U.S. patent application number 10/469440 was filed with the patent office on 2004-07-15 for chromatography of metal complexes.
Invention is credited to Naik, Arati, Riley, Dennis P., Slomczynska, Urszula J., Trawick, Bobby N..
Application Number | 20040137638 10/469440 |
Document ID | / |
Family ID | 32713653 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137638 |
Kind Code |
A1 |
Slomczynska, Urszula J. ; et
al. |
July 15, 2004 |
Chromatography of metal complexes
Abstract
A high performance liquid chromatography method to routinely and
reproducibly detect and quantitate metal complexes. The method
comprises loading a solution containing metal complexes onto a
column, eluting the metal complex from the column with a mobile
phase, the mobile phase comprising an excess of a salt of a
coordinating anion in a solvent system, and detecting the metal
complex with a detector. Eluting the complex from the column with
the mobile phase generates a metal complex in which the
coordinating anion (which is a competent ligand) out-competes all
other potential ligands present for the available coordination
sites on the metal. The metal complexes used in the method of the
invention can be different metal complexes, or they can be
stereoisomers of the same metal complexes. The high performance
liquid chromatography method of the present invention is suitable
for the separation of diastereomers of the same metal complexes.
Also provided is a chiral high performance liquid chromatography
method to separate enantiomers of metal complexes. In this chiral
high performance liquid chromatography method a chiral column is
employed to achieve the separation of the enantiomers of metal
complexes.
Inventors: |
Slomczynska, Urszula J.;
(Ballwin, MI) ; Trawick, Bobby N.; (Florissant,
MI) ; Riley, Dennis P.; (Chesterfield, MI) ;
Naik, Arati; (St. Louis, MI) |
Correspondence
Address: |
SONNENSCHEIN NATH & ROSENTHAL LLP
P.O. BOX 061080
WACKER DRIVE STATION, SEARS TOWER
CHICAGO
IL
60606-1080
US
|
Family ID: |
32713653 |
Appl. No.: |
10/469440 |
Filed: |
January 29, 2004 |
PCT Filed: |
March 4, 2002 |
PCT NO: |
PCT/US02/06521 |
Current U.S.
Class: |
436/161 |
Current CPC
Class: |
G01N 30/02 20130101;
G01N 30/96 20130101; G01N 30/74 20130101; B01D 15/3833 20130101;
G01N 30/02 20130101; B01D 2015/3838 20130101; G01N 30/02 20130101;
B01D 2015/3838 20130101; G01N 30/50 20130101; B01D 15/3828
20130101; G01N 2030/027 20130101; B01D 15/3833 20130101 |
Class at
Publication: |
436/161 |
International
Class: |
G01N 030/02 |
Claims
What is claimed is:
1. A high performance liquid chromatography method comprising:
loading a solution containing metal complexes onto a column,
wherein the metal complexes are selected from a group consisting of
superoxide dismutase mimetic compounds, MRI imaging enhancement
agents, catalase mimics, and peroxynitrite decomposition catalysts,
eluting the metal complexes from the column with a mobile phase,
said mobile phase comprising an excess of a salt of a coordinating
anion in a solvent system, and detecting the metal complexes with a
detector.
2. The method of claim 1 wherein the metal complexes comprise
superoxide dismutase mimetic compounds.
3. The method of claim 1 wherein the metal complexes comprise
different metal complexes.
4. The method of claim 1 wherein the metal complexes comprise
stereoisomers of the same metal complex.
5. The method of claim 1 wherein the metal complexes comprise
diastereomers of the same metal complex.
6. The method of claim 1 wherein the metal complexes comprise
enantiomers of the same metal complex.
7. The method of claim 1 wherein the metal complexes comprise
products of a reaction stream.
8. The method of claim 1 wherein the metal complexes are selected
from a group consisting of Fe.sup.III(salen) complexes,
Fe.sup.II(1,4,7,10,13-pe- ntaazacyclopentadecane) derivatives,
Fe.sup.III(porphyrinato) complexes, Mn.sup.III(porphyrinato)
complexes, M(salen) complexes, and
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
9. The method of claim 1 wherein the metal complexes are selected
from a group consisting of Mn.sup.III(porphyrinato) complexes,
Mn.sup.III(salen) complexes, and
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
10. The method of claim 1 wherein the metal complexes comprise
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
11. The method of claim 1 wherein the metal complexes comprise
stereoisomers of a Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane)
complex.
12. The method of claim 1 wherein the metal complexes comprise
stereoisomers of a metal complex having the following structure:
16
13. The method of claim 1 wherein the metal complexes comprise
diastereomers of a
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane)complex- .
14. The method of claim 1 wherein the metal complexes comprise
enantiomers of a
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane)complex.
15. The method of claim 14 wherein the enantiomers are selected
from the following structures: 17
16. The method of claim 1 wherein the coordinating anions comprise
chloride anions, thiocyanate anions, stearate anions, acetate
anions, trifluoroacetate anions, carboxylate anions, formate anions
or azide anions.
17. The method of claim 1 wherein the salt comprises sodium
chloride, lithium chloride, potassium chloride, ammonium chloride,
tetrabutylammonium chloride, sodium thiocyanate, potassium
thiocyanate, ammonium thiocyanate, lithium thiocyanate, potassium
acetate, sodium acetate, ammonium acetate, ammonium
trifluoroacetate, lithium acetate, potassium formate, sodium
formate, ammonium formate, lithium formate, sodium cyanate,
potassium cyanate, ammonium cyanate, potassium carboxylate, sodium
carboxylate, lithium stearate, sodium stearate, sodium azide,
potassium azide or lithium azide.
18. The method of claim 1 wherein the salt comprises sodium
chloride, lithium chloride or tetrabutylammonium chloride.
19. The method of claim 1 wherein the salt comprises ammonium
thiocyanate, sodium thiocyanate or potassium thiocyanate.
20. The method of claim 1 wherein the salt comprises a mixture of
salts.
21. The method of claim 20 wherein the mixture of salts comprises
two or more of sodium chloride, lithium chloride, potassium
chloride, ammonium chloride, tetrabutylammonium chloride, sodium
thiocyanate, potassium thiocyanate, ammonium thiocyanate, lithium
thiocyanate, potassium acetate, sodium acetate, ammonium acetate,
ammonium trifluoroacetate, lithium acetate, potassium formate,
sodium formate, ammonium formate, lithium formate, sodium cyanate,
potassium cyanate, ammonium cyanate, potassium carboxylate, sodium
carboxylate, lithium stearate, sodium stearate, sodium azide,
potassium azide, or lithium azide.
22. The method of claim 1 wherein the solvent system comprises a
solvent.
23. The method of claim 22 wherein the solvent comprises
acetonitrile, dioxane, ethanol, methanol, isopropanol,
tetrahydrofuran, or water.
24. The method of claim 1 wherein the solvent system comprises a
mixture of solvents.
25. The method of claim 24 wherein the mixture of solvents
comprises two or more of acetonitrile, dioxane, ethanol, methanol,
isopropanol, tetrahydrofuran, and water.
26. The method of claim 25 wherein the solvents comprise
acetonitrile and water.
27. The method of claim 25 wherein the solvents comprise methanol
and water.
28. The method of claim 1 wherein the salt is present in the mobile
phase at a concentration of between about 0.004 M to about 6 M.
29. The method of claim 1 wherein the salt is present in the mobile
phase at a concentration of between about 0.1 M to about 1 M.
30. The method of claim 1 wherein the salt is present in the mobile
phase at a concentration of between about 0.15 M to about 0.6
M.
31. The method of claim 18 wherein the sodium chloride is present
in the mobile phase at a concentration of between about 0.1 M to
about 1 M.
32. The method of claim 18 wherein the sodium chloride is present
in the mobile phase at a concentration of between about 0.3 M to
about 0.7 M.
33. The method of claim 18 wherein the sodium chloride is present
in the mobile phase at a concentration of between about 0.4 M to
about 0.6 M.
34. The method of claim 18 wherein the tetrabutylammonium chloride
is present in the mobile phase at a concentration of between about
0.005 M to about 0.15 M.
35. The method of claim 18 wherein the tetrabutylammonium chloride
is present in the mobile phase at a concentration of between about
0.01 M to about 0.13 M.
36. The method of claim 18 wherein the tetrabutylammonium chloride
is present in the mobile phase at a concentration of between about
0.05 M to about 0.125 M.
37. The method of claim 1 wherein the mobile phase comprises
acetonitrile in water containing between about 0.1 M to about 0.7 M
of salt.
38. The method of claim 1 wherein the mobile phase comprises
methanol in water containing between about 0.15 M to about 0.6 M of
salt.
39. The method of claim 1 wherein the mobile phase comprises
acetonitrile containing between about 0.3 M to about 0.7 M of
sodium chloride.
40. The method of claim 1 wherein the mobile phase comprises 5-15%
acetonitrile in water containing between about 0.01 M to about 0.13
M of tetrabutylammonium chloride.
41. The method of claim 1 wherein the mobile phase comprises 5-15%
acetonitrile in water containing between about 0.01 M to about 0.13
M of tetrabutylammonium chloride and between about 0.3 M to about
0.7 M lithium chloride.
42. The method of claim 1 wherein the mobile phase comprises 5-10%
acetonitrile in water containing between about 0.4 M to about 0.6 M
of sodium chloride.
43. The method of claim 1 wherein the mobile phase comprises 5-10%
acetonitrile in water containing between about 0.05 M to about
0.125 M of tetrabutylammonium chloride.
44. The method of claim 1 wherein the mobile phase comprises 5-10%
acetonitrile in water containing between about 0.05 M to about
0.125 M of tetrabutylammonium chloride and between about 0.4 M to
about 0.6 M lithium chloride.
45. The method of claim 1 wherein the mobile phase comprises 1-5%
methanol in water containing between about 0.1 M to about 2.5 M of
ammonium thiocyanate.
46. The method of claim 1 wherein the mobile phase comprises 1-5%
methanol in water containing between about 0.05 M to about 0.3 M of
tetrabutylammonium chloride.
47. The method of claim 1 wherein the mobile phase comprises 1-5%
methanol in water containing between about 0.2 M to about 0.3 M of
ammonium thiocyanate.
48. The method of claim 1 wherein the mobile phase comprises 1-5%
methanol in water containing between about 0.05 M to about 0.15 M
of tetrabutylammonium chloride.
49. The method of claim 1 wherein the column is selected from the
group consisting of a C1 modified column, a C3 modified column, a
C4 modified column, an octyl (C8) modified column, an octadecyl
(C18) modified column, a C18 polymer column, a phenyl column, and
an amino-cyano column.
50. The method of claim 1 wherein the column is selected from the
group consisting of an octadecyl column, a phenyl column, and an
amino-cyano column.
51. The method of claim 1 wherein the column comprises an octadecyl
column.
52. The method of claim 51 wherein the octadecyl column comprises a
YMC ODS-AQ S5 column.RTM., a Vydac column.RTM., or a Symmetry
Shield RP.sub.18 column.RTM..
53. The method of claim 1 wherein the column comprises a chiral
column.
54. The method of claim 53 wherein the chiral column comprises a
cellulose column or a Pirkle column.
55. The method of claim 54 wherein the cellulose column comprises a
Chiralcel-OD-RH column.RTM..
56. The method of claim 1 wherein the detector comprises a UV
detector.
57. A high performance liquid chromatography method comprising:
loading a solution containing metal complexes onto a column,
wherein the metal complexes are selected from a group consisting of
superoxide dismutase mimetic compounds, MRI imaging enhancement
agents, catalase mimics, and peroxynitrite decomposition catalysts,
eluting the metal complexes from the column with a mobile phase,
said mobile phase comprising an excess of a salt of a coordinating
anion in a solvent system, wherein eluting with said mobile phase
drives the substantial formation of metal complexes containing the
coordinating anion as ligands, and detecting the metal complexes
with a detector.
58. The method of claim 57 wherein the metal complexes comprise
superoxide dismutase mimetic compounds.
59. The method of claim 57 wherein the metal complexes are selected
from a group consisting of Fe.sup.III(salen) complexes,
Fe.sup.II(1,4,7,10,13-pe- ntaazacyclopentadecane) derivatives,
Fe.sup.III(porphyrinato) complexes, Mn.sup.III(porphyrinato)
complexes, M.sup.III(salen) complexes, and
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
60. The method of claim 57 wherein the metal complexes comprise
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
61. The method of claim 57 wherein the metal complexes comprise
stereoisomers of a Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane)
complex.
62. The method of claim 57 wherein the metal complexes comprise
stereoisomers of a metal complex having the following structure:
18
63. The method of claim 57 wherein the salt comprises sodium
chloride, lithium chloride, potassium chloride, ammonium chloride,
tetrabutylammonium chloride, sodium thiocyanate, potassium
thiocyanate, ammonium thiocyanate, lithium thiocyanate, potassium
acetate, sodium acetate, ammonium acetate, ammonium
trifluoroacetate, lithium acetate, potassium formate, sodium
formate, ammonium formate, lithium formate, sodium cyanate,
potassium cyanate, ammonium cyanate, potassium carboxylate, sodium
carboxylate, lithium stearate, sodium stearate, sodium azide,
potassium azide, or lithium azide.
64. The method of claim 57 wherein the salt comprises sodium
chloride, lithium chloride or tetrabutylammonium chloride, ammonium
thiocyanate or potassium thiocyanate.
65. The method of claim 57 wherein the salt is present in the
mobile phase at a concentration of between about 0.1 M to about 1
M.
66. The method of claim 57 wherein the salt is present in the
mobile phase at a concentration of between about 0.15 M to about
0.6 M.
67. The method of claim 57 wherein the column comprises a chiral
column.
68. A high performance liquid chromatography method comprising:
loading a solution containing metal complexes onto a column,
wherein the metal complexes are selected from a group consisting of
manganese complexes and iron complexes, eluting the metal complexes
from the column with a mobile phase, said mobile phase comprising
an excess of a salt of a coordinating anion in a solvent system,
and detecting the metal complexes with a detector.
69. The method of claim 68 wherein the metal complexes are selected
from a group consisting of superoxide dismutase mimetic compounds,
MRI imaging enhancement agents, catalase mimics, and peroxynitrite
decomposition catalysts.
70. The method of claim 68 wherein the metal complexes comprise
superoxide dismutase mimetic compounds.
71. The method of claim 68 wherein the metal complexes are selected
from a group consisting of Fe.sup.II(salen) complexes,
Fe.sup.III(1,4,7,10,13-pe- ntaazacyclopentadecane) derivatives,
Fe.sup.II(porphyrinato) complexes, Mn.sup.III(porphyrinato)
complexes, Mn.sup.III(salen) complexes, and
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
72. The method of claim 68 wherein the metal complexes are selected
from a group consisting of Mn.sup.III(porphyrinato) complexes,
Mn.sup.III(salen) complexes, and
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
73. The method of claim 68 wherein the metal complexes comprise
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
74. The method of claim 68 wherein the metal complexes comprise
stereoisomers of a Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane)
complex.
75. The method of claim 68 wherein the metal complexes comprise
stereoisomers of a metal complex having the following structure:
19
76. The method of claim 68 wherein the salt comprises sodium
chloride, lithium chloride, potassium chloride, ammonium chloride,
tetrabutylammonium chloride, sodium thiocyanate, potassium
thiocyanate, ammonium thiocyanate, lithium thiocyanate, potassium
acetate, sodium acetate, ammonium acetate, ammonium
trifluoroacetate, lithium acetate, potassium formate, sodium
formate, ammonium formate, lithium formate, sodium cyanate,
potassium cyanate, ammonium cyanate, potassium carboxylate, sodium
carboxylate, lithium stearate, sodium stearate, sodium azide,
potassium azide, or lithium azide.
77. The method of claim 68 wherein the salt comprises sodium
chloride, lithium chloride, tetrabutylammonium chloride, sodium
thiocyanate or potassium thiocyanate.
78. The method of claim 68 wherein the salt is present in the
mobile phase at a concentration of between about 0.1 M to about 1.0
M.
79. The method of claim 68 wherein the salt is present in the
mobile phase at a concentration of between about 0.15 M to about
0.6 M.
80. A high performance liquid chromatography method for the
analysis of metal complexes comprising: combining metal complexes
with an excess of a salt of a coordinating anion in an aqueous
solution, loading a solution containing the metal complexes onto a
column, eluting the metal complexes from the column with a mobile
phase, said mobile phase comprising an excess of a salt of a
coordinating anion in a solvent system, and detecting the metal
complexes with a detector.
81. The method of claim 80 wherein the salt comprises sodium
chloride, lithium chloride, potassium chloride, ammonium chloride,
tetrabutylammonium chloride, sodium thiocyanate, potassium
thiocyanate, ammonium thiocyanate, lithium thiocyanate, potassium
acetate, sodium acetate, ammonium acetate, ammonium
trifluoroacetate, lithium acetate, potassium formate, sodium
formate, ammonium formate, lithium formate, sodium cyanate,
potassium cyanate, ammonium cyanate, potassium carboxylate, sodium
carboxylate, lithium stearate, sodium stearate, sodium azide,
potassium azide, or lithium azide.
82. The method of claim 80 wherein the salt comprises sodium
thiocyanate, potassium thiocyanate, ammonium thiocyanate, or
lithium thiocyanate.
83. The method of claim 80 wherein the metal complexes are selected
from a group consisting of superoxide dismutase mimetic compounds,
catalase mimics, peroxynitrite decomposition catalysts, and MRI
imaging agents.
84. The method of claim 80 wherein the metal complexes comprise
superoxide dismutase mimetic compounds.
85. The method of claim 80 wherein the metal complexes are selected
from a group consisting of Fe.sup.III(salen) complexes,
Fe.sup.III(1,4,7,10,13-p- entaazacyclopentadecane) derivatives,
Fe.sup.II(porphyrinato) complexes, Mn.sup.II(porphyrinato)
complexes, Mn.sup.III(salen) complexes, and
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
86. The method of claim 80 wherein the metal complexes comprise
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
87. A high performance liquid chromatography method comprising:
loading a solution containing metal complexes onto a column,
eluting the metal complexes from the column with a mobile phase,
said mobile phase comprising an excess of a salt of a coordinating
anion in a solvent system, wherein eluting with said mobile phase
drives the formation of metal complexes containing the coordinating
anion as ligands, and detecting the metal complexes with a
detector.
88. The method of claim 87 wherein the metal complexes are selected
from the group consisting of superoxide dismutase mimetic
compounds, MRI imaging enhancement agents, catalase mimics, and
peroxynitrite decomposition catalysts.
89. The method of claim 87 wherein the metal complexes are selected
from a group consisting of Fe.sup.III(salen) complexes,
Fe.sup.II(1,4,7,10,13-pe- ntaazacyclopentadecane) derivatives,
Fe.sup.III(porphyrinato) complexes, Mn.sup.III(porphyrinato)
complexes, Mn.sup.III(salen) complexes, and
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) complexes.
90. The method of claim 87 wherein the metal complexes have the
following structure: 20
Description
FIELD OF THE INVENTION
[0001] This invention relates to a high performance liquid
chromatography method for the analytical detection and
quantification of metal complexes which have utility in
pharmaceutical and diagnostic applications.
BACKGROUND OF THE INVENTION
[0002] Superoxide dismutase (SOD) enzymes are enzymes that catalyze
the dismutation of the free radical superoxide, the one-electron
reduction product of molecular oxygen. The dismutation of the free
radical superoxide involves the conversion of this one-electron
reduction product of molecular oxygen to the nonradical molecular
oxygen. Superoxide dismutase enzymes are a class of oxidoreductases
which contain either Cu/Zn, Fe, or Mn at the active site.
Superoxide dismutase (SOD) mimetic compounds are low molecular
weight catalysts which mimic the natural enzyme function of the
superoxide dismutase enzymes. Thus, superoxide dismutase mimetic
compounds also catalyze the conversion of superoxide into oxygen
and hydrogen peroxide, rapidly eliminating the harmful biologically
generated superoxide species that are believed to contribute to
tissue pathology in a number of diseases and disorders. These
diseases and disorders include reperfusion diseases, such as those
following myocardial infarct or stroke, inflammatory disorders such
as arthritis, and neurological disorders such as Parkinson's
disease. Chem Reviews, 1999 vol 99, No. 9, 2573-2587.
[0003] Superoxide dismutase mimetic compounds possess several
advantages over the superoxide dismutase enzymes themselves in that
their chemical properties can be altered to enhance stability,
activity and biodistribution while still possessing the ability to
dismutase the harmful superoxide. Superoxide dismutase mimetic
compounds have generated intense interest and have been the focus
of considerable efforts to develop them as a therapeutic agent for
the treatment of a wide range of diseases and disorders, including
reperfusion injury, ischemic myocardium post-ischemic neuropathies,
inflammation, organ transplantation and radiation induced injury.
Most of the superoxide dismutase mimics currently being developed
as therapeutic agents are synthetic low molecular weight
manganese-based superoxide dismutase mimetic compounds. Chem
Reviews, 2576.
[0004] Superoxide dismutase mimetic compounds are metal complexes
in which the metal can coordinate axial ligands. Examples of such
metal complexes include, but are not limited to, complexes of the
metals Mn and Fe. Many of the complexes of the metals Mn and Fe do
not possess superoxide dismutase activity but possess properties
that enable them to be put to other therapeutic and diagnostic
uses. These therapeutic and diagnostic uses include MRI imaging
enhancement agents, peroxynitrite decomposition catalysts, and
catalase mimics. These metal complexes, however, share the
structural similarity of possessing a metal that can coordinate
exchangeable ligands. These metal complexes exist in water as a
mixture of species in which various ligands are possible. An
illustration of such a mixture is provided by M40403, a Mn(II)
complex of a nitrogen-containing fifteen membered macrocyclic
ligand, shown in Scheme 1. One of the forms for this metal complex
is the dichloro complex, which when dissolved in water another form
is generated where one of the chloride anions immediately
dissociates from the metal generating the [Mn(Cl)(aquo)]+ complex.
The problem in aqueous solvent systems or any solvent which has a
potential donor atom is that there are a variety of potential
ligands available to coordinate axially to the Mn(II) ion of the
complex. In conducting an analysis of a sample containing a metal
complex by high performance liquid chromatography (HPLC) the
chromatogram tends to be very broad and unresolved due to the
presence of the various species of complexes, as shown in Scheme 1.
This phenomena makes the identification and quantification of metal
complexes by standard HPLC techniques quite difficult. Therefore,
in light of the developing roles of metal complexes as therapeutics
in the treatment of various disorders and diagnostic agents, a
substantial need exists for an effective and workable high
performance liquid chromatography method for analyzing metal
complexes. 1
[0005] An additional complication which exists is the issue of the
acid stability of the metal complex. As the pH decreases, the rate
at which the complex becomes protonated and experiences instability
increases. This presents particular problems for the use of HPLC as
a method of detection and quantification of the metal complexes
because the mobile phase used for reverse phase HPLC frequently
contains mixtures of organic solvents and water in various
combinations with trifluoroacetic acid. The trifluoroacetic acid is
commonly present between about 0.1 to about 0.5% by weight. The
presence of the trifluoroacetic acid causes the complex to
dissociate. This dissociation destroys the potential of any such
method to be used for release testing for purity. Furthermore, the
trifluoroacetate anion causes the formation of some of the
trifluoroacetato complex which could possess a different retention
time from the chloro complexes thus, confusing the chromatography.
Thus, the phenomenon of ligand exchange, coupled with the acid
instability of the metal complexes, provides considerable
challenges to the effort to detect and quantify metal complexes
using HPLC. These challenges and needs have surprisingly been met
by the invention described below.
[0006] Analytical HPLC is a powerful method to obtain information
about a sample compound including information regarding
identification, quantification and resolution of a compound. HPLC
has been used particularly for the analysis of larger compounds and
for the analysis of inorganic ions for which liquid chromatography
is unsuitable. Skoog, D. A., West, M. A., Analytical Chemistry,
1986, p. 520. As an analytical tool HPLC takes advantage of the
differences in affinity that a particular compound of interest has
for the stationary phase and the mobile phase (the solvent being
continuously applied to the column). Those compounds having
stronger interactions with the mobile phase than with the
stationary phase will elute from the column faster and thus have a
shorter retention time. The mobile phase can be altered in order to
manipulate the interactions of the target compound and the
stationary phase. In normal-phase HPLC the stationary phase is
polar, such as silica, and the mobile phase is a nonpolar solvent
such as hexane or isopropyl ether. In reversed-phase HPLC the
stationary phase is non-polar, often a hydrocarbon, and the mobile
phase is a relatively polar solvent. Since 1974 when reversed-phase
packing materials became commercially available, the number of
applications for reversed-phase HPLC has grown, and reversed-phase
HPLC is now the most widely used type of HPLC. Reversed-phase
HPLC's popularity can be attributed to its ability to separate a
wide variety of organic compounds. Reversed-phase chromatography is
especially useful in separating the related components of reaction
mixtures, and therefore is a useful analytical tool for determining
the various compounds produced by reactions.
[0007] To create a non-polar stationary phase silica or synthetic
polymer based adsorbents are modified with hydrocarbons. The most
popular bonded phases are C1, C4, C8 and C18. Silica based
adsorbents modified with trimethylchlorosilane (C1) and
butyldimethylchlorosilane (C4) have a few applications in HPLC,
mainly for protein separation or purification. These adsorbents
show significant polar interactions. Octyl (C8) and octadecyl (C18)
modified adsorbents are the most widely used silica based
adsorbents, with almost 80% of all HPLC separations being developed
with these adsorbents.
[0008] The most important parameter in reversed-phase HPLC is the
mobile phase. The type of mobile phase employed in the HPLC will
have a significant effect on the retention of the analytes in the
sample, and varying the composition of the mobile phase allows the
chromatographer to adjust the retention times of target components
in the mixture to desired values. This ability provides the HPLC
method with flexibility. The mobile phase in reversed-phase
chromatography has to be polar and it also has to provide
reasonable competition for the adsorption sites for the analyte
molecules. Solvents that are commonly employed as eluent components
in reversed-phase HPLC are acetonitrile, dioxane, ethanol,
methanol, isopropanol, tetrahydrofuran, and water. In reversed
phase HPLC of high molecular weight biological compounds, the
solvents acetonitrile, isopropanol or propanol are most frequently
used. Popular additives to the mobile phase for the improvement of
resolution include mixtures of phosphoric acid and amines and
perfluorinated carboxylic acids, especially trifluoroacetic acid
(TFA).
[0009] HPLC exploits the differences in affinity that a particular
compound of interest has for the stationary phase and the mobile
phase. This phenomenon can be utilized to separate compounds based
on the differences in their physical properties. Thus, HPLC can be
used to separate stereoisomers, diastereomers, enantiomers, mirror
image stereoisomers, and impurities. Stereoisomers are those
molecules which differ from each other only in the way their atoms
are oriented in space. The particular arrangement of atoms that
characterize a particular stereoisomer is known as its optical
configuration, specified by known sequencing rules as, for example,
either + or - (also D or L) and/or R or S. Stereoisomers are
generally classified as two types, enantiomers or diastereomers.
Enantiomers are stereoisomers which are mirror-images of each
other. Enantiomers can be further classified as mirror-image
stereoisomers that cannot be superimposed on each other and
mirror-image stereoisomers that can be superimposed on each other.
Mirror-image stereoisomers that can be superimposed on each other
are known as meso compounds. Diastereomers are stereoisomers that
are not mirror images of each other. Diastereomers have different
physical properties such as melting points, boiling points,
solubilities in a given solvent, densities, refractive indices,
etc. Diastereomers can usually be readily separated from each other
by conventional methods, such as fractional distillation,
fractional crystallization, or chromatography, including HPLC.
[0010] Enantiomers, however, present special challenges because
their physical properties are identical. They generally cannot be
separated by conventional methods, especially if they are in the
form of a racemic mixture. Thus, they cannot be separated by
fractional distillation because their boiling points are identical
and they cannot be separated by fractional crystallization because
their solubilites are identical (unless the solvent is optically
active). They also cannot be separated by conventional
chromatography such as HPLC because (unless the adsorbent is
optically active) they are held equally onto the adsorbent. HPLC
methods employing chiral stationary phases are a very common
approach to the separation of enantiomers. To be able to separate
racemic mixtures of stereoisomers, the chiral phase has to form a
diastereomeric complex with one of the isomers, or has to have some
other type of stereospecific interaction. The exact mechanism of
chiral recognition is not yet completely understood. In
reversed-phase HPLC a common type of chiral bonded phase is chiral
cavity phases.
[0011] The ability to be able to separate diastereomers and
enantiomers by HPLC is a useful ability in evaluating the success
of synthetic schemes. It is often desirable to separate
stereoisomers as a means of evaluating the enantiomeric purity of
production samples. All references listed herein are hereby
incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to a high performance
liquid chromatography method to routinely and reproducibly detect
and quantitate metal complexes. The method comprises loading a
solution containing metal complexes onto a column, eluting the
metal complex from the column with a mobile phase, the mobile phase
comprising an excess of a salt of a coordinating anion in a solvent
system, and detecting the metal complex with a detector. Eluting
the complex from the column with the mobile phase generates a metal
complex in which the coordinating anion (which is a competent
ligand) out-competes all other potential ligands present for the
available coordination sites on the metal. Thus, the role of this
ligand is to, by the principles of mass action, occupy all the
available ligand sites, creating one species. The metal complexes
used in the method of the invention can be different metal
complexes, or they can be stereoisomers of the same metal
complexes. Thus, the HPLC method of the present invention is
suitable for the separation of diastereomers of the same metal
complexes.
[0013] Another embodiment of the present invention is directed to a
chiral HPLC method to separate enantiomers of metal complexes. In
this chiral HPLC method a chiral column is employed to achieve the
separation of the enantiomers of metal complexes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a HPLC chromatogram of M40403 using method 1.
[0015] FIG. 2 is a HPLC chromatogram of M40403 using method 2.
[0016] FIG. 3 is a HPLC chromatogram of M40403 using method 3.
[0017] FIG. 3a is a HPLC chromatogram of M40403 and related
compounds using method 3.
[0018] FIG. 4 is HPLC chromatogram of M40403 using method 4.
[0019] FIG. 4a is a HPLC chromatogram of M40403 and related
compounds using method 4.
[0020] FIG. 5a is a HPLC chromatogram of M40401 using method 1.
[0021] FIG. 6 is a HPLC chromatograms of M40401 with various NaCl
concentrations.
[0022] FIG. 7 is a HPLC chromatogram of M40401 using method 2.
[0023] FIG. 8 is a HPLC chromatogram of M40401 using method 3.
[0024] FIG. 9 is a HPLC chromatogram of M40401 using method 4.
[0025] FIG. 9a is a HPLC chromatogram of a mixture of M40401 and
related compounds.
[0026] FIG. 10 is a HPLC chromatogram of M40403-(HCOO.sup.-).sub.2
using formate anion.
[0027] FIG. 11 is a HPLC chromatogram of
M40403-(CH.sub.3COO.sup.-).sub.2 using acetate anion.
[0028] FIG. 12 is a HPLC Analysis of Diastereomers of M40403.
[0029] FIG. 13 is a Chiral HPLC profiles of the M40403 and M40419
bis(thiocyanato) enantiomers.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The methods of the present invention provide an excess of a
coordinating counterion in a mobile phase which will bind to the
ligand sites on the metal of metal complexes. Solutions containing
metal complexes are loaded onto a column and the metal complexes
are eluted with the mobile phase. By inclusion of the excess of the
counterion, the reaction is driven toward generating a single
species during the elution with the mobile phase so that only one
type of ligand, the counterion, is bound to the metal of the metal
complex and all or substantially all the ligand binding sites of
the metal are occupied by this one counterion. The formation of the
single species is shown in Scheme 2. The formation of a single
species of the metal complex(es) present allows the metal complexes
to be reliably detected by a detector without interference from
other species of complexes. The peaks on a chromatogram resulting
from this detection are sharper and more resolved than those of a
chromatogram resulting from a chromatography method in which a
traditional mobile phase is employed, as demonstrated in Examples
l(traditional mobile phase) and 2 (mobile phase containing excess
of salt of a coordinating anion). 2
[0031] Any metal complex possessing a metal that is capable of
coordinating a monodentate ligand can be used in the present
invention. Examples of such metal complexes include, but are not
limited to, complexes of the metals Mn and Fe. The metal complexes
of the invention preferably have therapeutic and diagnostic
utilities. These therapeutic and diagnostic utilities include, but
are not limited to, use as superoxide dismutase mimetic compounds,
MRI imaging enhancement agents, peroxynitrite decomposition
catalysts, and catalase mimics. The preferred metal complexes for
use in the invention are superoxide dismutase mimetic compounds.
Examples of such superoxide dismutase mimetic compounds include,
but are not limited to, the following complexes of the metals Mn
and Fe. Iron based superoxide dismutase millimetics include, but
are not limited to, Fe.sup.III(salen) complexes,
Fe.sup.II(1,4,7,10,13-pentaazacy- clopentadecane) derivatives and
Fe.sup.II(porphyrinato) complexes. Manganese based superoxide
dismutase mimetic compounds include, but are not limited to, metal
complexes containing manganese(II) or manganese(III). Examples of
manganese based superoxide dismutase mimetic compounds include
Mn.sup.III(porphyrinato) complexes, Mn.sup.III(salen) complexes,
and Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) derivatives.
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) derivatives are
more preferred for use in the invention. Examples of
Mn.sup.II(1,4,7,10,13-pentaazacyclopentadecane) derivatives
preferred for use in the invention include, but are not limited to,
M40403 and M40401, as shown in Scheme 3 below.
[0032] Furthermore, stereoisomers of all of the above metal
complexes can be used in the process of the present invention.
Diastereomers of the same metal complexes can also be detected and
separated by the method of the present invention. As it is often
desirable to separate stereoisomers as a means of evaluating the
chemical and optical purity of production samples, the metal
complexes can also comprise products of a reaction stream.
Enantiomers of any of the metal complexes referenced above can be
used in the chiral HPLC method of the invention for the separation
of enantiomers of a metal complex. 3
[0033] The ligand is a coordinating anion that binds to the metal
cation of the metal complex. The coordinating anion can serve as an
axial ligand for a superoxide dismutase mimetic compound. Examples
of such anions include, but are not limited to, chloride anions,
thiocyanate anions, stearate anions, acetate anions,
trifluoroacetate anions, carboxylate anions, formate anions, or
azide anions. Preferred anions include chloride anions, thiocyanate
anions, and formate anions. More preferred anions are chloride
anions. The more preferred anions in the chiral HPLC embodiment of
the invention are thiocyanate anions. When present in an excess,
the thiocyanate anions bind to the coordinating metal of the
complexes preferentially to the chloride anions. An excess of
thiocyanate anions will produce the bis(thiocyanato) complexes of
M40403 and M40419 as shown in Scheme 4. 4
[0034] An example of the use of the acetate anion as the
coordinating anion with M40403 is shown in Scheme 5 below. Scheme 6
illustrates the use of the formate anion as the coordinating anion
with M40403. 5 6
[0035] The coordinating anion is supplied by a salt of the
coordinating anion. Salts of the chloride anion include, but are
not limited to, sodium chloride, lithium chloride, potassium
chloride, ammonium chloride, or tetraalkylammonium chloride.
Preferred salts of the chloride anion include sodium chloride,
lithium chloride and tetrabutylammonium chloride. Salts of the
thiocyanate anion include, but are not limited to, sodium
thiocyanate, potassium thiocyanate, ammonium thiocyanate, or
lithium thiocyanate. Preferred salts of the thiocyanate anion
include sodium thiocyanate and potassium thiocyanate. Salts of the
acetate anion include, but are not limited to, potassium acetate,
sodium acetate, ammonium acetate, ammonium trifluoroacetate and
lithium acetate. Preferred salts of the acetate anion include
ammonium acetate. Salts of the formate anion include, but are not
limited to, potassium formate, sodium formate, ammonium formate and
lithium formate. Preferred salts of the formate anion include
ammonium formate. Salts of the cyanate anion include but are not
limited to, sodium cyanate, potassium cyanate, or ammonium cyanate.
Salts of the carboxylate anion include, but are not limited to,
potassium carboxylate, ammonium carboxylate and sodium carboxylate.
Salts of the stearate anion include, but are not limited to,
lithium stearate and sodium stearate. Salts of the azide anion
include, but are not limited to, sodium azide, potassium azide, and
lithium azide. The salt added to the mobile phase can also be a
mixture of any of these salts. Examples include a mixture of
tetrabutylammonium chloride and lithium chloride.
[0036] The solvent system can comprise any solvent employed in HPLC
procedures. The solvent system can comprise a single solvent or a
mixture of solvents as long as salts of the coordinating anion are
soluble in the solvent system. Examples of suitable solvents
include, but are not limited to, acetonitrile, dioxane, ethanol,
methanol, isopropanol, tetrahydrofuran, and water. Preferred
solvents for all embodiments of the invention include acetonitrile,
isopropanol, propanol, water, and methanol. More preferred solvents
are acetonitrile, water and methanol. Suitable mixtures of solvents
can be, for example, mixtures of acetonitrile and water or mixtures
of methanol and water. The more preferred solvent for the chiral
HPLC embodiment of the invention is methanol.
[0037] The solvent system containing the excess of salt and
optionally a base or acid for pH adjustment comprises the mobile
phase. The composition of the mobile phase is important to the
success of the method of the present invention. The amount of the
salt of the coordinating anion should be in sufficient excess for
the coordinating anion to saturate substantially all, and
preferably, all of the exchangeable ligand binding sites on the
metal of the metal complexes, driving the formation of a single
species during elution with the mobile phase. The single species is
substantially formed during elution with the mobile phase. A
substantial formation of a single species, a complex in which the
coordinating anion of the salt comprises the ligands, is that
amount of single species that produces enhanced resolution and
improved peak shape in a chromatogram compared to a chromatogram
from an HPLC in which either no salt or an insufficient amount of
salt has been used in the mobile phase. This enhanced resolution
and improved peak shape is illustrated in the FIG. 2 chromatogram
of Example 2 and should occur without the metal complex
dissociating. An example of a chromatogram from an HPLC in which
either no salt or an insufficient amount of salt has been used in
the mobile phase is FIG. 1 in Example 1.
[0038] The salt of the coordinating anion is present in excess in
the mobile phase. The concentration of salt in the mobile phase can
vary considerably, depending on the composition of the mobile phase
and the particular type of salt employed. Upper limits on the
concentration of salt in the mobile phase are set by the solubility
of the salt in the mobile phase. Lower limits on the concentration
of salt in the mobile phase are set by the concentration of salt
that provides an amount of a coordinating anion sufficient to cause
the formation of a metal complex possessing substantially only the
coordinating anion as ligands. The lower limit of the concentration
of salt in the mobile phase is at least greater than a
stoichiometrical amount. Preferably, the salt is present in the
mobile phase at a concentration that maximizes the formation of a
single species. Generally, however, the concentration of the salt
in the mobile phase varies from between about 0.004 M to about 6 M.
Preferably, the concentration of the salt in the mobile phase
varies from between about 0.1 M to about 1 M. More preferably, the
concentration of the salt in the mobile phase varies from between
about 0.15 M to about 0.6 M.
[0039] These ranges will differ depending upon the type of salt
employed in the mobile phase. For example, the concentration of
sodium chloride in the mobile phase ranges from between about 0.1 M
to about 1 M NaCl. Preferably, the concentration of sodium chloride
in the mobile phase ranges from between about 0.3 M to about 0.7 M.
More preferably, the concentration of sodium chloride in the mobile
phase ranges from between about 0.4 to about 0.6 M. The
concentration of lithium chloride in the mobile phase ranges from
between about 0.1 M to about 1 M NaCl. Preferably, the
concentration of lithium chloride in the mobile phase ranges from
between about 0.3 M to about 0.7 M. More preferably, the
concentration of lithium chloride in the mobile phase ranges from
between about 0.4 M to about 0.6 M. The concentration of
tetrabutylammonium chloride in the mobile phase ranges from between
about 0.005 M to about 0.15 M. Preferably, the concentration of
tetrabutylammonium chloride in the mobile phase ranges from between
about 0.01 M to about 0.13 M. More preferably, the concentration of
tetrabutylammonium chloride in the mobile phase ranges from between
about 0.05 M to about 0.125 M.
[0040] The mobile phase should have a pH that is appropriate to the
metal complexes employed in the method and the column employed in
the method. A pH that is appropriate to the metal complexes
employed in the method is a pH at which the metal complex is
stable, i.e. that does not cause the metal complex to dissociate. A
pH that is appropriate for the column employed in the method of the
invention is a pH at which the column is stable and functions
properly. The pH of the mobile phase can be adjusted by the
addition of a base or an acid. The need for an adjustment of the pH
will depend on many factors, including the particular metal
complexes used, the type of column employed and the composition of
the mobile phase. Thus, the pH of the mobile phase could be
anywhere from 2-14.
[0041] However, for the preferred metal complexes of the invention
the pH is preferably between 6-8. A pH between 6-8 minimizes
complex dissociation. Proton assisted complex dissociation is a
phenomenon whereby the metal complex becomes protonated and due to
this protonation more readily dissociates. The complex experiences
more protonation at lower pH values. More preferably, the pH of the
mobile phase is between about 6.0 to about 7.5. Even more
preferably, the pH of the mobile phase is between about 6.4 to
about 7.2. The pH of the mobile phase can be adjusted to these
preferred values using any appropriate base. Examples of suitable
bases include, but are not limited to, sodium hydroxide, lithium
hydroxide, potassium hydroxide, and ammonium hydroxide. Preferably,
the cation of the base corresponds to the cation of the salt in the
mobile phase. For example, a preferred base for use in a solvent
system containing sodium chloride would be sodium hydroxide.
Similarly, a preferred base for use in a mobile phase containing
tetrabutylammonium chloride would be ammonium hydroxide. The amount
of base in the mobile phase will be that amount needed to adjust
the mobile phase to the appropriate pH.
[0042] Thus, an example of a suitable mobile phase is acetonitrile
in water containing between about 0.1 M to about 0.7 M of a salt at
a pH of between about 6 to about 8. Another suitable mobile phase
is 1-5% methanol in water containing between about 0.15 M to about
0.6 M of salt at a pH of between about 6 to about 8. A preferred
mobile phase would be acetonitrile containing between about 0.3 M
to about 0.7 M of sodium chloride at a pH of between about 6.0 to
about 7.5. Another preferred mobile phase would be 5-15%
acetonitrile in water containing between about 0.01 M to about 0.13
M of tetrabutylammonium chloride at a pH of between about 6.0 to
about 7.5. Still another preferred mobile phase would be 5-15%
acetonitrile in water containing a mixture of between about 0.01 M
to about 0.13 M of tetrabutylammonium chlroide and between about
0.3 M to about 0.7 M lithium chloride at a pH of between about 6.0
to about 7.5.
[0043] A more preferred mobile phase would be 5-10% acetonitrile in
water containing between about 0.4 M to about 0.6 M of sodium
chloride at a pH of between about 6.4 to about 7.2. Another more
preferred mobile phase would be 5-10% acetonitrile in water
containing between about 0.05 M to about 0.125 M of
tetrabutylammonium chloride at a pH of between about 6.4 to about
7.2. Still another preferred mobile phase would be 5-10%
acetonitrile in water containing a mixture of between about 0.05 M
to about 0.125 M of tetrabutylammonium chloride and between about
0.4 M to about 0.6 M lithium chloride at a pH of between about 6.4
to about 7.2.
[0044] For the chiral HPLC embodiment of the invention a preferred
mobile phase would include 1-5% methanol in water containing
between about 0.1 M to about 2.5 M of ammonium thiocyanate. Another
preferred mobile phase would be 1-5% methanol in water containing
between about 0.05 M to about 0.3 M of tetrabutylammonium chloride.
A more preferred mobile phase for the chiral HPLC embodiment would
be 1-5% methanol in water containing between about 0.2 M to about
0.3 M of ammonium thiocyanate. Another more preferred mobile phase
would be 1-5% methanol in water containing between about 0.05 M to
about 0.15 M of tetrabutylammonium chloride.
[0045] In the first step of the analytical method a solution
containing the metal complex is loaded onto the column. The loading
can be accomplished by injection or another suitable means of
placing the solution containing the metal complex onto the column.
Preferably, the solution containing the metal complex is loaded on
the column by injection through an injector. The process of
injection can be manual or it may be automated. The preparation of
the metal complex for injection could occur in several ways.
Preferably, the metal complex is directly dissolved in the mobile
phase. However, depending on the solubility of the metal complex,
the metal complex can also be dissolved in a solvent and then the
mobile phase could be added to it. Another way of accomplishing the
combining step is to dissolve the metal complex in a solvent with a
salt of the same coordinating anion that is present in the mobile
phase and then dilute with a mobile phase. Suitable solvents in
which the metal complexes could be dissolved include organic
solvents such as methanol, ethanol, and propanol. The solvent in
which the metal complexes can be dissolved does not have to be the
same solvent or solvents that comprise the solvent system in the
mobile phase. However, it is preferred that the solvent in which
the metal complexes are dissolved be the same solvent or solvents
that comprise the solvent system in the mobile phase. Thus, the
solution containing the metal complexes can be the mobile phase, a
suitable solvent that dissolves the metal complexes, or a suitable
solvent that dissolves the metal complexes that has been further
diluted with mobile phase.
[0046] An additional optional step in the method of both the chiral
HPLC and achiral HPLC embodiment of the invention is to form a
metal complex containing only one type of coordinating anion as
ligands before combining the metal complex with the mobile phase.
The single species is formed by combining an excess of the salt of
a coordinating anion with a metal complex in an aqueous solution to
generate a single species of the metal complex. Following the
addition of the excess of the salt of the coordinating anion to the
aqueous solution, the solution is agitated to form a homogenous
solution. The agitation ensures that all of the ligand binding
sites of the metal of the metal complex are occupied by the
coordinating anions to form a single species. The agitation
continues for a period of time ranging from a few minutes to
several hours until a homogeneous solution is achieved. For
example, a bis(thiocyanato) complex could be formed from the metal
complex by combining an excess of potassium thiocyanate with the
metal complex in water. The resulting solution or suspension is
extracted with methylene chloride to provide the thiocyanate
complex of the metal complex. The metal complex can then be
combined with a thiocyanate salt in the solvent system as a
bis(thiocyanato) complex.
[0047] Suitable stationary phases for use in the method of the
invention include the columns commonly used in HPLC methods. Any
HPLC column can be utilized provided that it can provide successful
separation of metal complexes. Columns typically range from 2-5 mm
in diameter with particles of size ranging from 3-10 mm. Examples
of suitable columns include C1 modified columns, C3 modified
columns, C4 modified columns, octyl (C8) modified columns,
octadecyl (C18) modified columns, C18 polymer column, phenyl
columns, and amino-cyano columns. Preferred types of columns
include octadecyl modified columns, phenyl columns, and amino-cyano
columns. More preferred types of columns include octadecyl modified
columns. Examples of these more preferred octadecyl modified
columns include the YMC ODS-AQ S5 column.RTM. available from Waters
Corporation, Vydac columns available from Vydac, and the Symmetry
Shield RP.sub.18 columns available from Waters Corporation.
[0048] In the chiral HPLC embodiment of the invention a chiral
stationary phase should be employed. Any type of chiral column
utilized in HPLC can be employed in the invention provided it
successfully separates enantiomers of metal complexes. Chiral
columns employed with high performance liquid chromatography are
preferred for use in the invention. Thus, preferred columns
typically range from 2-5 mm in diameter with particles of size
ranging from 3-10 mm. Examples of suitable chiral stationary phases
include cellulose based columns and Pirkle columns. A preferred
chiral column is the Chiralcel-OD-RH column.RTM. available from
Chiral Technology.
[0049] Eluting the complex from the column with the mobile phase
generates a metal complex in which the coordinating anion (which is
a competent ligand) out-competes all other potential ligands
present for the available coordination sites on the metal. The
composition of the mobile phase can be varied during the elution to
meet the objectives of a particular chromatography experiment.
Thus, isocratic or gradient elution can be employed with the method
of the invention. The mobile phase is passed through the column at
a determined flow rate. Evaluating these factors and arriving at an
appropriate flow rate for the objectives of the HPLC method can be
accomplished by one of ordinary skill in the art. The rate at which
the compound will elute from the column will depend on the metal
complex's affinity for the mobile phase relative to its affinity
for the column. This will in turn depend on the type of column
employed in the method of the invention, the composition of the
mobile phase, and the flow rate of the mobile phase through the
column. The appropriate flow rate for a column will depend on the
nature of the column, including the column's length and tolerance
of pressure, the particular metal complexes being eluted from the
column, and the composition of the mobile phase. Generally, the
flow rate can range from about 0.1 to about 10.0 m/min. A preferred
flow rate would be between about 0.5 to about 3 ml/min. For
example, for a 25 cm SymmetryShield RP.sub.18 column.RTM. a typical
flow rate will range from about 0.5 to about 2 ml/min. A preferred
flow rate would be between about 0.9 to about 1.2 ml/min. The
typical flow rate for a YMC ODS-AQ S5 column.RTM. that is 5 cm in
length ranges from 0.5 to 4 ml/min. The preferred flow rate for a
YMC ODS-AQ S5 column.RTM. ranges from 2 to 3 ml/min.
[0050] After the metal complex is eluted from the column the
compound is detected by a detector. The detecting can be performed
by any detector appropriate to meet the objectives of the HPLC
procedure. The detecting may be performed by an "on-line" detector
or an "off-line" detector. An "on-line" detector, as utilized
herein, is a detector that is directly coupled to the column and
detects the metal complex as it elutes from the column. An
"off-line" detector, as utilized herein, is a detector that is not
directly coupled to the column, but detects the metal complex after
it has been collected and manually transferred to the detector.
Thus, the detecting may be manual or automated. On-line detectors
are preferred for use in the invention. Examples of suitable
detectors for the metal complexes include, but are not limited to,
refractive index detectors, radiochemical detectors,
electrochemical detectors, and mass spectroscopy detectors.
Ultraviolet/visible absorption detectors are a preferred type of
detector for use in the chromatographic method of the invention.
Ultraviolet/visible absorption detectors include fixed wavelength
detectors, variable wavelength detectors, and diode array
detectors. Fixed wavelength detectors measure at one wavelength,
typically 254 or 264 nm. Variable wavelength detectors measure at
one wavelength at a time, while diode array detectors measure a
spectrum of wavelengths simultaneously. Fixed wavelength UV
detectors are preferred for use in the invention.
[0051] High performance liquid chromatography procedures are widely
used analytical methods that are very familiar to those of ordinary
skill in the art. Selecting the appropriate equipment and
parameters for a particular HPLC procedure and making the
appropriate variations in the procedure to meet the objective of a
particular experiment are readily accomplished by one of ordinary
skill in the art. The methods of each embodiment of the invention
can be used with any HPLC machine, provided that a chiral column is
employed in the chiral HPLC embodiment of the invention. The method
of the present invention can be used with either normal phase or
reverse phase HPLC depending upon the selection of solvents and
columns. Furthermore, the methods of the present invention are not
limited to any particular scale, however, it is preferred that the
method be operated using metal complex sample sizes similar to
those employed in high performance liquid chromatography.
[0052] Thus, in each embodiment of the present invention there is
provided a HPLC method in which a single species of the metal
complex(es) present can be reliably generated so that detection and
quantification of the metal complexes can proceed without
interference from other complexes. The following examples are
intended to illustrate but not to limit the invention.
EXAMPLES
[0053] Experimental For Examples 1-8
[0054] Chemicals, Solvents and Materials
[0055] All solvents used in the study were HPLC grade or
equivalent. All chemicals were ACS reagent grade or equivalent.
[0056] HPLC System and Data Analysis
[0057] The HPLC chromatography was performed using a Gilson system
Model 306 pump, Model 155 UV-V detector, Model 215 liquid handler,
Unipoint Software, Win98), a Varian system (Model 310 pump, Model
340 UV-V detector, Model 410 autosampler Star Workstation, Win98)
or SSI system (Acuflow Series IV pump, Acutect 500 UV-V detector,
Alcott Model 718 autosampler, HP Model 3395 integrator).
Example 1
HPLC Analysis of M40403 Using Method 1
[0058] 7
[0059] Method 1: Analytical Column: Waters YMC ODS-AQ S5 120 .ANG.
(4.6.times.50 mm); System A: 0.1% trifluoroacetic acid in H.sub.2O;
System B: 0.08% trifluoroacetic acid in acetonitrile; Gradient:
10-50% system B over 10 min; Flow rate: 3 ml/min; Detector
wavelength: 265. Injected 20 .mu.l of stock solution of M40403
prepared by dissolving 1 mg in 1 ml of water and diluting with 1 ml
of system A. The HPLC chromatogram of M40403 using method 1 is
shown in FIG. 1.
Example 2
HPLC Analysis of M40403 Using Method 2
[0060] Method 2: Analytical Column: Waters YMC 9DS-AQ S5 120 .ANG.
(4.6.times.50 MM); System A: 0.5 N aqueous NaCl; System B: 1:4
water/CH.sub.3CN; Gradient: 10-50% system B over 9 min; Flow rate:
3 mL/min; Detector wavelength: 265 nm. Injected 20 .mu.l of stock
solution of M40403 prepared by dissolving 1 mg in 1 ml of system A.
The HPLC chromatogram of M40403 using method 2 is shown in FIG.
2.
Example 3
HPLC Analysis of M40403 Using Method 3
[0061] Method 3: Analytical Column: Waters Symmetry Shield RP18, 5
.mu.m, 250.times.4.6 mm; Mobile Phase: Acetonitrile: 0.125 M
Tetrabutylammonium Chloride in water (pH 6.5), 5%: 95%
H.sub.2O(v/v); Flow rate: 1 mL/min; Detection wavelength: 265 nm.
Injected 20 .mu.l of stock solution of M40403 prepared by
dissolving 1 mg in 1 ml of mobile phase. The HPLC chromatogram of
M40403 using method 3 is shown in FIG. 3.
[0062] The HPLC chromatogram of M40403 and related compounds using
method 3 is shown in FIG. 3a. Method 3 allows a separation of
M40402 (bisimine of M40403), M40414 (monoimine of M40403) and
M40475 (free ligand of M40403) (see chromatogram in FIG. 3a).
Example 4
HPLC Analysis of M40403 Using Method 4
[0063] Method 4: Analytical Column: Waters Symmetry Shield RP18, 5
.mu.m, 250.times.4.6 mm; Mobile Phase: Acetonitrile: 0.125 M
Tetrabutylammonium Chloride and 0.5 M LiCl in water (pH 6.5), 5%:
95% H.sub.2O (v/v); Flow rate: 1 mL/min; Detection wavelength: 265
nm. Injected 20 .mu.l of stock solution of M40403 prepared by
dissolving 1 mg in 1 ml of system A. The HPLC chromatogram of
M40403 using method 4 is shown in FIG. 4.
[0064] The HPLC chromatogram of M40403 and related compounds using
method 4 is shown in FIG. 4a. Method 4 allows a separation of
M40402 (bisimine of M40403), M40414 (monoimine of M40403) and
M40475 (free ligand of M40403) and all diastereomers of M40403 (see
chromatogram in FIG. 4a).
Example 5
HPLC Analysis of M40401 using Method 1
[0065] 8
[0066] Method 1: Analytical Column: Waters YMC ODS-AQ S5 120 .ANG.
(4.6.times.50 mm); System A: 0.1% trifluoroacetic acid in H.sub.2O;
System B: 0.08% trifluoroacetic acid in acetonitrile; Gradient:
10-50% system B over 10 min; Flow rate: 3 ml/min; Detector
wavelength: 265. Injected 20 .mu.l of stock solution of M40401
prepared by dissolving 1 mg in 1 ml of water and diluting with 1 ml
of system A. The HPLC chromatogram of M40401 using method 1 is
shown in FIG. 5.
Example 6
HPLC With Various NaCl Concentrations
[0067] An HPLC was taken of M40401 with various concentrations of
NaCl. Analytical Column: Waters YMC 9DS-AQ S5 120 .ANG.
(4.6.times.50 mm); System A: (A) H.sub.2O (no NaCl); (B) 0.01 M
NaCl in water; (C) 0.5 M NaCl in water; System B: acetonitrile;
Gradient: 0-100% system B over 10 min; Flow: 3 ml/min; Detector
wavelength: 265 nm. Injected 20 .mu.l of stock solution of M40401
prepared by dissolving 1 mg in 1 ml of system A. The HPLC
chromatogram of M40401 using various NaCl concentrations is shown
in FIG. 6.
Example 7
HPLC Analysis of M40401 Using Method 2
[0068] Method 2: Analytical Column: Waters YMC ODS-AQ S5 120 .ANG.
(4.6.times.50 MM); System A: 0.5 N aqueous NaCl; System B: 1:4
water/CH.sub.3CN; Gradient 1: 10-50% system B over 9 min; Flow
rate: 3 mL/min; Detector wavelength: 265 nm. Injected 20 .mu.l of
stock solution of M40403 prepared by dissolving 1 mg in 1 ml of
system A.
[0069] The HPLC chromatogram of M40401 using method 2 is shown in
FIG. 7. Method 2 allows a separation of M40472 (bisimine of
M40401), M40473 (monoimine of M40401), free ligand of M40403 and
two isomers of M40401 (M40406, M40404).
Example 8
HPLC Analysis of M40401 Using Method 3
[0070] Method 3: Analytical Column: Waters Symmetry Shield RP18, 5
m, 250.times.4.6 mm; Mobile Phase: Acetonitrile: 0.125 M
Tetrabutylammonium Chloride in H.sub.2O (pH 6.5), 5:95% H.sub.2O
(v/v); Flow rate: 1 mL/min; Detection wavelength: 265 nm.
[0071] The HPLC chromatogram of M40401 using method 3 is shown in
FIG. 8. Method 3 allows a separation of M40472 (bisimine of
M40401), M40473 (monoimine of M40401), free ligand of M40403 and
two isomers of M40401 (M40406, M40404).
Example 9
HPLC Analysis of M40401 Using Method 4
[0072] Method 4: Analytical Column: Waters Symmetry Shield RP18, 5
.mu.m, 250.times.4.6 mm; Mobile Phase: Acetonitrile: 0.125 M
Tetrabutylammonium Chloride and 0.5 M LiCl in water (pH 6.5), 5:
95% H.sub.2O (v/v); Flow rate: 1 ml/min; Detection wavelength: 265
nm; Injected 20 .mu.l of stock solution of M40401 prepared by
dissolving 1 mg in 1 ml of a mobile phase. The HPLC chromatogram of
M40401 using method 4 is shown in FIG. 9.
[0073] The HPLC chromatogram of M40401 and related compounds using
method 4 is shown in FIG. 9a. Method 4 allows a separation of
M40472 (bisimine of M40401), M40473 (monoimine of M40401), free
ligand of M40403 and two isomers of M40401 (M40406, M40404).
Example 10
HPLC of M40403-(HCOO.sup.-).sub.2 Using Formate Anion
[0074] An HPLC of M40403 employing the formate anion was taken.
Analytical Column: Waters YMC 9DS-AQ S5 120 .ANG. (4.6.times.50
mm); System A: 0.025 M ammonium formate in water; System B: 1:
4=0.125 M ammonium formate in water/acetonitrile; Gradient: 0-100%
system B over 10 min; Flow: 3 ml/min;
[0075] Detector wavelength: 265 nm. Injected 20 .mu.l of stock
solution of M40403-(Formate).sub.2 prepared by dissolving 1 mg in 1
ml of system A.
[0076] The HPLC chromatogram of M40403-(HCOO.sup.-).sub.2 is shown
in FIG. 10.
Example 11
HPLC of M40403-(OAc).sub.2 Using Acetate Anion
[0077] An HPLC of M40403 employing the acetate anion was taken.
Analytical Column: Waters YMC 9DS-AQ S5 120 .ANG. (4.6.times.50
mm); System A: 0.025 M ammonium acetate in water; System B: 1:
4=0.125 M ammonium acetate in water/acetonitrile; Gradient: 0-100%
system B over 10 min; Flow: 3 ml/min;
[0078] Detector wavelength: 265 nm. Injected 20 .mu.l of stock
solution of M40403-(OAc).sub.2 prepared by dissolving 1 mg in 1 ml
of system A.
[0079] The HPLC chromatogram of M40403-(OAc).sub.2 is shown in FIG.
11.
Example 12
[0080] An HPLC method to separate the diastereomers of superoxide
dismutase mimetic compound M40403. Four stereoisomer mixtures were
prepared (Part A) as shown in Schemes 5-9 and then separated (Part
B) via reversed-phase high performance liquid chromatography.
Part A: Synthesis of Stereoisomers of M40403
[0081] M40403 is synthesized from its single-isomer, tetra-amine
precursor M40400 in the reaction shown in Scheme 7. 9
[0082] The various stereoisomers of M40403 are synthesized from the
various isomers of 1,2-diaminocyclohexane which provides the chiral
carbon centers in M40403. The 1,2-diaminocyclohexane isomers used
to prepare the (R,R+R,S) M40403 stereoisomer mixture of Set 1 are
shown in Scheme 6. Similarly, the 1,2-diaminocyclohexane isomers
used to prepare the (R,R+S,S) M40403 stereoisomer mixture of Set 2
are shown in Scheme 7. The 1,2-diaminocyclohexane isomers used to
prepare the (R,S+R,S) M40403 stereoisomer mixture of Set 3 are
shown in Scheme 8. The 1,2-diaminocyclohexane isomers used to
prepare the (S,S+R,S) M40403 stereoisomer mixture of Set 4 are
shown in Scheme 9. As shown in Schemes 6-9 the M40403 diastereomers
are prepared by template cyclization, followed by reduction with
sodium borohydride. 10 11 12 13
1TABLE 1 M40403 Stereoisomer Mixtures Enantiomeric Compound ID
Relation Predicted HPLC Chromatogram SET 1 SET 1 1 1 and 8 R,R, +
R,S R,R,R,R = S,R,R,R 2 2 and 9 5 peaks R,R,S,R = R,S,R,R 3 3 and
10 (3 single stereoisomers R,S,R,S = S,R,S,R 4 4 - meso 1 meso
isomer, R,S,S,R 5 5 and 6 1 pair of enantiomers) S,R,R,S 6 SET 2
SET 2 R,R,R,R 1 2 peaks R,R + S,S R,R,S,S = S,S,R,R 7 7 - meso (1
pair of enantiomers, S,S,S,S 8 1 meso isomer) SET 3 SET 3 R,S,R,S =
S,R,S,R 4 2 peaks R,S + R,S R,S,S,R 5 (1 meso isomer, S,R,R,S 6 1
pair of enantiomers) SET 4 SET 4 S,S,S,S 8 5 peaks S,S + R,S
S,S,S,R = R,S,S,S 9 (3 single stereoisomers, S,S,R,S = S,R,S,S 10 1
meso isomer, R,S,R,S = S,R,S,R 4 1 pair of enantiomers) R,S,S,R 5
S,R,R,S 6 4 CHIRAL CARBON CENTERS = 2.sup.4 = 16 THEORETICAL
STEREOISOMERS DUE TO THE C.sub.2 SYMMETRY OF THE MOLECULE - 10
STEREOISOMERS POSSIBLE 4 PAIRS IN ENATIOMERIC RELATION AND 2 MESO
ISOMERS HPLC (Regular): SET 1 and SET 4 - the same chromatography
profile (number peaks and retention times) SET 1 minus SET 3 -
isomers with one S chiral center can be assigned
Part B: Separation of Stereoisomer Mixtures
[0083] Chemicals, Materials, and Methods
[0084] Tetrabutylammonium chloride hydrate (98%, 34,585-7) was
purchased from Aldrich Chemical Company. Sodium chloride (99.6%,
S-9888) was purchased from Sigma Chemical Company. All other
solvents (HPLC-grade unless otherwise indicated) and reagents were
purchased from Fisher Scientific and were of the finest grade
available. The SymmetryShield.RTM. RP.sub.18 column (4.6
mm.times.250 mm, 5 .mu.m particle size) and its corresponding guard
column were purchased from Waters Corporation.
[0085] Reversed-Phase HPLC Experiments
[0086] Preparation of Standard Solutions
[0087] HPLC Mobile phase A was an aqueous solution consisting of
0.125 M tetrabutylammonium chloride (TBAC) and 0.5 M LiCl, prepared
by adding tetrabutylammonium chloride hydrate (36.99 g) and solid
LiCl (21.2 g) to a 1 L volumetric flask, diluting to volume with
Millipore water, and inverting the flask several times to obtain a
homogeneous solution. The resulting solution was filtered through a
0.45 .mu.m nylon filter prior to use. Mobile phase B was HPLC-grade
acetonitrile. Samples of each diastereoisomer set for HPLC-UV
analysis were prepared at concentrations of -3.0 mg/mL in a 50:50
mixture of 0.5 M LiCl in MeOH: Mobile Phase A.
[0088] Chromatographic Conditions
[0089] The column used for the HPLC experiments was the
SymmetryShield RP8 column.RTM., 4.6 mm.times.250 mm, 5 .mu.m
particle size (Waters Corporation). Separations were achieved under
isocratic flow conditions using a mobile phase composed of 95%
Mobile Phase A and 5% Acetonitrile (Mobile Phase B on a dual pump
system). The flow rate was 1.0 mL/min. using a 15 minute runtime,
and the wavelength for detection was 265 nm. The injection volume
of each sample was 20 .mu.L.
[0090] FIG. 12 contains the chromatograms resulting from the HPLC
analysis of the diastereomeric mixtures of Set 1-4. Table 1
summarized the predicted results from the HPLC analysis of the
diastereomeric mixtures of Set 1-4. The chromatogram resulting from
the HPLC analysis of the Set 1 (R,R+R,S) M40403 stereoisomer
mixture shows only five peaks, although there are a total of six
stereoisomers produced. However, of the six stereoisomers produced,
compounds 5 and 6 are enantiomerically related. As a result they
have the same adsorption characteristics and they elute as one
peak.
[0091] The chromatogram resulting from the HPLC analysis of the Set
2 (RR+R,S) M40403 stereoisomer mixture shows only two peaks,
although there are a total of three stereoisomers produced. Of the
three stereoisomers produced, compounds 1 and 8 are
enantiomerically related. As a result they have the same adsorption
properties and they elute as one peak.
[0092] Similarly, the chromatogram resulting from the HPLC analysis
of the Set 3 (R,S+R,S) M40403 stereoisomer mixture shows only two
peaks, although there are a total of three stereoisomers produced.
Of the three stereoisomers produced, compounds 5 and 6 are
enantiomerically related and elute as one peak.
[0093] The chromatogram resulting from the HPLC analysis of the Set
4 (S,S+R,S) M40403 stereoisomer mixture shows only five peaks,
although there are a total of six stereoisomers produced. Of the
six stereoisomers produced, compounds 5 and 6 are enantiomerically
related and elute as one peak.
Example 13
[0094] The following chiral HPLC method was used to separate the
all-R and all-S enantiomers M40403 and M40419: 14
[0095] The complexes were first converted to their corresponding
bis(thiocyanato) forms via ligand exchange reactions with KSCN, as
shown in Scheme 10. The resultant M40403 and M40419 (SCN).sub.2
derivatives were then separated via chiral HPLC. 15
[0096] Chemicals, Materials, and Methods
[0097] Preparation of Bis(thiocyanato) Complexes of M40403 and
M40419, Respectively
[0098] M40403 (SCN).sub.2: The M40403 complex (5.0 mg, 0.01 mmol)
was dissolved in 1.0 mL H.sub.2O. To this solution was added KSCN
(49.0 mg, 0.5 mmol) to bring its concentration to 0.5 M. A white
precipitate formed immediately upon addition of the KSCN. The
reaction was mixed vigorously for 60 minutes at room temperature,
and the resulting suspension was extracted with CH.sub.2Cl.sub.2
(3.times.1.0 mL). The combined CH.sub.2Cl.sub.2 extracts were then
dried over MgSO.sub.4, filtered, and evaporated to yield the
product as a white solid. The product was dissolved in MeOH (2.5
mL), and aliquots of the MeOH solution were mixed in a 1:1 ratio
with the HPLC mobile phase (0.26 M NH.sub.4SCN in MeOH) prior to
injection.
[0099] M40419 (SCN).sub.2: The M40419 complex (5.0 mg, 0.01 mmol)
was dissolved in 1.0 mL H.sub.2O. To this solution was added KSCN
(49.0 mg, 0.5 mmol) to bring its concentration to 0.5 M. A white
precipitate formed immediately upon addition of the KSCN. The
reaction was mixed vigorously for 60 minutes at room temperature,
and the resulting suspension was extracted with CH.sub.2Cl.sub.2
(3.times.1.0 mL). The combined CH.sub.2Cl.sub.2 extracts were then
dried over MgSO.sub.4, filtered, and evaporated to yield the
product as a white solid. The product was dissolved in MeOH (2.5
mL), and aliquots of the MeOH solution were mixed in a 1:1 ratio
with the HPLC mobile phase (0.26 M NH.sub.4SCN in MeOH) prior to
injection.
[0100] Preparation of Standard Solutions
[0101] The HPLC mobile phase was a 0.2 M solution of NH.sub.4SCN in
MeOH, prepared by adding 2.0 g NH.sub.4SCN to 100 mL MeOH(HPLC
grade). The mobile phase solution was filtered through a 0.45 .mu.m
nylon filter (Osmonics) prior to use.
[0102] Chromatographic Conditions
[0103] The column used for the chiral HPLC experiment was the
Chiralcel OD-RH column, 4.6 mm.times.150 mm, 5 .mu.m particle size
(Chiral Technologies). Separations were achieved using a simple
isocratic flow at a rate of 0.5 mL/min. The wavelength for
detection was 265 nm, and the injection volume of each sample was
20 .mu.L.
[0104] The chiral HPLC profiles of the M40403 and M40419
bis(thiocyanato) enantiomers are shown in FIG. 13. The chiral HPLC
profiles for the M4043-(SCN).sub.2 and M40419(SCN).sub.2
enantiomers are shown separately in Profiles A and B. As revealed
in the profiles, the all-R M40403-(SCN).sub.2 enantiomer has a
retention time (t.sub.R) of 6.8 min, while its all-S
M40419-(SCN).sub.2 mirror-image has a retention time of 6.5
minutes. The enantiomers were then analyzed by co-injection
experiment to confirm that they are truly resolved under these
chromatographic conditions. The resulting HPLC profile for the
co-injected enantiomers is shown in Profile C. The enantiomers were
separated by approximately 0.3 minutes, thereby confirming the
initial results.
* * * * *