lysophospholipid capture.
[0077] The following example illustrates a comparison between
target analyte and phospholipid recoveries from spiked plasma
extracted by protein precipitation, as compared to similar samples
processed after protein precipitation through a phospholipotropic
multivalent cation-containing sorbent. The example also illustrates
extraction selectivity for the phospholipids as compared to the
target analytes.
Example 17
[0078] a. A stock solution was prepared to contain the following
target analytes in methanol: triprolidine, quinidine, ketoconazole
and reserpine. Levels of each analyte were adjusted so as to each
give a similar signal level by ESI-LC/MS/MS with a 20 microliter
injection in positive ionization mode using the following four
respective transitions: 279.2.fwdarw.208.0; 325.2.fwdarw.81.2;
531.2.fwdarw.82.0; 609.6.fwdarw.194.8.
[0079] b. The following five transitions were monitored by
ESI-LC/MS/MS as representative of extract phospholipid content:
496.4.fwdarw.184.0; 524.4.fwdarw.184.0; 704.5.fwdarw.184.0;
758.6.fwdarw.184.0; 806.5.fwdarw.184.0.
[0080] c. (Experiment performed in triplicate, and recoveries
averaged.) 200 microliters of stock solution (step "a." above) was
added to each of two tubes and brought to dryness. To each tube was
then added 400 microliters of plasma, and the tubes were vortexed
for two minutes. An additional 1.5 milliliters of acetonitrile was
then added to each tube, to precipitate proteins. The samples were
vortexed, centrifuged, decanted, and the supernatant brought to
dryness. To one tube was added 400 microliters of methanol
("PPTA"), and to the other was added 200 microliters of methanol
("PPTB 1"). The tubes were vortexed again.
[0081] d. Extract "PPTB1" was further passed through a column (see
Example 13 above) containing a 100 mg bed of a phospholipotropic
multivalent cation-containing sorbent (see Example 3 above) and
collected (Fraction 1). The column was washed with an additional
200 milliliters of 1:1::water:methanol, and the wash combined with
Fraction 1, to give sample "PPTB".
[0082] e. 10 microliter injections of samples "PPTA" and "PPTB"
were analyzed for recoveries of phospholipids and target analytes
via positive ion MRM ESI-LC/MS/MS, using the transitions listed
above in steps "a." and "b." Recoveries for components of sample
"PPTB" in Table II below are shown as percentages of "PPTA"
component recoveries, to illustrate the effect of the extraction
column. Values under the phospholipid parent ions and target
analyte names in Table II below are raw area counts for the
respective chromatographic peaks. TABLE-US-00002 TABLE II Sample
Tripro Quin Keto Reserp PPTA 1.12E+05 100.0% 1.45E+05 100.0%
9.12E+04 100.0% 1.19E+05 100.0% PPTB 9.89E+04 87.9% 9.02E+04 62.2%
7.94E+04 87.2% 8.58E+04 72.2% 496 524 704 758 806 PPTA 3.05E+06
100.0% 1.23E+06 100.0% 1.07E+05 100.0% 3.45E+06 100.0% 5.99E+05
100.0% PPTB 1.32E+04 0.4% 4.73E+02 0.0% 2.19E+03 2.0% 5.03E+04 1.5%
5.19E+03 0.9%
[0083] As illustrated in Table II above, the extraction column used
removed >97% of the monitored phospholipids as compared to
protein precipitation alone. Some removal of target analytes was
also exhibited; however, the amounts were much lower (<40%),
indicating selectivity of the extraction mechanism for
phospholipids.
[0084] The following example illustrates a comparison between
target analyte and phospholipid recoveries from spiked plasma
extracted by liquid/liquid extraction with methyl t-butyl ether
("MTBE extraction"), as compared to similar samples processed after
MTBE extraction through a phospholipotropic multivalent
cation-containing sorbent.
Example 18
[0085] a. A stock solution was prepared to contain the following
analytes in methanol: triprolidine, quinidine, ketoconazole and
reserpine. Levels of each analyte were adjusted so as to each give
a similar signal level by ESI-LC/MS/MS with a 20 microliter
injection in positive ionization mode using the following
respective transitions: 279.2.fwdarw.208.0; 325.2.fwdarw.81.2;
531.2.fwdarw.82.0; 609.6.fwdarw.194.8.
[0086] b. The following five transitions were monitored by
ESI-LC/MS/MS as representative of extract phospholipid content:
496.4.fwdarw.184.0; 524.4.fwdarw.184.0; 704.5.fwdarw.184.0;
758.6.fwdarw.184.0; 806.5.fwdarw.184.0.
[0087] c. (Experiment performed in triplicate, and recoveries
averaged.) 200 microliters of stock solution (step "a." above) was
added to each of two tubes and brought to dryness. To each tube was
then added 400 microliters of plasma, and the tubes were vortexed
for two minutes. Then 4 milliliters of MTBE was added, the samples
were vortex mixed, the MTBE removed via freeze-pour technique, and
the MTBE supernatant brought to dryness. To one tube was added 400
microliters of methanol ("LLA"), and to the other was added 200
microliters of methanol ("LLB1"). The tubes were vortexed
again.
[0088] d. Extract "LLB1" was further passed through a column (see
Example 13 above) containing a 100 mg bed of a phospholipotropic
multivalent cation-containing sorbent (see Example 3 above) and
collected (Fraction 1). The column was washed with an additional
200 milliliters of 1:1::water:methanol, and the wash combined with
Fraction 1, to give sample "LLB".
[0089] e. 10 microliter injections of samples "LLA" and "LLB" were
analyzed for recoveries of phospholipids and target analytes via
positive ion MRM ESI-LC/MS/MS, using the transitions listed above
in steps "a." and "b." Recoveries for components of sample "LLB" in
Table III below are shown as percentages of "LLA" component
recoveries, to illustrate the effect of the extraction column.
Values under the phospholipid parent ions and target analyte names
in Table III below are raw area counts for the respective
chromatographic peaks. TABLE-US-00003 TABLE III Sample Tripro Quin
Keto Reserp LL A 1.32E+05 100.0% 1.26E+05 100.0% 8.30E+04 100.0%
1.17E+05 100.0% LL B 4.86E+04 36.7% 2.13E+04 16.9% 7.69E+04 92.7%
3.35E+04 28.7% 496 524 704 758 806 LL A 2.97E+05 100.0% 1.55E+05
100.0% 1.47E+05 100.0% 2.32E+06 100.0% 2.75E+05 100.0% LL B
6.54E+02 0.2% 1.00E+02 0.1% 1.17E+03 0.8% 3.01E+04 1.3% 2.93E+03
1.1%
[0090] As illustrated in Table III above, the column used for
extraction removed >98% of the monitored phospholipids. Some
removal of target analytes was also exhibited; however, the amounts
were much lower than those for the phospholipids (.about.7-83%),
indicating selectivity of the extraction mechanism for
phospholipids.
[0091] The following example illustrates a comparison between
target analyte and phospholipid recoveries from spiked plasma
extracted by protein precipitation, as compared to similar samples
processed after protein precipitation through a phospholipotropic
multivalent cation-containing sorbent. The example also illustrates
extraction selectivity for the phospholipids as compared to the
target analytes. Further, the addition of formic acid to the column
wash improves recovery of target analytes without affecting removal
of phospholipids, thus enhancing selectivity further.
Example 19
[0092] a. A stock solution was prepared to contain the following
target analytes in methanol: triprolidine, quinidine, and
ketoconazole. Levels of each analyte were adjusted so as to each
give a similar signal level by ESI-LC/MS/MS with a 20 microliter
injection in positive ionization mode using the following four
respective transitions: 279.2.fwdarw.208.0; 325.2.fwdarw.81.2;
531.2.fwdarw.82.0.
[0093] b. The following five transitions were monitored by
ESI-LC/MS/MS as representative of extract phospholipid content:
496.4.fwdarw.184.0; 524.4.fwdarw.184.0; 704.5.fwdarw.184.0;
758.6.fwdarw.184.0; 806.5.fwdarw.184.0.
[0094] c. (Experiment performed in triplicate, and recoveries
averaged.) 200 microliters of stock solution (step "a." above) was
added to each of two tubes and brought to dryness. To each tube was
then added 400 microliters of plasma, and the tubes were vortexed
for two minutes. An additional 1.5 milliliters of acetonitrile was
then added to each tube, to precipitate proteins. The samples were
vortexed, centrifuged, decanted, and the supernatant brought to
dryness. To one tube was added 400 microliters of methanol
("PPTA"), and to the other was added 200 microliters of methanol
("PPTB1"). The tubes were vortexed again.
[0095] d. Extract "PPTB1" was further passed through a column (see
Example 13 above) containing a 100 mg bed of a phospholipotropic
multivalent cation-containing sorbent (see Example 3 above) and
collected (Fraction 1). The column was washed with an additional
200 milliliters of 1:1::water:methanol containing 1% formic acid,
and the wash combined with Fraction 1, to give sample "PPTB".
[0096] e. 10 microliter injections of samples "PPTA" and "PPTB"
were analyzed for recoveries of phospholipids and target analytes
via positive ion MRM ESI-LC/MS/MS, using the transitions listed
above in steps "a." and "b." Recoveries for components of sample
"PPTB" in Table II below are shown as percentages of "PPTA"
component recoveries, to illustrate the effect of the extraction
column. Values under the phospholipid parent ions and target
analyte names in Table IV below are raw area counts for the
respective chromatographic peaks. TABLE-US-00004 TABLE IV Sample
Tripro Quin Keto PPTA 8.49E+05 6.59E+05 6.25E+05 PPTB 7.15E+05
84.2% 5.66E+05 86.0% 5.77E+05 92.4% 496 524 704 758 806 PPTA
5.30E+06 4.54E+06 8.81E+05 2.31E+07 3.93E+06 PPTB 1.57E+04 0.3%
4.54E+03 0.1% 9.84E+03 1.1% 1.82E+05 0.8% 2.13E+04 0.5%
[0097] As illustrated in Table IV, the extraction column used
removed .about.99% of the monitored phospholipids as compared to
protein precipitation alone. Some removal of target analytes was
also exhibited; however, the amounts were much lower (<16%),
indicating improved selectivity of the extraction mechanism for
phospholipids.
[0098] The following example illustrates a comparison between
target analyte and phospholipid recoveries from spiked plasma
extracted by liquid/liquid extraction with methyl t-butyl ether
("MTBE extraction"), as compared to similar samples processed after
MTBE extraction by addition of a bulk phospholipotropic multivalent
cation-containing inorganic salt.
Example 20
[0099] a. A stock solution was prepared to contain the following
analytes in methanol: triprolidine, quinidine, PPT, ketoconazole
and reserpine. Levels of each analyte were adjusted so as to each
give a similar signal level by ESI-LC/MS/MS with a 20 microliter
injection in positive ionization mode using the following
respective transitions: 279.2.fwdarw.208.0; 325.2.fwdarw.81.2;
415.2.fwdarw.247.0; 531.2.fwdarw.82.0; 609.6.fwdarw.194.8.
[0100] b. The following five transitions were monitored by
ESI-LC/MS/MS as representative of extract phospholipid content:
496.4.fwdarw.184.0; 524.4.fwdarw.184.0; 704.5.fwdarw.184.0;
758.6.fwdarw.184.0; 806.5.fwdarw.184.0.
[0101] c. (Experiment performed in triplicate, and recoveries
averaged.) 200 microliters of stock solution (step "a." above) was
added to each of two tubes and brought to dryness. To each tube was
then added 400 microliters of plasma, and the tubes were vortexed
for two minutes. Then 2 milliliters of MTBE was added, the samples
were vortex mixed for 5 minutes, centrifuged to separate the
phases, and the MTBE removed via freeze-pour technique. To one of
these was added 100 milligrams of cerium oxalate (see Example 8
above) to give sample "CeOx", and to the other nothing additional
was added, to give sample "STD". The tubes were vortexed again for
2 minutes, spun down by centrifugation, the supernatants removed,
dried, and reconstituted in 800 microliters of acetone.
[0102] d. 10 microliter injections of samples "STD" and "CeOx" were
analyzed for recoveries of phospholipids and target analytes via
positive ion MRM ESI-LC/MS/MS, using the transitions listed above
in steps "a." and "b." Recoveries for components of sample "CeOx"
in Table V below are shown as percentages of "STD" component
recoveries, to illustrate the effect of the bulk cerium oxalate
addition. Values under the phospholipid parent ions and target
analyte names in Table V below are raw area counts for the
respective chromatographic peaks. TABLE-US-00005 TABLE V Sample
Triprp Quin PPT Keto Reserp STD 1.09E+05 100.0% 8.40E+04 100.0%
1.00E+00 100.0% 1.40E+05 100.0% 9.82E+04 100.0% CeOx 1.10E+05
100.6% 8.54E+04 101.7% 1.00E+00 100.0% 1.43E+05 102.5% 9.57E+04
97.5% 496 524 704 758 806 STD 1.26E+04 100.0% 6.26E+03 100.0%
1.01E+05 100.0% 2.32E+06 100.0% 3.00E+05 100.0% CeOx 6.44E+02 5.1%
2.37E+02 3.8% 2.98E+03 3.0% 1.22E+05 5.3% 1.21E+04 4.1%
[0103] As illustrated in Table V, the addition of bulk cerium
oxalate removed >94% of the monitored phospholipids, while
removing <3% of the respective target analytes, illustrating
significant selectivity of the extraction mechanism for
phospholipids.
[0104] It is to be understood that the above-referenced
arrangements are only illustrative of certain embodiments of the
present invention. Numerous modifications and alternative
arrangements can be devised by those of ordinary skill in the art
without departing from the spirit and scope of the present
invention, such as modifications in size, shape, materials, etc.,
and the appended claims are intended to cover such modifications
and arrangements.
* * * * *