U.S. patent application number 11/946017 was filed with the patent office on 2009-05-28 for methods for detecting estradiol by mass spectrometry.
Invention is credited to Nigel J. Clarke, Mildred M. Goldman, Richard E. Reitz.
Application Number | 20090134325 11/946017 |
Document ID | / |
Family ID | 40668902 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090134325 |
Kind Code |
A1 |
Goldman; Mildred M. ; et
al. |
May 28, 2009 |
METHODS FOR DETECTING ESTRADIOL BY MASS SPECTROMETRY
Abstract
Provided are methods for determining the amount of estradiol in
a sample using mass spectrometry. The methods generally involve
ionizing estradiol in a sample and detecting and quantifying the
amount of the ion to determine the amount of estradiol in the
sample.
Inventors: |
Goldman; Mildred M.; (Laguna
Niguel, CA) ; Clarke; Nigel J.; (Oceanside, CA)
; Reitz; Richard E.; (San Clemente, CA) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Family ID: |
40668902 |
Appl. No.: |
11/946017 |
Filed: |
November 27, 2007 |
Current U.S.
Class: |
250/283 |
Current CPC
Class: |
H01J 49/004 20130101;
G01N 30/7233 20130101; G01N 33/6851 20130101; H01J 49/0031
20130101; G01N 33/743 20130101; H01J 49/0431 20130101 |
Class at
Publication: |
250/283 |
International
Class: |
H01J 49/26 20060101
H01J049/26 |
Claims
1. A method for determining the amount of estradiol in a test
sample said method comprising: (a) purifying estradiol from said
test sample by liquid chromatography; (b) ionizing the purified
estradiol from said test sample to produce one or more ions
detectable by tandem mass spectrometry; and (c) detecting the
amount of the estradiol ion(s) by tandem mass spectrometry, wherein
the amount of the estradiol ion(s) is related to the amount of
estradiol in said test sample; wherein the limit of quantitation of
the method is less than or equal to 80 pg/mL and estradiol is not
derivatized prior to mass spectrometry.
2. The method of claim 1, wherein said liquid chromatography is
high performance liquid chromatography (HPLC).
3. The method of claim 1, wherein said purifying further comprises
high turbulence liquid chromatography (HTLC) followed by high
performance liquid chromatography (HPLC).
4. The method of claim 1, wherein said estradiol ions comprise one
or more ions selected from the group consisting of ions with a
mass/charge ratio of 271.14.+-.0.5, 255.07.+-.0.5, 183.10.+-.0.5,
159.20.+-.0.5, 145.10.+-.0.5, and 133.20.+-.0.5.
5. The method of claim 1, wherein said ionizing comprises
generating a precursor ion selected from the group consisting of
ions with a mass/charge ratio of 271.14.+-.0.5 and 255.07.+-.0.5,
and generating one or more fragment ions selected from the group
consisting of ions with a mass/charge ratio of 183.10.+-.0.5,
159.20.+-.0.5, 145.10.+-.0.5, and 133.20.+-.0.5.
6. The method of claim 1, wherein an agent is added to said test
sample in an amount sufficient to free estradiol from a protein
that may be present in said test sample prior to said ionizing
step.
7. The method of claim 6, wherein said agent is added in an amount
sufficient to acidify said test sample.
8. The method of claim 7, wherein said agent is formic acid.
9. The method of claim 6, wherein said agent is a salt.
10. The method of claim 9, wherein said salt is ammonium
sulfate.
11. The method of claim 1, wherein both glucuronidated and
non-glucuronidated estradiol present in said test sample are
detected and measured by the method.
12. The method of claim 1, wherein said test sample is body
fluid.
13. A method for determining the amount of estradiol in a body
fluid sample by tandem mass spectrometry said method comprising:
(a) purifying estradiol from said body fluid sample by liquid
chromatography; (b) generating a precursor ion of said estradiol
having a mass/charge ratio of 255.07.+-.0.5; (c) generating one or
more fragment ions of said precursor ion, wherein at least one of
said one or more fragment ions comprise an ion fragment having a
mass/charge ratio of 133.20.+-.0.5: and (d) detecting the amount of
one or more of said ions generated in step (b) or (c) or both and
relating the detected ions to the amount of said estradiol in said
body fluid sample.
14. The method of claim 13, wherein said method has a limit of
quantitation less than or equal to 80 pg/mL.
15. The method of claim 13, wherein said estradiol is not
derivatized prior to mass spectrometry.
16. The method of claim 13, wherein said liquid chromatography is
high performance liquid chromatography (HPLC).
17. The method of claim 13, wherein said purifying further
comprises high turbulence liquid chromatography (HTLC) followed by
high performance liquid chromatography (HPLC).
18. The method of claim 13, wherein said precursor ion has a
mass/charge ratio of 255.07.+-.0.5, and said one or more fragment
ions comprise a fragment ion with a mass/charge ratio of
159.20.+-.0.5.
19. The method of claim 13, wherein an agent is added to said body
fluid sample in an amount sufficient to free estradiol from a
protein that may be present in said body fluid sample prior to said
ionizing step.
20. The method of claim 19, wherein said agent is added in an
amount sufficient to acidify said body fluid sample.
21. The method of claim 20, wherein said agent is formic acid.
22. The method of claim 13, wherein both glucuronidated and
non-glucuronidated estradiol present in said body fluid are
detected and measured by the method.
23. A method for determining the amount of estradiol in a body
fluid sample by tandem mass spectrometry said method comprising:
(a) purifying estradiol from said body fluid sample by liquid
chromatography; (b) generating a precursor ion of said estradiol
having a mass/charge ratio of 271.14.+-.0.5; (c) generating one or
more fragment ions of said precursor ion, wherein at least one of
said one or more fragment ions comprise an ion fragment having a
mass/charge ratio of 183.10.+-.0.5; and (d) detecting the amount of
one or more of said ions generated in step (b) or (c) or both and
relating the detected ions to the amount of said estradiol in said
body fluid sample.
24. The method of claim 23, wherein said method has a limit of
quantitation less than or equal to 80 pg/mL.
25. The method of claim 23, wherein said estradiol is not
derivatized prior to mass spectrometry.
26. The method of claim 23, wherein said liquid chromatography is
high performance liquid chromatography (HPLC).
27. The method of claim 23, wherein said purifying further
comprises high turbulence liquid chromatography (HTLC) followed by
high performance liquid chromatography (HPLC).
28. The method of claim 23, wherein said precursor ion has a
mass/charge ratio of 271.14.+-.0.5, and said one or more fragment
ions comprise a fragment ion with a mass/charge ratio of
145.10.+-.0.5.
29. The method of claim 23, wherein an agent is added to said body
fluid sample in an amount sufficient to free estradiol from a
protein that may be present in said test sample prior to said
ionizing step.
30. The method of claim 29, wherein said agent is a salt.
31. The method of claim 30, wherein said salt is ammonium
sulfate.
32. The method of claim 23, wherein both glucuronidated and
non-glucuronidated estradiol present in said body fluid sample are
detected and measured by the method.
33. A method for determining the amount of estradiol in a test
sample said method comprising: (a) acidifying said test sample with
an agent in an amount sufficient to free estradiol from a protein
that may be present in said test sample; (b) purifying estradiol
from said test sample by liquid chromatography; (c) ionizing the
purified estradiol from said test sample to produce one or more
ions detectable by tandem mass spectrometry; and (d) detecting the
amount of the estradiol ion(s) by tandem mass spectrometry in
positive ion mode, wherein the amount of the estradiol ion(s) is
related to the amount of estradiol in said test sample.
34. The method of claim 33, wherein said method has a limit of
quantitation less than or equal to 80 pg/mL.
35. The method of claim 33, wherein said estradiol is not
derivatized prior to mass spectrometry.
36. The method of claim 33, wherein said liquid chromatography is
high performance liquid chromatography (HPLC).
37. The method of claim 33, wherein said purifying further
comprises high turbulence liquid chromatography (HTLC) followed by
high performance liquid chromatography (HPLC).
38. The method of claim 33, wherein said acidifying agent is formic
acid.
39. The method of claim 33, wherein said estradiol ions comprise
one or more ions selected from the group consisting of ions with a
mass/charge ratio of 255.07.+-.0.5, 159.20.+-.0.5, and
133.20.+-.0.5.
40. The method of claim 33, wherein said ionizing comprises
generating a precursor ion with a mass/charge ratio of
255.07.+-.0.5, and generating one or more fragment ions selected
from the group consisting of ions with a mass/charge ratio of
159.20.+-.0.5, and 133.20.+-.0.5.
41. The method of claim 33, wherein both glucuronidated and
non-glucuronidated estradiol present in said test sample are
detected and measured by the method.
42. The method of claim 33, wherein said test sample is body
fluid.
43. A method for determining the amount of estradiol in a test
sample said method comprising: (a) adding an agent to said test
sample in an amount sufficient to free estradiol from a protein
that may be present in said test sample; (b) purifying estradiol
from said test sample by liquid chromatography; (c) ionizing the
purified estradiol from said test sample to produce one or more
ions detectable by tandem mass spectrometry; and (d) detecting the
amount of the estradiol ion(s) by tandem mass spectrometry in
negative ion mode, wherein the amount of the estradiol ion(s) is
related to the amount of estradiol in said test sample.
44. The method of claim 43, wherein said method has a limit of
quantitation less than or equal to 80 pg/mL.
45. The method of claim 43, wherein said estradiol is not
derivatized prior to mass spectrometry.
46. The method of claim 43, wherein said liquid chromatography is
high performance liquid chromatography (HPLC).
47. The method of claim 43, wherein said purifying further
comprises high turbulence liquid chromatography (HTLC) followed by
high performance liquid chromatography (HPLC).
48. The method of claim 43, wherein said agent is a salt.
49. The method of claim 48, wherein said salt is ammonium
sulfate.
50. The method of claim 43, wherein said estradiol ions comprise
one or more ions selected from the group consisting of ions with a
mass/charge ratio of 271.14.+-.0.5, 183.10.+-.0.5, and
145.10.+-.0.5.
51. The method of claim 43, wherein said ionizing comprises
generating a precursor ion with a mass/charge ratio of
271.14.+-.0.5, and generating one or more fragment ions selected
from the group consisting of ions with a mass/charge ratio of
183.10.+-.0.5, and 145.10.+-.0.5.
52. The method of claim 43, wherein both glucuronidated and
non-glucuronidated estradiol present in said test sample are
detected and measured by the method.
53. The method of claim 43, wherein said test sample is body fluid.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the detection of estradiol. In a
particular aspect, the invention relates to methods for detecting
estradiol by mass spectrometry.
BACKGROUND OF THE INVENTION
[0002] The following description of the background of the invention
is provided simply as an aid in understanding the invention and is
not admitted to describe or constitute prior art to the
invention.
[0003] Estradiol [17.beta.-estradiol or
Estra-1,3,5(10)-triene-3,17-diol,(17-beta)] or E2 is a C.sub.18
steroid hormone with a molecular weight of 272.38 daltons. It is a
sex hormone labeled as the "female" hormone (also present in
males), which is the most potent estrogen of a group of endogenous
steroids, which include estrone (E1) and estriol (E3). In women,
mainly the ovaries produce estradiol with secondary production by
the adrenal glands and conversion of steroid precursors into
estrogens in fat tissue. Estradiol is responsible for growth of the
breast and reproductive epithelia and development of secondary
sexual characteristics. Normal levels of estradiol provide for
proper ovulation, conception and pregnancy, in addition to
promoting healthy bone structure and regulating cholesterol levels
in females. In men, the testes and the adrenal glands are the
principal source of estradiol. Estradiol is needed for hormonal
balance and the function of other glands.
[0004] Methods for detecting specific estradiol ions using mass
spectrometry have been described. For example Nelson R, et al.,
Clinical Chem 2004, 50(2):373-84; Mellon-Nussbaum S, et al., J Biol
Chem 1982, 257(10):5678-5684; and Xu X, et al., Nature Protocols
2007, 2(6):1350-1355 disclose methods for detecting various
estradiol ions using liquid chromatography and mass spectrometry.
These methods derivatize estradiol prior to detection by mass
spectrometry. Methods to detect underivatized estradiol by liquid
chromatography/mass spectrometry are disclosed in Guo T, et al.,
Arch Pathol Lab Med 2004, 128:469-475; Sun Y, et al., J Am Soc Mass
Spectrom 2005, 16(2):271-279; and Diaz-Cruz S, et al., J Mass
Spectrom 2003, 38:917-923. Methods to detect estradiol by gas
chromatography/mass spectrometry are disclosed in Nachtigall L, et
al., Menopause: J of N. Amer. Menopause Society 2000, 7(4):243-250;
Santen R, et al., Steroids 2007, 72:666-671; Dorgan J, et al.,
Steroids 2002, 67:151-158; Biancotto G, et al., J Mass Spectrom
2002, 37(12): 1266-1271; Fedeniuk R W, et al., J Chromatogr B
Analyt Technol Biomed Life Sci 2004, 802(2):307-315; and Biddle S,
et al., Anal Chim Acta, 2007, 586(1-2);115-121.
SUMMARY OF THE INVENTION
[0005] The present invention provides methods for detecting the
amount of estradiol in a sample by mass spectrometry, including
tandem mass spectrometry.
[0006] In one aspect, methods are provided for determining the
amount of estradiol in a test sample. The methods may include: (a)
purifying estradiol in the test sample by liquid chromatography;
(b) ionizing estradiol in the test sample; and (c) detecting the
amount of the estradiol ion(s) by mass spectrometry and relating
the amount of the detected estradiol ion(s) to the amount of
estradiol in the test sample. In certain preferred embodiments of
this aspect, the limit of quantitation of the methods is less than
or equal to 80 pg/mL; and preferably, estradiol is not derivatized
prior to mass spectrometry. In some preferred embodiments, the
methods include generating one or more precursor ions of estradiol
in which at least one of the precursor ions has a mass/charge ratio
of 271.14.+-.0.5 or 255.07.+-.0.5. In related preferred
embodiments, the methods may include generating one or more
fragment ions of an estradiol precursor ion in which at least one
of the fragment ions has a mass/charge ratio of 183.10.+-.0.5,
159.20.+-.0.5, 145.10.+-.0.5, or 133.20.+-.0.5; preferably one or
more fragment ions are selected from the group consisting of ions
with a mass/charge ratio of 183.10.+-.0.5 and 133.20.+-.0.5. In
certain preferred embodiments of the aspect, the test sample is
body fluid. In some preferred embodiments, the methods may include
adding an agent to the test sample in an amount sufficient to free
estradiol from a protein that may be present in the test sample. In
related preferred embodiments, the methods may include acidifying
the test sample; preferably acidifying before ionizing; more
preferably acidifying before purifying; preferably acidifying with
formic acid. In different preferred embodiments, the methods may
include adding a salt to the test sample in an amount sufficient to
free estradiol from a protein that may be present in the test
sample; preferably adding before ionizing; more preferably adding
before purifying; preferably adding ammonium sulfate. In
particularly preferred embodiments, 100 mM ammonium sulfate is
added to the test sample for a final concentration of .about.57 mM
ammonium sulfate in the test sample.
[0007] As used herein, unless otherwise stated, the singular forms
"a," "an," and "the" include plural reference. Thus, for example, a
reference to "a protein" includes a plurality of protein
molecules.
[0008] As used herein, the term "purification" or "purifying" does
not refer to removing all materials from the sample other than the
analyte(s) of interest. Instead, purification refers to a procedure
that enriches the amount of one or more analytes of interest
relative to other components in the sample that may interfere with
detection of the analyte of interest. Samples are purified herein
by various means to allow removal of one or more interfering
substances, e.g., one or more substances that would interfere with
the detection of selected estradiol parent and daughter ions by
mass spectrometry.
[0009] As used herein, the term "test sample" refers to any sample
that may contain estradiol. As used herein, the term "body fluid"
means any fluid that can be isolated from the body of an
individual. For example, "body fluid" may include blood, plasma,
serum, bile, saliva, urine, tears, perspiration, and the like.
[0010] As used herein, the term "derivatizing" means reacting two
molecules to form a new molecule. Derivatizing agents may include
isothiocyanate groups, dinitro-fluorophenyl groups,
nitrophenoxycarbonyl groups, and/or phthalaldehyde groups, and the
like.
[0011] As used herein, the term "chromatography" refers to a
process in which a chemical mixture carried by a liquid or gas is
separated into components as a result of differential distribution
of the chemical entities as they flow around or over a stationary
liquid or solid phase.
[0012] As used herein, the term "liquid chromatography" or "LC"
means a process of selective retardation of one or more components
of a fluid solution as the fluid uniformly percolates through a
column of a finely divided substance, or through capillary
passageways. The retardation results from the distribution of the
components of the mixture between one or more stationary phases and
the bulk fluid, (i.e., mobile phase), as this fluid moves relative
to the stationary phase(s). Examples of "liquid chromatography"
include reverse phase liquid chromatography (RPLC), high
performance liquid chromatography (HPLC), and high turbulence
liquid chromatography (HTLC).
[0013] As used herein, the term "high performance liquid
chromatography" or "HPLC" refers to liquid chromatography in which
the degree of separation is increased by forcing the mobile phase
under pressure through a stationary phase, typically a densely
packed column.
[0014] As used herein, the term "high turbulence liquid
chromatography" or "HTLC" refers to a form of chromatography that
utilizes turbulent flow of the material being assayed through the
column packing as the basis for performing the separation. HTLC has
been applied in the preparation of samples containing two unnamed
drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et
al., J. Chromatogr. A 854: 23-35 (1999); see also, U.S. Pat. Nos.
5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further
explain HTLC. Persons of ordinary skill in the art understand
"turbulent flow". When fluid flows slowly and smoothly, the flow is
called "laminar flow". For example, fluid moving through an HPLC
column at low flow rates is laminar. In laminar flow the motion of
the particles of fluid is orderly with particles moving generally
in straight lines. At faster velocities, the inertia of the water
overcomes fluid frictional forces and turbulent flow results. Fluid
not in contact with the irregular boundary "outruns" that which is
slowed by friction or deflected by an uneven surface. When a fluid
is flowing turbulently, it flows in eddies and whirls (or
vortices), with more "drag" than when the flow is laminar. Many
references are available for assisting in determining when fluid
flow is laminar or turbulent (e.g., Turbulent Flow Analysis:
Measurement and Prediction, P. S. Bernard & J. M. Wallace, John
Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow,
Jean Mathieu & Julian Scott, Cambridge University Press
(2001)).
[0015] As used herein, the term "gas chromatography" or "GC" refers
to chromatography in which the sample mixture is vaporized and
injected into a stream of carrier gas (as nitrogen or helium)
moving through a column containing a stationary phase composed of a
liquid or a particulate solid and is separated into its component
compounds according to the affinity of the compounds for the
stationary phase.
[0016] As used herein, the term "large particle column" or
"extraction column" refers to a chromatography column containing an
average particle diameter greater than about 35 .mu.m. As used in
this context, the term "about" means .+-.10%. In a preferred
embodiment the column contains particles of about 60 .mu.m in
diameter.
[0017] As used herein, the term "analytical column" refers to a
chromatography column having sufficient chromatographic plates to
effect a separation of materials in a sample that elute from the
column sufficient to allow a determination of the presence or
amount of an analyte. Such columns are often distinguished from
"extraction columns", which have the general purpose of separating
or extracting retained material from non-retained materials in
order to obtain a purified sample for further analysis. As used in
this context, the term "about" means .+-.10%. In a preferred
embodiment the analytical column contains particles of about 4
.mu.m in diameter.
[0018] As used herein, the term "on-line" or "inline", for example
as used in "on-line automated fashion" or "on-line extraction"
refers to a procedure performed without the need for operator
intervention. In contrast, the term "off-line" as used herein
refers to a procedure requiring manual intervention of an operator.
Thus, if samples are subjected to precipitation, and the
supernatants are then manually loaded into an autosampler, the
precipitation and loading steps are off-line from the subsequent
steps. In various embodiments of the methods, one or more steps may
be performed in an on-line automated fashion.
[0019] As used herein, the term "mass spectrometry" or "MS" refers
to an analytical technique to identify compounds by their mass. MS
refers to methods of filtering, detecting, and measuring ions based
on their mass-to-charge ratio, or "m/z". MS technology generally
includes (1) ionizing the compounds to form charged compounds; and
(2) detecting the molecular weight of the charged compounds and
calculating a mass-to-charge ratio. The compounds may be ionized
and detected by any suitable means. A "mass spectrometer" generally
includes an ionizer and an ion detector. In general, one or more
molecules of interest are ionized, and the ions are subsequently
introduced into a mass spectrographic instrument where, due to a
combination of magnetic and electric fields, the ions follow a path
in space that is dependent upon mass ("m") and charge ("z"). See,
e.g., U.S. Pat. No. 6,204,500, entitled "Mass Spectrometry From
Surfaces;" U.S. Pat. No. 6,107,623, entitled "Methods and Apparatus
for Tandem Mass Spectrometry;" U.S. Pat. No. 6,268,144, entitled
"DNA Diagnostics Based On Mass Spectrometry;" U.S. Pat. No.
6,124,137, entitled "Surface-Enhanced Photolabile Attachment And
Release For Desorption And Detection Of Analytes;" Wright et al.,
Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and
Merchant and Weinberger, Electrophoresis 21:1164-67 (2000).
[0020] As used herein, the term "operating in negative ion mode"
refers to those mass spectrometry methods where negative ions are
generated and detected. The term "operating in positive ion mode"
as used herein, refers to those mass spectrometry methods where
positive ions are generated and detected.
[0021] As used herein, the term "ionization" or "ionizing" refers
to the process of generating an analyte ion having a net electrical
charge equal to one or more electron units. Negative ions are those
having a net negative charge of one or more electron units, while
positive ions are those having a net positive charge of one or more
electron units.
[0022] As used herein, the term "electron ionization" or "EI"
refers to methods in which an analyte of interest in a gaseous or
vapor phase interacts with a flow of electrons. Impact of the
electrons with the analyte produces analyte ions, which may then be
subjected to a mass spectrometry technique.
[0023] As used herein, the term "chemical ionization" or "CI"
refers to methods in which a reagent gas (e.g. ammonia) is
subjected to electron impact, and analyte ions are formed by the
interaction of reagent gas ions and analyte molecules.
[0024] As used herein, the term "fast atom bombardment" or "FAB"
refers to methods in which a beam of high energy atoms (often Xe or
Ar) impacts a non-volatile sample, desorbing and ionizing molecules
contained in the sample. Test samples are dissolved in a viscous
liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl
alcohol, 18-crown-6 crown ether, 2-nitrophenyloctyl ether,
sulfolane, diethanolamine, and triethanolamine. The choice of an
appropriate matrix for a compound or sample is an empirical
process.
[0025] As used herein, the term "matrix-assisted laser desorption
ionization" or "MALDI" refers to methods in which a non-volatile
sample is exposed to laser irradiation, which desorbs and ionizes
analytes in the sample by various ionization pathways, including
photo-ionization, protonation, deprotonation, and cluster decay.
For MALDI, the sample is mixed with an energy-absorbing matrix,
which facilitates desorption of analyte molecules.
[0026] As used herein, the term "surface enhanced laser desorption
ionization" or "SELDI" refers to another method in which a
non-volatile sample is exposed to laser irradiation, which desorbs
and ionizes analytes in the sample by various ionization pathways,
including photo-ionization, protonation, deprotonation, and cluster
decay. For SELDI, the sample is typically bound to a surface that
preferentially retains one or more analytes of interest. As in
MALDI, this process may also employ an energy-absorbing material to
facilitate ionization.
[0027] As used herein, the term "electrospray ionization" or "ESI,"
refers to methods in which a solution is passed along a short
length of capillary tube, to the end of which is applied a high
positive or negative electric potential. Solution reaching the end
of the tube is vaporized (nebulized) into a jet or spray of very
small droplets of solution in solvent vapor. This mist of droplets
flows through an evaporation chamber, which is heated slightly to
prevent condensation and to evaporate solvent. As the droplets get
smaller the electrical surface charge density increases until such
time that the natural repulsion between like charges causes ions as
well as neutral molecules to be released.
[0028] As used herein, the term "atmospheric pressure chemical
ionization" or "APCI," refers to mass spectroscopy methods that are
similar to ESI; however, APCI produces ions by ion-molecule
reactions that occur within a plasma at atmospheric pressure. The
plasma is maintained by an electric discharge between the spray
capillary and a counter electrode. Then ions are typically
extracted into the mass analyzer by use of a set of differentially
pumped skimmer stages. A counterflow of dry and preheated N.sub.2
gas may be used to improve removal of solvent. The gas-phase
ionization in APCI can be more effective than ESI for analyzing
less-polar species.
[0029] The term "Atmospheric Pressure Photoionization" or "APPI" as
used herein refers to the form of mass spectroscopy where the
mechanism for the photoionization of molecule M is photon
absorption and electron ejection to form the molecular ion M+.
Because the photon energy typically is just above the ionization
potential, the molecular ion is less susceptible to dissociation.
In many cases it may be possible to analyze samples without the
need for chromatography, thus saving significant time and expense.
In the presence of water vapor or protic solvents, the molecular
ion can extract H to form MH+. This tends to occur if M has a high
proton affinity. This does not affect quantitation accuracy because
the sum of M+ and MH+ is constant. Drug compounds in protic
solvents are usually observed as MH+, whereas nonpolar compounds
such as naphthalene or testosterone usually form M+. Robb, D. B.,
Covey, T. R. and Bruins, A. P. (2000): See, e.g., Robb et al.,
Atmospheric pressure photoionization: An ionization method for
liquid chromatography-mass spectrometry. Anal Chem. 72(15):
3653-3659.
[0030] As used herein, the term "inductively coupled plasma" or
"ICP" refers to methods in which a sample interacts with a
partially ionized gas at a sufficiently high temperature such that
most elements are atomized and ionized.
[0031] As used herein, the term "field desorption" refers to
methods in which a non-volatile test sample is placed on an
ionization surface, and an intense electric field is used to
generate analyte ions.
[0032] As used herein, the term "desorption" refers to the removal
of an analyte from a surface and/or the entry of an analyte into a
gaseous phase.
[0033] As used herein, the term "limit of quantification", "limit
of quantitation" or "LOQ" refers to the point where measurements
become quantitatively meaningful. The analyte response at this LOQ
is identifiable, discrete and reproducible with a precision of 20%
and an accuracy of 80% to 120%.
[0034] As used herein, the term "limit of detection" or "LOD" is
the point at which the measured value is larger than the
uncertainty associated with it. The LOD is defined arbitrarily as 2
standard deviations (SD) from the zero concentration.
[0035] As used herein, an "amount" of estradiol in a body fluid
sample refers generally to an absolute value reflecting the mass of
estradiol detectable in volume of body fluid. However, an amount
also contemplates a relative amount in comparison to another
estradiol amount. For example, an amount of estradiol in a body
fluid can be an amount which is greater than a control or normal
level of estradiol normally present.
[0036] In a second aspect, methods are provided for determining the
amount of estradiol in a body fluid sample by tandem mass
spectrometry that include: (a) purifying estradiol in the body
fluid sample by liquid chromatography; (b) generating a precursor
ion of estradiol having a mass/charge ratio of 255.07.+-.0.5; (c)
generating one or more fragment ions of the precursor ion in which
at least one of the fragment ions has a mass/charge ratio of
133.20.+-.0.5; and (d) detecting the amount of one or more of the
ions generated in step (b) or (c) or both and relating the detected
ions to the amount of estradiol in the body fluid sample. In some
preferred embodiments, the limit of quantitation of the methods is
less than or equal to 80 pg/mL. In other preferred embodiments,
estradiol is not derivatized prior to mass spectrometry. In certain
preferred embodiments, the methods may further include generating
one or more fragment ions of an estradiol precursor ion in which at
least one of the fragment ions has a mass/charge ratio of
159.20.+-.0.5. In some preferred embodiments, the methods may
include adding an agent to the body fluid sample in an amount
sufficient to free estradiol from a protein that may be present in
the body fluid sample. In related preferred embodiments, the
methods may include acidifying the body fluid sample; preferably
acidifying before ionizing; more preferably acidifying before
purifying; preferably acidifying with formic acid.
[0037] In a third aspect, methods are provided for determining the
amount of estradiol in a body fluid sample by tandem mass
spectrometry that include: (a) purifying estradiol in the body
fluid sample by liquid chromatography; (b) generating a precursor
ion of estradiol having a mass/charge ratio of 271.14.+-.0.5; (c)
generating one or more fragment ions of the precursor ion in which
at least one of the fragment ions has a mass/charge ratio of
183.10.+-.0.5; and (d) detecting the amount of one or more of the
ions generated in step (b) or (c) or both and relating the detected
ions to the amount of estradiol in the body fluid sample. In some
preferred embodiments, the limit of quantitation of the methods is
less than or equal to 80 pg/mL. In other preferred embodiments,
estradiol is not derivatized prior to mass spectrometry. In certain
preferred embodiments, the methods may further include generating
one or more fragment ions of an estradiol precursor ion in which at
least one of the fragment ions has a mass/charge ratio of
145.10.+-.0.5. In some preferred embodiments, the methods may
include adding an agent to the body fluid sample in an amount
sufficient to free estradiol from a protein that may be present in
the body fluid sample. In related preferred embodiments, the
methods may include adding a salt agent to the body fluid sample;
preferably adding before ionizing; more preferably adding before
purifying; preferably adding ammonium sulfate. In particularly
preferred embodiments, 100 mM ammonium sulfate is added to the body
fluid sample for a final concentration of .about.57 mM ammonium
sulfate in the body fluid sample.
[0038] In a fourth aspect, methods are provided for determining the
amount of estradiol in a test sample that include: (a) acidifying
the test sample with an agent in an amount sufficient to free
estradiol from a protein that may be present in the test sample;
(b) purifying estradiol in the test sample by liquid
chromatography; (c) ionizing estradiol in the test sample to
produce one or more ions detectable by tandem mass spectrometry;
and (d) detecting the amount of the estradiol ion(s) by tandem mass
spectrometry in positive ion mode and relating the amount of the
detected estradiol ion(s) to the amount of estradiol in the test
sample. In certain preferred embodiments, the test sample is body
fluid. In some preferred embodiments, the limit of quantitation of
the methods is less than or equal to 80 pg/mL. In other preferred
embodiments, estradiol is not derivatized prior to mass
spectrometry. In some preferred embodiments, the methods include
generating one or more precursor ions of estradiol in which at
least one of the precursor ions has a mass/charge ratio of
255.07.+-.0.5. In related preferred embodiments, the methods may
include generating one or more fragment ions of an estradiol
precursor ion in which at least one of the fragment ions has a
mass/charge ratio of 159.20.+-.0.5 or 133.20.+-.0.5. In some
preferred embodiments, the methods may include acidifying the test
sample before ionizing; more preferably acidifying before
purifying; preferably acidifying with formic acid.
[0039] In a fifth aspect, methods are provided for determining the
amount of estradiol in a test sample that include: (a) adding an
agent to the test sample in an amount sufficient to free estradiol
from a protein that may be present in the test sample; (b)
purifying estradiol in the test sample by liquid chromatography;
(c) ionizing the purified estradiol in the test sample to produce
one or more ions detectable by tandem mass spectrometry; and (d)
detecting the amount of the estradiol ion(s) by tandem mass
spectrometry in negative ion mode and relating the amount of the
detected estradiol ion(s) to the amount of estradiol in the test
sample. In certain preferred embodiments, the test sample is body
fluid. In some preferred embodiments, the limit of quantitation of
the methods is less than or equal to 80 pg/mL. In other preferred
embodiments, estradiol is not derivatized prior to mass
spectrometry. In some preferred embodiments, the methods include
generating one or more precursor ions of estradiol in which at
least one of the precursor ions has a mass/charge ratio of
271.14.+-.0.5. In related preferred embodiments, the methods may
include generating one or more fragment ions of an estradiol
precursor ion in which at least one of the fragment ions has a
mass/charge ratio of 183.10.+-.0.5 or 145.10.+-.0.5. In other
preferred embodiments, the methods may include adding a salt agent
to the test sample, preferably adding before ionizing; more
preferably adding before purifying, preferably adding ammonium
sulfate. In particularly preferred embodiments, 100 mM ammonium
sulfate is added to the test sample for a final concentration of
.about.57 mM ammonium sulfate in the test sample.
[0040] In some preferred embodiments, estradiol may be derivatized
prior to mass spectrometry, however, in certain preferred
embodiments; sample preparation excludes the use of
derivatization.
[0041] In certain preferred embodiments of the above aspects,
liquid chromatography is performed using HTLC and HPLC, preferably
HTLC is used in conjunction with HPLC, however other methods can be
used that include for example, protein precipitation and
purification in conjunction with HPLC.
[0042] Preferred embodiments utilize high performance liquid
chromatography (HPLC), alone or in combination with one or more
purification methods, for example HTLC or protein precipitation, to
purify estradiol in samples.
[0043] In certain preferred embodiments of the methods disclosed
herein, mass spectrometry is performed in negative ion mode.
Alternatively, mass spectrometry is performed in positive ion mode.
In particularly preferred embodiments, estradiol is measured using
both positive and negative ion mode. In certain preferred
embodiments, estradiol is measured using APCI or ESI in either
positive or negative mode.
[0044] In preferred embodiments of the above aspects, both
glucuronidated and non-glucuronidated estradiol present in the body
fluid sample are detected and measured.
[0045] In preferred embodiments, the estradiol ions detectable in a
mass spectrometer are selected from the group consisting of ions
with a mass/charge ratio (m/z) of 271.14.+-.0.5, 255.07.+-.0.5,
183.10.+-.0.5, 159.20.+-.0.5, 145.10.+-.0.5, and 133.20.+-.0.5; the
latter four being fragment ions of the precursor ions. In
particularly preferred embodiments, the precursor ions have a
mass/charge ratio of 271.14.+-.0.5 or 255.07.+-.0.5, and the
fragment ions have amass/charge ratio of 183.10.+-.0.5 or
133.20.+-.0.5.
[0046] In preferred embodiments, a separately detectable internal
estradiol standard is provided in the sample, the amount of which
is also determined in the sample. In these embodiments, all or a
portion of both the endogenous estradiol and the internal standard
present in the sample is ionized to produce a plurality of ions
detectable in a mass spectrometer, and one or more ions produced
from each are detected by mass spectrometry.
[0047] A preferred internal estradiol standard is
2,4,16,16,17-d.sub.5 estradiol. In preferred embodiments, the
internal estradiol standard ions detectable in a mass spectrometer
are selected from the group consisting of ions with a mass/charge
ratio of 276.15.+-.0.5, 260.10.+-.0.5, 187.10.+-.0.5,
161.10.+-.0.5, 147.10.+-.0.5, and 135.10.+-.0.5. In particularly
preferred embodiments, a precursor ion of the internal estradiol
standard is selected from the group consisting of ions having a
mass/charge ratio of 276.15.+-.0.5 and 260.10.+-.0.5; and one or
more fragment ions is selected from the group consisting of ions
having a mass/charge ratio of 187.10.+-.0.5, 161.10.+-.0.5,
147.10.+-.0.5, and 135.10.+-.0.5.
[0048] In preferred embodiments, the presence or amount of the
estradiol ion is related to the presence or amount of estradiol in
the test sample by comparison to a reference such as
2,4,16,16,17-d.sub.5 estradiol.
[0049] In one embodiment, the methods involve the combination of
liquid chromatography with mass spectrometry. In a preferred
embodiment, the liquid chromatography is HPLC. A preferred
embodiment utilizes HPLC alone or in combination with one or more
purification methods such as for example HTLC or protein
purification, to purify estradiol in samples. In another preferred
embodiment, the mass spectrometry is tandem mass spectrometry
(MS/MS).
[0050] In certain preferred embodiments of the aspects disclosed
herein, the limit of quantitation (LOQ) of estradiol is less than
or equal to 80 pg/mL; preferably less than or equal to 75 pg/mL;
preferably less than or equal to 50 pg/mL; preferably less than or
equal to 25 pg/mL; preferably less than or equal to 10 pg/mL;
preferably less than or equal to 5 pg/mL; preferably less than or
equal to 4.5 pg/mL; preferably less than or equal to 4 pg/mL;
preferably less than or equal to 3.5 pg/mL; preferably less than or
equal to 3 pg/mL; preferably less than or equal to 2.5 pg/mL;
preferably 2 pg/mL.
[0051] The term "about" as used herein in reference to quantitative
measurements not including the measurement of the mass of an ion,
refers to the indicated value plus or minus 10%. Mass spectrometry
instruments can vary slightly in determining the mass of a given
analyte. The term "about" in the context of the mass of an ion or
the mass/charge ratio of an ion refers to .+-.0.5 atomic mass
unit.
[0052] The summary of the invention described above is non-limiting
and other features and advantages of the invention will be apparent
from the following detailed description of the invention, and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 shows the linearity of the quantitation of estradiol
in serially diluted stock samples using an LC-MS/MS assay. Details
are described in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0054] Methods are described for detecting and quantifying
estradiol in a test sample. The methods utilize liquid
chromatography (LC), most preferably HTLC in conjunction with HPLC,
to perform an initial purification of selected analytes, and
combine this purification with unique methods of mass spectrometry
(MS), thereby providing a high-throughput assay system for
detecting and quantifying estradiol in a test sample. The preferred
embodiments are particularly well suited for application in large
clinical laboratories. Estradiol methods are provided that have
enhanced specificity and are accomplished in less time and with
less sample preparation than required in other estradiol
assays.
[0055] In preferred embodiments, the limit of detection (LOD) of
estradiol in test samples is less than or equal to 80 pg/mL;
preferably less than or equal to 75 pg/mL; preferably less than or
equal to 50 pg/mL; preferably less than or equal to 25 pg/mL;
preferably less than or equal to 10 pg/mL; preferably less than or
equal to 5 pg/mL; preferably less than or equal to 4.5 pg/mL;
preferably less than or equal to 4 pg/mL; preferably less than or
equal to 3.5 pg/mL; preferably less than or equal to 3 pg/mL;
preferably less than or equal to 2.5 pg/mL; preferably 2 pg/mL.
[0056] Suitable test samples include any test sample that may
contain the analyte of interest. For example, samples obtained
during the manufacture of synthetic estradiol may be analyzed to
determine the composition and yield of the manufacturing process.
In some preferred embodiments, a sample is a biological sample;
that is, a sample obtained from any biological source, such as an
animal, a cell culture, an organ culture, etc. In certain preferred
embodiments samples are obtained from a mammalian animal, such as a
dog, cat, horse, etc. Particularly preferred mammalian animals are
primates, most preferably male or female humans. Particularly
preferred samples include blood, plasma, serum, hair, muscle,
urine, saliva, tear, cerebrospinal fluid, or other tissue sample.
Such samples may be obtained, for example, from a patient; that is,
a living person, male or female, presenting oneself in a clinical
setting for diagnosis, prognosis, or treatment of a disease or
condition. The test sample is preferably obtained from a patient,
for example, blood serum.
Sample Preparation for Mass Spectrometry
[0057] In women, approximately 1.3% of estradiol circulates in free
form with the remainder bound to sex hormone binding globulin
(SHBG) and to albumin. Estradiol binds to SHBG with high affinity
(approximately 40%) or to albumin with lower affinity.
[0058] Methods that may be used to enrich in estradiol relative to
other components in the sample (e.g. protein) include for example,
filtration, centrifugation, thin layer chromatography (TLC),
electrophoresis including capillary electrophoresis, affinity
separations including immunoaffinity separations, extraction
methods including ethyl acetate extraction and methanol extraction,
and the use of chaotropic agents or any combination of the above or
the like.
[0059] Various methods may be used to disrupt the interaction
between estradiol and protein prior to chromatography and or MS
sample analysis so that the analysis can be directed to the total
amount of estradiol in the sample (e.g., free estradiol and
estradiol bound to protein). Protein precipitation is one preferred
method of preparing a test sample, especially a biological test
sample, such as serum or plasma. Such protein purification methods
are well known in the art, for example, Polson et al., Journal of
Chromatography B 785:263-275 (2003), describes protein
precipitation techniques suitable for use in the methods. Protein
precipitation may be used to remove most of the protein from the
sample leaving estradiol in the supernatant. The samples may be
centrifuged to separate the liquid supernatant from the
precipitated proteins. The resultant supernatant may then be
applied to liquid chromatography and subsequent mass spectrometry
analysis. In certain embodiments, the use of protein precipitation
such as for example, acetonitrile protein precipitation, obviates
the need for high turbulence liquid chromatography (HTLC) or other
on-line extraction prior to HPLC and mass spectrometry. Accordingly
in such embodiments, the method involves (1) performing a protein
precipitation of the sample of interest; and (2) loading the
supernatant directly onto the HPLC-mass spectrometer without using
on-line extraction or high turbulence liquid chromatography
(HTLC).
[0060] In other preferred embodiments, estradiol may be released
from a protein without having to precipitate the protein. For
example, formic acid or 40% ethanol in water may be added to the
sample to disrupt interaction between a protein and estradiol.
Alternatively, ammonium sulfate may be added to the sample to
disrupt ionic interactions between a carrier protein and estradiol
without precipitating the carrier protein.
[0061] In some preferred embodiments, HTLC, alone or in combination
with one or more purification methods, may be used to purify
estradiol prior to mass spectrometry. In such embodiments samples
may be extracted using an HTLC extraction cartridge which captures
the analyte, then eluted and chromatographed on a second HTLC
column or onto an analytical HPLC column prior to ionization.
Because the steps involved in these chromatography procedures can
be linked in an automated fashion, the requirement for operator
involvement during the purification of the analyte can be
minimized. This feature can result in savings of time and costs,
and eliminate the opportunity for operator error.
[0062] It is believed that turbulent flow, such as that provided by
HTLC columns and methods, may enhance the rate of mass transfer,
improving separation characteristics. HTLC columns separate
components by means of high chromatographic flow rates through a
packed column containing rigid particles. By employing high flow
rates (e.g., 3-5 mL/min), turbulent flow occurs in the column that
causes nearly complete interaction between the stationary phase and
the analyte(s) of interest. An advantage of using HTLC columns is
that the macromolecular build-up associated with biological fluid
matrices is avoided since the high molecular weight species are not
retained under the turbulent flow conditions. HTLC methods that
combine multiple separations in one procedure lessen the need for
lengthy sample preparation and operate at a significantly greater
speed. Such methods also achieve a separation performance superior
to laminar flow (HPLC) chromatography. HTLC allows for direct
injection of biological samples (plasma, urine, etc.). Direct
injection is difficult to achieve in traditional forms of
chromatography because denatured proteins and other biological
debris quickly block the separation columns. HTLC also allows for
very low sample volume of less than 1 mL, preferably less than 0.5
mL, preferably less than 0.2 mL, preferably 0.1 mL.
[0063] Examples of HTLC applied to sample preparation prior to
analysis by mass spectrometry have been described elsewhere. See,
e.g., Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also,
U.S. Pat. Nos. 5,968,367; 5,919,368; 5,795,469; and 5,772,874. In
certain embodiments of the method, samples are subjected to protein
precipitation as described above prior to loading on the HTLC
column; in alternative preferred embodiments, the samples may be
loaded directly onto the HTLC without being subjected to protein
precipitation. Preferably, HTLC is used in conjunction with HPLC to
extract and purify estradiol without the sample being subjected to
protein precipitation. In related preferred embodiments, the
purifying step involves (i) applying the sample to an HTLC
extraction column, (ii) washing the HTLC extraction column under
conditions whereby estradiol is retained by the column, (iii)
eluting retained estradiol from the HTLC extraction column, (iv)
applying the retained material to an analytical column, and (v)
eluting purified estradiol from the analytical column. The HTLC
extraction column is preferably a large particle column. In various
embodiments, one of more steps of the methods may be performed in
an on-line, automated fashion. For example, in one embodiment,
steps (i)-(v) are performed in an on-line, automated fashion. In
another, the steps of ionization and detection are performed
on-line following steps (i)-(v).
[0064] Liquid chromatography (LC) including high-performance liquid
chromatography (HPLC) relies on relatively slow, laminar flow
technology. Traditional HPLC analysis relies on column packings in
which laminar flow of the sample through the column is the basis
for separation of the analyte of interest from the sample. The
skilled artisan will understand that separation in such columns is
a diffusional process. HPLC has been successfully applied to the
separation of compounds in biological samples but a significant
amount of sample preparation is required prior to the separation
and subsequent analysis with a mass spectrometer (MS), making this
technique labor intensive. In addition, most HPLC systems do not
utilize the mass spectrometer to its fullest potential, allowing
only one HPLC system to be connected to a single MS instrument,
resulting in lengthy time requirements for performing a large
number of assays.
[0065] Various methods have been described for using HPLC for
sample clean-up prior to mass spectrometry analysis. See, e.g.,
Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000); and
Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000).
[0066] One of skill in the art may select HPLC instruments and
columns that are suitable for use with estradiol. The
chromatographic column typically includes a medium (i.e., a packing
material) to facilitate separation of chemical moieties (i.e.,
fractionation). The medium may include minute particles. The
particles include a bonded surface that interacts with the various
chemical moieties to facilitate separation of the chemical
moieties. One suitable bonded surface is a hydrophobic bonded
surface such as an alkyl bonded surface. Alkyl bonded surfaces may
include C-4, C-8, C-12, or C-18 bonded alkyl groups, preferably
C-18 bonded groups. The chromatographic column includes an inlet
port for receiving a sample and an outlet port for discharging an
effluent that includes the fractionated sample. In one embodiment
the sample (or pre-purified sample) is applied to the column at the
inlet port, eluted with a solvent or solvent mixture, and
discharged at the outlet port. Different solvent modes may be
selected for eluting the analyte(s) of interest. For example,
liquid chromatography may be performed using a gradient mode, an
isocratic mode, or a polytyptic (i.e. mixed) mode. During
chromatography, the separation of materials is effected by
variables such as choice of eluent (also known as a "mobile
phase"), elution mode, gradient conditions, temperature, etc.
[0067] In certain embodiments, an analyte may be purified by
applying a sample to a column under conditions where the analyte of
interest is reversibly retained by the column packing material,
while one or more other materials are not retained. In these
embodiments, a first mobile phase condition can be employed where
the analyte of interest is retained by the column, and a second
mobile phase condition can subsequently be employed to remove
retained material from the column, once the non-retained materials
are washed through. Alternatively, an analyte may be purified by
applying a sample to a column under mobile phase conditions where
the analyte of interest elutes at a differential rate in comparison
to one or more other materials. Such procedures may enrich the
amount of one or more analytes of interest relative to one or more
other components of the sample.
[0068] In one preferred embodiment, the HTLC may be followed by
HPLC on a hydrophobic column chromatographic system. In certain
preferred embodiments, a TurboFlow Cyclone P.RTM. polymer-based
column from Cohesive Technologies (60 .mu.m particle size,
50.times.1.0 mm column dimensions, 100 .ANG. pore size) is used. In
related preferred embodiments, a Synergi Polar-RP.RTM. ether-linked
phenyl, analytical column from Phenomenex Inc (4 .mu.m particle
size, 150.times.2.0 mm column dimensions, 80 .ANG. pore size) with
hydrophilic endcapping is used. In certain preferred embodiments,
HTLC and HPLC are performed using HPLC Grade Ultra Pure Water and
100% methanol as the mobile phases.
[0069] By careful selection of valves and connector plumbing, two
or more chromatography columns may be connected as needed such that
material is passed from one to the next without the need for any
manual steps. In preferred embodiments, the selection of valves and
plumbing is controlled by a computer pre-programmed to perform the
necessary steps. Most preferably, the chromatography system is also
connected in such an on-line fashion to the detector system, e.g.,
an MS system. Thus, an operator may place a tray of samples in an
autosampler, and the remaining operations are performed under
computer control, resulting in purification and analysis of all
samples selected.
[0070] In certain preferred embodiments, estradiol present in a
test sample may be purified prior to ionization. In particularly
preferred embodiments the chromatography is not gas chromatography.
Preferably, the methods are performed without subjecting estradiol,
to gas chromatography prior to mass spectrometric analysis.
Detection and Quantitation by Mass Spectrometry
[0071] In various embodiments, estradiol present in a test sample
may be ionized by any method known to the skilled artisan. Mass
spectrometry is performed using a mass spectrometer, which includes
an ion source for ionizing the fractionated sample and creating
charged molecules for further analysis. For example ionization of
the sample may be performed by electron ionization, chemical
ionization, electrospray ionization (ESI), photon ionization,
atmospheric pressure chemical ionization (APCI), photoionization,
atmospheric pressure photoionization (APPI), fast atom bombardment
(FAB), liquid secondary ionization (LSI), matrix assisted laser
desorption ionization (MALDI), field ionization, field desorption,
thermospray/plasmaspray ionization, surface enhanced laser
desorption ionization (SELDI), inductively coupled plasma (ICP) and
particle beam ionization. The skilled artisan will understand that
the choice of ionization method may be determined based on the
analyte to be measured, type of sample, the type of detector, the
choice of positive versus negative mode, etc.
[0072] In preferred embodiments, estradiol is ionized by
electrospray ionization (ESI) in positive or negative mode. In
related preferred embodiments, estradiol ion is in a gaseous state
and the inert collision gas is argon or nitrogen. In alternative
preferred embodiments, estradiol is ionized by atmospheric pressure
chemical ionization (APCI) in positive or negative mode.
[0073] After the sample has been ionized, the positively charged or
negatively charged ions thereby created may be analyzed to
determine a mass-to-charge ratio. Suitable analyzers for
determining mass-to-charge ratios include quadrupole analyzers, ion
traps analyzers, and time-of-flight analyzers. The ions may be
detected using several detection modes. For example, selected ions
may be detected i.e., using a selective ion monitoring mode (SIM),
or alternatively, ions may be detected using a scanning mode, e.g.,
multiple reaction monitoring (MRM) or selected reaction monitoring
(SRM). Preferably, the mass-to-charge ratio is determined using a
quadrupole analyzer. For example, in a "quadrupole" or "quadrupole
ion trap" instrument, ions in an oscillating radio frequency field
experience a force proportional to the DC potential applied between
electrodes, the amplitude of the RF signal, and the mass/charge
ratio. The voltage and amplitude may be selected so that only ions
having a particular mass/charge ratio travel the length of the
quadrupole, while all other ions are deflected. Thus, quadrupole
instruments may act as both a "mass filter" and as a "mass
detector" for the ions injected into the instrument.
[0074] One may enhance the resolution of the MS technique by
employing "tandem mass spectrometry," or "MS/MS". In this
technique, a precursor ion (also called a parent ion) generated
from a molecule of interest can be filtered in an MS instrument,
and the precursor ion is subsequently fragmented to yield one or
more fragment ions (also called daughter ions or product ions) that
are then analyzed in a second MS procedure. By careful selection of
precursor ions, only ions produced by certain analytes are passed
to the fragmentation chamber, where collisions with atoms of an
inert gas produce the fragment ions. Because both the precursor and
fragment ions are produced in a reproducible fashion under a given
set of ionization/fragmentation conditions, the MS/MS technique may
provide an extremely powerful analytical tool. For example, the
combination of filtration/fragmentation may be used to eliminate
interfering substances, and may be particularly useful in complex
samples, such as biological samples.
[0075] The mass spectrometer typically provides the user with an
ion scan; that is, the relative abundance of each ion with a
particular mass/charge over a given range (e.g., 100 to 1000 amu).
The results of an analyte assay, that is, a mass spectrum, may be
related to the amount of the analyte in the original sample by
numerous methods known in the art. For example, given that sampling
and analysis parameters are carefully controlled, the relative
abundance of a given ion may be compared to a table that converts
that relative abundance to an absolute amount of the original
molecule. Alternatively, molecular standards may be run with the
samples, and a standard curve constructed based on ions generated
from those standards. Using such a standard curve, the relative
abundance of a given ion may be converted into an absolute amount
of the original molecule. In certain preferred embodiments, an
internal standard is used to generate a standard curve for
calculating the quantity of estradiol. Methods of generating and
using such standard curves are well known in the art and one of
ordinary skill is capable of selecting an appropriate internal
standard. For example, an isotope of estradiol may be used as an
internal standard; in certain preferred embodiments the standard is
d.sub.5-estradiol. Numerous other methods for relating the amount
of an ion to the amount of the original molecule will be well known
to those of ordinary skill in the art.
[0076] One or more steps of the methods may be performed using
automated machines. In certain embodiments, one or more
purification steps are performed on-line, and more preferably all
of the purification and mass spectrometry steps may be performed in
an on-line fashion.
[0077] In certain embodiments, such as MS/MS, where precursor ions
are isolated for further fragmentation, collision activation
dissociation is often used to generate the fragment ions for
further detection. In CAD, precursor ions gain energy through
collisions with an inert gas, and subsequently fragment by a
process referred to as "unimolecular decomposition". Sufficient
energy must be deposited in the precursor ion so that certain bonds
within the ion can be broken due to increased vibrational
energy.
[0078] In particularly preferred embodiments, estradiol is detected
and/or quantified using MS/MS as follows. The samples are subjected
to liquid chromatography, preferably HTLC followed by HPLC, the
flow of liquid solvent from the chromatographic column enters the
heated nebulizer interface of an MS/MS analyzer and the
solvent/analyte mixture is converted to vapor in the heated tubing
of the interface. The analyte (e.g., estradiol), contained in the
nebulized solvent, is ionized by the corona discharge needle of the
interface, which applies a large voltage to the nebulized
solvent/analyte mixture. The ions, e.g. precursor ions, pass
through the orifice of the instrument and enter the first
quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters,
allowing selection of ions (i.e., "precursor" and "fragment" ions)
based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is the
collision cell, where ions are fragmented. The first quadrupole of
the mass spectrometer (Q1) selects for molecules with the mass to
charge ratios of estradiol. Precursor ions with the correct
mass/charge ratios of estradiol are allowed to pass into the
collision chamber (Q2), while unwanted ions with any other
mass/charge ratio collide with the sides of the quadrupole and are
eliminated. Precursor ions entering Q2 collide with neutral argon
gas molecules and fragment. This process is called collision
activated dissociation (CAD). The fragment ions generated are
passed into quadrupole 3 (Q3), where the fragment ions of estradiol
are selected while other ions are eliminated.
[0079] The methods may involve MS/MS performed in either positive
or negative ion mode. Using standard methods well known in the art,
one of ordinary skill is capable of identifying one or more
fragment ions of a particular precursor ion of estradiol that may
be used for selection in quadrupole 3 (Q3).
[0080] If the precursor ion of estradiol includes an alcohol or
amine group, fragment ions are commonly formed that represent
dehydration or deamination of the precursor ion, respectfully. In
the case of precursor ions that include an alcohol group, such
fragment ions formed by dehydration are caused by a loss of one or
more water molecules from the precursor ion (i.e., where the
difference in mass to charge ratio between the precursor ion and
fragment ion is about 18 for the loss of one water molecule, or
about 36 for the loss of two water molecules, etc.). In the case of
precursor ions that include an amine group, such fragment ions
formed by deamination are caused by a loss of one or more ammonia
molecules (i.e. where the difference in mass to charge ratio
between the precursor ion and fragment ion is about 17 for the loss
of one ammonia molecule, or about 34 for the loss of two ammonia
molecules, etc.). Likewise, precursor ions that include one or more
alcohol and amine groups commonly form fragment ions that represent
the loss of one or more water molecules and/or one or more ammonia
molecules (i.e., where the difference in mass to charge ratio
between the precursor ion and fragment ion is about 35 for the loss
of one water molecule and the loss of one ammonia molecule).
Generally, the fragment ions that represent dehydrations or
deaminations of the precursor ion are not specific fragment ions
for a particular analyte. Accordingly, in preferred embodiments of
the invention, MS/MS is performed such that at least one fragment
ion of estradiol is detected that does not represent only a loss of
one or more water molecules and/or a loss of one or more ammonia
molecules from the precursor ion.
[0081] As ions collide with the detector they produce a pulse of
electrons that are converted to a digital signal. The acquired data
is relayed to a computer, which plots counts of the ions collected
versus time. The resulting mass chromatograms are similar to
chromatograms generated in traditional HPLC methods. The areas
under the peaks corresponding to particular ions, or the amplitude
of such peaks, are measured and the area or amplitude is correlated
to the amount of the analyte (estradiol) of interest. In certain
embodiments, the area under the curves, or amplitude of the peaks,
for fragment ion(s) and/or precursor ions are measured to determine
the amount of estradiol. As described above, the relative abundance
of a given ion may be converted into an absolute amount of the
original analyte, e.g., estradiol, using calibration standard
curves based on peaks of one or more ions of an internal molecular
standard, such as d.sub.5-estradiol.
[0082] The following examples serve to illustrate the invention.
These examples are in no way intended to limit the scope of the
methods.
EXAMPLES
Example 1
Sample and Reagent Preparation
[0083] Blood was collected in a Vacutainer with no additives and
allowed to clot 30 minutes at room temperature, 18.degree. to
25.degree. C. Samples that exhibited gross hemolysis and/or lipemia
were excluded.
[0084] An estradiol stock standard of 1 mg/mL in methanol was
prepared and further diluted in methanol to prepare an estradiol
intermediate stock standard of 1,000,000 pg/mL, which was used to
prepare two estradiol working standards of 10,000 pg/mL, diluted in
either methanol for standard A or in stripped serum for standard
B.
[0085] Deuterated methanol (methyl-d.sub.1 alcohol; Fisher Cat. No.
AC29913-1000 or equivalent) was used to prepare a 1 mg/mL
d.sub.5-estradiol stock standard (2,4,16,16,17-d.sub.5 estradiol),
which was used to prepare a 1,000,000 pg/mL intermediate stock
standard in deuterated methanol. The d.sub.5-estradiol intermediate
stock standard was used to prepare a working d.sub.5-estradiol
internal standard of 5000 pg/mL in DI water: 1 mL of the
d.sub.5-estradiol intermediate stock standard was diluted to volume
with DI water in a 200 mL volumetric flask.
[0086] A 20% formic acid solution was prepared by adding 50 mL of
formic acid (.about.98% pure Aldrich Cat. No. 06440 or equivalent)
to a 250 mL volumetric flask, which was diluted to volume with
ultrapure HPLC-grade water. A 100 mM ammonium sulfate solution in
DI water was prepared by adding 13.2 gram of ammonium sulfate
powder (CAS#7783-20-2 Fisher Cat. No: A702-500) to a 1000 mL
volumetric flask, which was diluted to volume with DI water.
[0087] All calibrators/standards used in each run were prepared
fresh weekly from series of dilutions of frozen aliquots of 10,000
pg/mL estradiol standard in stripped serum. The standards were
prepared from highest concentration to the lowest with a final
total volume for each standard of 10 mL.
Example 2
Extraction of Estradiol from Serum Using Liquid Chromatography
[0088] Liquid chromatography (LC) samples were prepared by
pipetting 200 .mu.L of standards, controls, or patient samples into
a 96-well plate. If run on positive ion mode, 300 .mu.L of 20%
formic acid were delivered to each well for a final concentration
of .about.11% (V/V). If run on negative ion mode, 300 .mu.L of 100
mM ammonium sulfate solution were added to each well for a final
concentration of .about.57 mM ammonium sulfate. In addition, 25
.mu.L of the 5000 pg/mL d.sub.5-estradiol standard were added to
each well. The samples were incubated at room temperature for 30 to
45 minutes prior to LC.
[0089] Liquid chromatography was performed with a Cohesive
Technologies Aria TX-4 HTLC system using Aria OS V 1.5 or newer
software. An autosampler wash solution was prepared using 30%
acetonitrile, 30% methanol, 30% isopropanol, and 10% acetone
(v/v).
[0090] The HTLC system automatically injected 75 .mu.L of the above
prepared samples into a TurboFlow column (50.times.1.0 mm, 60 .mu.m
Cyclone P column from Cohesive Technologies) packed with large
particles. The samples were loaded at a high flow rate (5 mL/min,
loading reagent 100% DI) to create turbulence inside the extraction
column. This turbulence ensured optimized binding of estradiol to
the large particles in the column and the passage of residual
protein and debris to waste.
[0091] Following loading, the flow direction was reversed and the
sample eluted off to the analytical column (Phenomenex analytical
column, Synergi Polar-RP.RTM. 150.times.2.0 mm, 4 .mu.m column)
with 200 .mu.L of 100% methanol in the loop. A binary HPLC gradient
was applied to the analytical column, to separate estradiol from
other analytes contained in the sample. Mobile phase A was Ultra
Pure Water (HPLC grade) and mobile phase B was 100% methanol. The
HPLC gradient started with a 40% organic gradient which ramped to
100% in approximately 5.33 minutes. The analytes eluted off the
HPLC column at 77% methanol gradient. The separated sample was then
subjected to MS/MS for quantitation of estradiol.
[0092] To determine interference with other molecules, blank sera
was spiked with 1000 pg/mL each of steroids of the same weight such
as 16-.beta. estradiol and 17-.alpha. estradiol as well as the
following steroids: estrone, estriol, testosterone, 17-.alpha.
hydroxyprogesterone, progesterone, androstenedione, 17-.beta.
estradiol glucuronide, 17-.beta. estradriol sulfate, and
dihydroxytestosterone. The samples were subject to LC. None of the
steroids co-eluted with estradiol except for 17-.alpha. estradiol,
which partially interfered with 17-.beta. estradiol but had
sufficiently different retention time to allow the operator to
determine its presence readily.
Example 3
Detection and Quantitation of Estradiol by MS/MS
[0093] MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS
system (Thermo Electron Corporation). The following software
programs all from ThermoElectron were used in the Examples
described herein: Tune Master V 1.2 or newer, Xcalibur V 2.0 SRI or
newer, TSQ Quantum 1.4 or newer, LCQuan V 2.0 or newer, and XReport
1.0 or newer. Liquid solvent analyte exiting the analytical HPLC
column flowed to the heated nebulizer interface of a Thermo
Finnigan MS/MS analyzer. The solvent/analyte mixture was converted
to vapor in the heated tubing of the interface. Analytes in the
nebulized solvent were ionized by the corona discharge needle of
the interface, which applied voltage to the nebulized
solvent/analyte mixture.
[0094] Ions passed to the first quadrupole (Q1), which selected
ions with a mass to charge ratio of either 271.14.+-.0.5 m/z or
255.07.+-.0.5 m/z. Ions entering Quadrupole 2 (Q2) collided with
argon gas to generate ion fragments, which were passed to
quadrupole 3 (Q3) for further selection. Simultaneously, the same
process using isotope dilution mass spectrometry was carried out
with an internal standard, a 5-deuterated estradiol molecule. The
following mass transitions were used for detection and quantitation
during validation on positive polarity.
TABLE-US-00001 TABLE 1 Mass Transitions for Estradiol (Positive
Polarity) Analyte Precursor Ion (m/z) Product Ion (m/z) Estradiol
255.07 133.20 &159.20 2,4,16,16,17-d.sub.5 Estradiol 260.10
135.10 &161.10
[0095] The following mass transitions were used for detection and
quantitation during validation on negative polarity.
TABLE-US-00002 TABLE 2 Mass Transitions for Estradiol (Negative
Polarity) Analyte Precursor Ion (m/z) Product Ion (m/z) Estradiol
271.14 145.10 &183.10 2,4,16,16,17-d.sub.5 Estradiol 276.15
147.10 &187.10
Example 4
Intra-Assay and Inter-Assay Precision and Accuracy
[0096] Three quality control (QC) pools were prepared from charcoal
stripped serum, spiked with estradiol to a concentration of 10,
200, and 800 pg/mL.
[0097] Ten aliquots from each of the three QC pools were analyzed
in a single assay to determine the reproducibility (CV) of a sample
within an assay. The following values were determined:
TABLE-US-00003 TABLE 3 Intra-Assay Variation and Accuracy Level I
Level II Level III (10 pg/mL) (200 pg/mL) (800 pg/mL) Mean 12 203
804 Stdev 0.8 14.8 24.2 CV 7.4% 7.3% 3.0% Accuracy 115% 102%
101%
[0098] Ten aliquots from each of the three QC pools were assayed
over 5 days to determine the reproducibility (RSD %) between
assays. The following values were determined:
TABLE-US-00004 TABLE 4 Inter-Assay Variation and Accuracy Level 1
Level II Level III (10 pg/mL) (200 pg/mL) (800 pg/mL Mean 11 209
797 Stdev 2.0 26.8 81.6 RSD (%) 17.6 12.8 10.2 Accuracy (%) 112.9
104.3 99.7
Example 5
Analytical Sensitivity: Limit of Detection (LOD) and Limit of
Quantitation (LOQ)
[0099] The estradiol zero standard was run in 10 replicates to
determine the limit of detection of the assay, which is the point
at which the measured value is larger than the uncertainty
associated with it. The LOD was defined arbitrarily as 2 standard
deviations (SD) from the zero concentration. The resulting peak
area ratios for the zero standard were statistically analyzed with
a mean value of 0.021 and a SD of 0.006. The LOD for the estradiol
assay was 2.0 pg/mL.
[0100] To determine the limit of quantitation with a precision of
20% and an accuracy of 80% to 120%, five different samples at
concentrations close to the expected LOQ were assayed and the
reproducibility determined for each. The LOQ for the estradiol
assay was defined at 2.0 pg/mL
Example 6
Assay Reportable Range and Linearity
[0101] To establish the linearity of estradiol detection in the
assay, one blank assigned as zero standard and 11 spiked serum
standards were prepared and analyzed on 5 separate days. A
quadratic regression from five consecutive runs yielded coefficient
correlations of 0.995 or greater, with an accuracy of .+-.20%
revealing a quantifiable linear range of 2 to 2000 pg/mL.
Example 7
Matrix Specificity
[0102] Matrix specificity was evaluated using water, stripped
serum, and pooled serum to determine whether patient samples could
be diluted in a linear fashion. The samples were run in duplicate
following a calibration run. The accuracy was as follows:
TABLE-US-00005 TABLE 5 Matrix Specificity Accuracy Stripped Pooled
Water Serum Serum 1:2 Dilution 120% 94% 147% 1:4 Dilution 38% 85%
94%
Example 8
Recovery
[0103] To determine the ability to recover estradiol from spiked
samples, two patient samples of known concentrations were spiked
with 3 levels (low, medium, high) of estradiol. The recovery was
calculated by dividing the spiked amount by the expected
concentration. The mean recoveries were 92%, 108% and 98% for low,
mid and high levels respectively.
[0104] The contents of the articles, patents, and patent
applications, and all other documents and electronically available
information mentioned or cited herein, are hereby incorporated by
reference in their entirety to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other physical and electronic documents.
[0105] The methods illustratively described herein may suitably be
practiced in the absence of any element or elements, limitation or
limitations, not specifically disclosed herein. Thus, for example,
the terms "comprising", "including," containing", etc. shall be
read expansively and without limitation. Additionally, the terms
and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof. It is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the invention embodied therein herein disclosed may be
resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0106] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
methods. This includes the generic description of the methods with
a proviso or negative limitation removing any subject matter from
the genus, regardless of whether or not the excised material is
specifically recited herein.
[0107] Other embodiments are within the following claims. In
addition, where features or aspects of the methods are described in
terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
* * * * *