Note: Descriptions are shown in the official language in which they were submitted.
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HPLC ANALYSIS OF IMPURITIES IN DIANHYDROGALACTITOL
by
Xiaoyun Lu and Mike Li
CROSS-REFERENCES
[0001] This application claims the benefit of United States Patent Application
Serial No. 14/083,135 by Xiaoyun Lu and Mike Li, entitled "Improved Analytical
Methods
for Analyzing and Determining Impurities in Dianhydrogalactitol," and filed on
November
18, 2013, which claimed the benefit and is a continuation-in-part of United
States Patent
Application Serial No. 13/933,844, by Xiaoyun Lu, entitled "Improved
Analytical Methods
for Analyzing and Determining Impurities in Dianhydrogalactitol," and filed on
July 2,
2013, which in turn claimed the benefit and is a continuation-in-part of PCT
Application
Serial No. PCT/162013/000793, by Xiaoyun Lu, entitled "Improved Analytical
Methods
for Analyzing and Determining Impurities in Dianhydrogalactitol," and filed on
February
26, 2013, designating the United States, which in turn, claimed the benefit of
United
States Provisional Patent Application Serial No. 61/603,464, entitled
"Improved
Analytical Methods for Analyzing and Determining Impurities in
Dianhydrogalactitol" by
Xiaoyun Lu, filed February 27, 2012. The contents of all of these applications
are
incorporated herein in their entirety by this reference.
FIELD OF THE INVENTION
[0002] This invention is directed to improved analytical methods for
dianhydrogalactitol, especially involving high performance liquid
chromatography
(H PLC).
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BACKGROUND OF THE INVENTION
[0003] Dianhydrogalactitol (1,2:5,6 dianhydrogalactitol or DAG) is one of a
number of hexitols or hexitol derivatives having significant pharmacological
activity,
including chemotherapeutic activity. In particular, dianhydrogalactitol has
been
suggested for use in chemotherapy, such as in United States Patent No.
7,157,059 to
Nielsen et al., incorporated herein by this reference.
[0004] Dianhydrogalactitol has activity against a number of neoplasms.
However, if dianhydrogalactitol is to be used successfully as a therapeutic
agent, an
extremely high degree of purity and the removal of impurities is essential.
The
presence of impurities can lead to undesirable side effects. One example
occurred a
number of years ago, when impurities present in a batch of the amino acid
tryptophan, a
normal constituent of protein, were responsible for a significant outbreak of
eosinophilia-
myalgia syndrome, which caused a large number of cases of permanent disability
and
at least 37 deaths. This is particularly important if the therapeutic agent
such as
dianhydrogalactitol is to be employed in patients with compromised immune
systems or
liver or kidney dysfunction, or in elderly patients. Such patients may
experience a
greater incidence of undesirable side effects owing to their sensitivity to
contaminants.
[0005] One of the impurities found in preparations of dianhydrogalactitol is
dulcitol. Other impurities exist in preparations of dianhydrogalactitol as
well, depending
on their method of preparation.
[0006] Therefore, there is a need for improved analytical methods to detect
impurities and degradation products in preparations of dianhydrogalactitol to
provide
preparations of greater purity that are less likely to induce side effects
when
dianhydrogalactitol is administered for therapeutic purposes.
SUMMARY OF THE INVENTION
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[0007] An improved analytical method for determining the purity of
dianhydrogalactitol and detecting impurities and degradation products in
preparations of
dianhydrogalactitol that meets these needs is described herein.
[0008] In general, this analytical method employs high performance liquid
chromatography (H PLC), in particular, HPLC with refractive index (RI)
detection.
[0009] In one alternative, an analytical method for analyzing the presence and
quantity of impurities present in a preparation of dianhydrogalactitol
comprises the steps
of:
(1) analyzing a preparation of dianhydrogalactitol by subjecting the
preparation to high performance liquid chromatography using elution with a
mobile
phase gradient to separate dianhydrogalactitol from dulcitol and other
contaminants of
the preparation; and
(2) determining the relative concentration of one or more peaks
resolved by high performance liquid chromatography that represent compounds
other
than dianhydrogalactitol itself.
[0010] The compounds other than dianhydrogalactitol itself can be at least one
of: (1) dulcitol; (2) an impurity other than dulcitol; and (3) a degradation
product of
dianhydrogalactitol.
[0011] In one alternative of this method, elution is with a gradient of NaOH
from
about 2.5 mM to about 0.1 mM. Preferably, elution is with a gradient of NaOH
from
about 1.5 mM to about 0.1 mM. More preferably, elution is with a gradient of
NaOH
from about 1 mM to about 0.1 mM.
[0012] In another alternative of this method, elution is with a gradient of a
combination of ammonium hydroxide and a volatile ammonium salt selected from
the
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group consisting of ammonium formate and ammonium acetate and the total
concentration of the ammonium formate and ammonium acetate is from about 2.5
mM
to about 0.1 mM. Preferably, the total concentration of the ammonium hydroxide
and
the volatile ammonium salt selected from the group consisting of ammonium
formate
and ammonium acetate is from about 1.5 mM to about 0.1 mM. More preferably,
the
total concentration of the ammonium hydroxide and the volatile ammonium salt
selected
from the group consisting of ammonium formate and ammonium acetate is from
about 1
mM to about 0.1 mM. Typically, the proportion of ammonium hydroxide and the
volatile
ammonium salt selected from the group consisting of ammonium formate and
ammonium acetate is varied from about 100:1 at the beginning of elution to
about 1:100
at the end of elution.
[0013] Typically, in this method, the step of determining the relative
concentration of one or more peaks resolved by high performance liquid
chromatography that represent compounds other than dianhydrogalactitol itself
is
performed by evaporative light scattering detection. Typically, the
evaporative light
scattering detection is compatible with electrospray LC/MS. Typically, the
evaporative
light scattering detection comprises post-column addition of a volatile
solvent to
enhance evaporation of the 100% aqueous mobile phase. Typically, the volatile
solvent
is selected from the group consisting of methanol, ethanol, isopropanol, and
acetonitrile.
[0014] In one alternative, an electrospray tandem mass spectrometer is
installed
and connected on-line to an HPLC system with ELSD. Typically, in this
alternative,
mass spectral data providing chemical information for each of the impurities
that may be
present in a preparation of dianhydrogalactitol is collected. Also, typically,
in this
alternative, tandem mass spectral data providing structural information for
each of the
impurities that may be present in a preparation of dianhydrogalactitol is
collected.
[0015] The method can further comprise the step of performing preparative
HPLC collection of at least one specific substance peak present in a
preparation of
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dianhydrogalactitol. The at last one substance peak present in the preparation
of
dianhydrogalactitol can be an impurity.
[0016] In another alternative, instead of gradient elution, isocratic elution
can be
used. When isocratic elution is used, in general, the method comprises the
steps of:
(1) analyzing a preparation of dianhydrogalactitol by subjecting the
preparation to high performance liquid chromatography using elution with an
isocratic
mobile phase to separate dianhydrogalactitol from dulcitol and other
contaminants of
the preparation; and
(2) determining the relative concentration of one or more peaks
resolved by high performance liquid chromatography that represent compounds
other
than dianhydrogalactitol itself.
[0017] In one alternative, when isocratic elution is used, the isocratic
mobile
phase is NaOH, and the concentration of NaOH is from about 5 mM to 0.1 mM.
Preferably, the concentration of NaOH is from about 2.5 mM to about 0.1 mM.
More
preferably, the concentration of NaOH is about 1 mM.
[0018] In another alternative, when isocratic elution is used, the isocratic
mobile
phase is a combination of ammonium hydroxide and a volatile ammonium salt
selected
from the group consisting of ammonium formate and ammonium acetate and the
total
concentration of the ammonium hydroxide and the volatile ammonium salt
selected from
the group consisting of ammonium formate and ammonium acetate is from about 5
mM
to 0.1 mM. Preferably, the total concentration of the ammonium hydroxide and
the
volatile ammonium acetate is from about 2.5 mM to about 0.1 mM. More
preferably, the
total concentration of the ammonium hydroxide and the volatile ammonium salt
selected
from the group consisting of ammonium formate and ammonium acetate is about 1
mM.
Typically, the proportion of ammonium hydroxide and the volatile ammonium salt
selected from the group consisting of ammonium formate and ammonium acetate is
about 50:50.
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[0019] Typically, in this alternative, the step of determining the relative
concentration of one or more peaks resolved by high performance liquid
chromatography that represent compounds other than dianhydrogalactitol itself
is
performed by evaporative light scattering detection (ELSD), as described
above.
Typically, the evaporative light scattering detection is compatible with
electrospray
LC/MS. Typically, the evaporative light scattering detection comprises post-
column
addition of a volatile solvent to enhance evaporation of the 100% aqueous
mobile
phase. Typically, the volatile solvent is selected from the group consisting
of methanol,
ethanol, isopropanol, and acetonitrile.
[0020] In this alternative as well, an electrospray tandem mass spectrometer
can be installed and connected on-line to an HPLC system with ELSD. Typically,
in this
alternative, mass spectral data providing chemical information for each of the
impurities
that may be present in a preparation of dianhydrogalactitol is collected.
Also, typically,
in this alternative, tandem mass spectral data providing structural
information for each
of the impurities that may be present in a preparation of dianhydrogalactitol
is collected.
[0021] This alternative of a method according to the present invention can
further comprise the step of performing HPLC collection of at least one
specific
substance peak present in a preparation of dianhydrogalactitol. The at last
one
substance peak present in the preparation of dianhydrogalactitol can be an
impurity.
[0022] In still another alternative, an analytical method for analyzing the
presence and quantity of impurities present in a preparation of
dianhydrogalactitol
comprises the step of analyzing a preparation of dianhydrogalactitol by
subjecting the
preparation to high performance liquid chromatography (HPLC) on an HPLC column
using elution with a mobile phase gradient to separate dianhydrogalactitol
from dulcitol
and other contaminants of the preparation;
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wherein the high performance liquid chromatography employs evaporative light
scattering detection (ELSD).
[0023] Typically, the HPLC column is a silica gel column bonded to 018
compounds and finished with an endcapping procedure employing Lewis acid-Lewis
base chemistry.
[0024] Typically, elution is performed with a gradient of 95% water/5`)/0
acetonitrile to 70% water/30% acetonitrile, returning to 95% water/5`)/0
acetonitrile.
Typically, the time schedule for varying the eluant is as follows: 0 minutes,
95%
water/5`)/0 acetonitrile; 15 minutes, 95% water/5`)/0 acetonitrile; 15.1
minutes, 70%
water/30% acetonitrile; 20 minutes, 70% water/30% acetonitrile; 20.1 to 35
minutes,
95% water/5`)/0 acetonitrile. Typically, the method detects a monoepoxide
degradation
product of dianhydrogalactitol, a monoepoxide dimer, and dulcitol. Preferably,
the
method also detects a dimer of dianhydrogalactitol and condensed products.
[0025] Typically, the peaks resulting from HPLC are analyzed by LC-MS.
[0026] Typically, the method further comprises a step of determining the
relative
concentration of one or more peaks resolved by high performance liquid
chromatography that represent compounds other than dianhydrogalactitol itself.
[0027] In an alternative for detection by ELSD, typically, the column
temperature
is about 30 C.
[0028] Typically, the flow rate is about 0.5 mL/min. Typically, the ELSD
detector
is operated in cooling mode with the drift tube temperature of 35 C and gain
400, 2
pps, 45 PSI. Typically, in this alternative, Mobile Phase A and Mobile Phase B
are
employed, with Mobile Phase A being 0.05% formic acid in water and Mobile
Phase B
being 100% methanol. Typically, with these mobile phases, elution is performed
from 0
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minutes to 25 minutes with 100% of 0.05% formic acid in water, from 25 minutes
to 25.1
minutes with 90% of 0.05% formic acid in water and 10% of 100% methanol, from
25.1
minutes to 35 minutes with 10% of 0.05% formic acid in water and 90% of 100%
methanol, and from 35.1 minutes to 50 minutes with 100% of 0.05% formic acid
in
water.
[0029] The method can further comprise the preparation of an external
calibration standard curve for an impurity. The impurity can be selected from
the group
consisting of dulcitol, a monoepoxide degradation product of
dianhydrogalactitol, and a
dimer of dianhydrogalactitol. The method can estimate the content of an
unknown
impurity by using a calibration standard curve established by chromatography
of
dianhydrogalactitol reference material.
[0030] Another alternative employing HPLC and ELSD employs a dual elution
sequence. The dual elution sequence is as follows: a first part of the elution
sequence
in which elution is performed from 0 minutes to 25 minutes with 100% of 0.05%
formic
acid in water, from 25 minutes to 25.1 minutes with 90% of 0.05% formic acid
in water
and 10% of 100% methanol, from 25.1 minutes to 35 minutes with 10% of 0.05%
formic
acid in water and 90% of methanol, and from 35.1 minutes to 50 minutes with
100% of
0.05% formic acid in water, and a second part of the elution sequence in which
elution
is performed as follows: from 0 minutes to 7.5 minutes with 100% of 0.05%
formic acid;
from 7.5 minutes to 7.6 minutes with 97% of 0.05% formic acid and 3% of
methanol;
and from 7.6 minutes to 20 minutes with 100% of 0.05% formic acid. In this
alternative,
typically the column temperature for HPLC is about 30 C, the sample
temperature for
HPLC is about 5 C, the flow rate for HPLC is about 0.5 mL/min, and the
injection
volume is about 10-100 L. In this alternative, typically, for ELSD, the gain
is about
400, the drift tube temperature is about 45 C, the gas pressure is about 35
PSI of
nitrogen, the nebulizer is set to cooling, the data rate is 2 points per
second, and the
Rayleigh factor is about 6Ø Typically, in this alternative, standards of
dulcitol at 0.1,
0.08, 0.05, 0.03, 0.01, 0.005 mg/mL are employed to determine the sensitivity
and
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linearity of the system. Typically, in this alternative, the retention time
for dulcitol is
about 6.4 minutes and the retention time for dianhydrogalactitol is about 12.1
minutes.
In this alternative, the amount and percentage of a dulcitol impurity can be
determined
from the results of HPLC and ELSD. Also, in this alternative, the amount and
percentage of an unknown impurity other than dulcitol can be determined from
the
results of HPLC and ELSD.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] These and other features, aspects, and advantages of the present
invention will become better understood with reference to the following
description,
appended claims, and accompanying drawings where:
[0032] Figure 1 is a representative HPLC/RI chromatogram of a preparation of
dianhydrogalactitol, showing resolution of dulcitol and an unknown related
substance at
RRT ¨0.6 in the bulk drug and drug product.
[0033] Figure 2 shows representative HPLC chromatograms showing resolution
of dianhydrogalactitol and dulcitol in a standard, and, for comparison, a
water blank; in
Figure 2, the dianhydrogalactitol-dulcitol standard is shown in the top panel,
and the
water blank is shown in the bottom panel.
[0034] Figure 3 is a HPLC chromatogram of a dianhydrogalactitol clinical
sample
using an evaporative light scattering detector for detection showing the
existence of a
possible dianhydrogalactitol dimer and possible condensed products, along with
the
monoepoxide and dulcitol as degradation products.
[0035] Figure 4 is a mass spectroscopy profile of the impurity peak occurring
at
22.6 minutes of the HPLC chromatogram of Figure 3.
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[0036] Figure 5 is a chromatogram of a sample of dianhydrogalactitol as
performed in Example 3 employing 0.05% formic acid in water as Mobile Phase A
and
100% methanol as Mobile Phase B.
[0037] Figure 6 is an example chromatogram of a blank solution as performed in
Example 4.
[0038] Figure 7 is an example chromatogram of an 0.10% dulcitol solution as
performed in Example 4.
[0039] Figure 8 is an example chromatogram of a test solution as performed in
Example 4.
DETAILED DESCRIPTION OF THE INVENTION
[0040] This invention is directed to improved analytical methods for
determining
the purity of dianhydrogalactitol and determining the existence and
concentration of
impurities present in preparations of dianhydrogalactitol.
[0041] The structure of dianhydrogalactitol is shown below in Formula (I).
HO OH
(I)
[0042] One of the significant impurities present in dianhydrogalactitol
preparations is dulcitol. The structure of dulcitol is shown below in Formula
(II). Other
impurities are known to exist in dianhydrogalactitol preparations.
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OH OH
HO(l- OH
OH OH
(II)
[0043] An improved method of analyzing dianhydrogalactitol preparations is
based on HPLC (high performance liquid chromatography) with evaporative light
scattering detection (ELSD). In one alternative, to detect and identify all
significant
components present in such dianhydrogalactitol preparations, HPLC is combined
with
mass spectroscopy (MS).
[0044] The theory and practice of HPLC are described in L.R. Snyder et al.,
"Introduction to Modern Liquid Chromatography" (3rd ed., John Wiley & Sons,
New York,
2009). The theory and practice of MS are described in E. de Hoffmann & V.
Stroobant,
"Mass Spectroscopy: Principles and Applications" (3rd ed., John Wiley & Sons,
New
York, 2007).
[0045] The HPLC method has demonstrated resolution of a synthetic
intermediate, dulcitol, in preparations of dianhydrogalactitol, in addition to
resolution of
an unknown related substance observed at RRT 0.6 (Figure 1). Figure 1 is a
representative HPLC/RI chromatogram of a preparation of dianhydrogalactitol,
showing
resolution of dulcitol and an unknown related substance at RRT ¨0.6 in the
bulk drug
and drug product. Representative HLPC chromatograms showing resolution of
dianhydrogalactitol and dulcitol in a standard, and, for comparison, a water
blank, are
shown in Figure 2. In Figure 2, the dianhydrogalactitol-dulcitol standard is
shown in the
top panel, and the water blank is shown in the bottom panel.
[0046] The present application describes improved HPLC chromatographic
conditions for resolution of potentially co-eluting substances. A thermally
stressed
dianhydrogalactitol product sample is evaluated to provide confirmation of the
chromatographic conditions appropriate for resolution of dulcitol and other
related
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impurities and degradation products. Subsequently, LC/MS and LC/MS/MS is
performed to characterize the unknown DAG-related substance at RRT ¨ 0.6 to
provide
mass spectral characterization and determination of the chemical structure of
this
unidentified component.
[0047] Previously employed HPLC conditions involve isocratic elution of
dianhydrogalactitol and its related substances using a 50 mM NaOH mobile
phase. In
an improvement on these conditions, employed as part of the method disclosed
herein,
a gradient mobile phase is employed. One alternative is the use of NaOH in a
concentration gradient. If NaOH is employed in a concentration gradient,
typically
elution is with a gradient of NaOH from about 2.5 mM to about 0.1 mM.
Preferably,
elution is with a gradient of NaOH from about 1.5 mM to about 0.1 mM. More
preferably, elution is with a gradient of NaOH from about 1 mM to about 0.1
mM.
[0048] In another alternative, a combination of ammonium hydroxide and a
volatile ammonium salt selected from the group consisting of ammonium formate
and
ammonium acetate can be used as eluant. In this alternative, the total
concentration of
the ammonium formate and ammonium acetate is from about 2.5 mM to about 0.1
mM.
Preferably, the total concentration of the ammonium hydroxide and the volatile
ammonium salt selected from the group consisting of ammonium formate and
ammonium acetate is from about 1.5 mM to about 0.1 mM. More preferably, the
total
concentration of the ammonium hydroxide and the volatile ammonium salt
selected from
the group consisting of ammonium formate and ammonium acetate is from about 1
mM
to about 0.1 mM. Typically, the proportion of ammonium hydroxide and the
volatile
ammonium salt selected from the group consisting of ammonium formate and
ammonium acetate is varied from about 100:1 at the beginning of elution to
about 1:100
at the end of elution.
[0049] Other gradient elution schemes are known in the art.
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[0059] Typically, in HPLC analytical methods according to the present
invention,
detection is by means of evaporative light scattering (ELSD). An evaporative
light
scattering detector (ELSD) atomizes the column eluate, shines light on the
resulting
particulate components, and detects the resulting scattered light.
Theoretically, an
ELSD can detect any nonvolatile component. The evaporative light scattering
detection
of a non-chromogenic compound is based on nebulization of the HPLC eluant and
evaporation of mobile-phase solvents to produce atomizing solute particles for
light
scattering detection. This nebulization and solvent evaporation process to
produce
atomizing analyte solute particles is comparable to the electrospray LC/MS
procedure.
Typically, the ELSD detection is compatible with electrospray LC/MS.
[0051] Implementation of an HPC method with ELSD detection that is
compatible with electrospray LC/MS application involves post-column addition
of a
volatile solvent to enhance evaporation of the 100% aqueous mobile phase. The
volatile solvent is typically selected from the group consisting of methanol,
ethanol,
isopropanol, and acetonitrile.
[0052] Accordingly, in methods according to the present invention, an
electrospray tandem mass spectrometer is installed and connected on-line to an
HPLC
system with ELSD. Mass spectral data providing molecular information and
tandem
mass spectral data providing chemical structural information for each of the
impurities
that may be present in a preparation of dianhydrogalactitol can be collected.
Mass
spectroscopy in tandem with HPLC will provide molecular ion information and
possible
chemical structures having a molecular weight consistent with the molecular
ion
information for each of the observed impurities and degradation products.
[0053] In another alternative, preparative HPLC collection of specific DAG-
related substance peaks, including impurities present in a preparation of DAG,
can be
performed.
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[0054] Accordingly, one analytical method for analyzing the presence and
quantity of impurities present in a preparation of dianhydrogalactitol
comprises the steps
of:
(1) analyzing a preparation of dianhydrogalactitol by subjecting the
preparation to high performance liquid chromatography using elution with a
mobile
phase gradient to separate dianhydrogalactitol from dulcitol and other
contaminants of
the preparation; and
(2) determining the relative concentration of one or more peaks
resolved by high performance liquid chromatography that represent compounds
other
than dianhydrogalactitol itself.
[0055] The compounds other than dianhydrogalactitol itself can be at least one
of: (1) dulcitol; (2) an impurity other than dulcitol; and (3) a degradation
product of
dianhydrogalactitol.
[0056] Typically, in one alternative, in this method, the mobile phase
gradient is
a gradient of sodium hydroxide.
[0057] In another alternative, in this method, the mobile phase gradient is a
gradient of a combination of ammonium hydroxide and a volatile ammonium salt
selected from the group consisting of ammonium formate and ammonium acetate.
[0058] Typically, in this method, detection is by evaporative light
scattering.
Typically, when evaporative light scattering is employed, the method further
comprises
the step of post-column addition of a volatile solvent to enhance evaporation
of
components of the mobile phase.
[0059] Typically, the present invention further comprises the step of
analyzing
one or more peaks eluting from the high performance liquid chromatography by
electrospray tandem mass spectroscopy.
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[0060] In one alternative, the present invention further comprises the step of
preparative HPLC collection of at least one specific DAG-related substance
peak.
[0061] If an impurity or degradation product (other than dulcitol) exists, the
unknown impurity or degradation product can be identified by separation by
column
chromatography followed by at least one purification procedure to yield a
solid unknown
sample which can then be characterized for identification by at least one
standard
analytical procedure selected from the group consisting of nuclear magnetic
resonance
(NMR), mass spectroscopy (MS), Fourier transform infrared spectroscopy (FT-
IR),
elemental analysis, determination of purity by HPLC, and determination of
water content
by the Karl Fischer titration method. These methods are well known in the art.
[0062] In another alternative, the method comprises:
(1) analyzing a preparation of dianhydrogalactitol by subjecting the
preparation to high performance liquid chromatography using elution with an
isocratic
mobile phase to separate dianhydrogalactitol from dulcitol and other
contaminants of
the preparation; and
(2) determining the relative concentration of one or more peaks
resolved by high performance liquid chromatography that represent compounds
other
than dianhydrogalactitol itself.
[0063] As in the method employing gradient elution, the compounds other than
dianhydrogalactitol itself can be at least one of: (1) dulcitol; (2) an
impurity other than
dulcitol; and (3) a degradation product of dianhydrogalactitol.
[0064] In this alternative, the elution with the isocratic mobile phase can
either
be elution with sodium hydroxide or elution with a combination of ammonium
hydroxide
and a volatile ammonium salt selected from the group consisting of ammonium
formate
and ammonium acetate. If the isocratic mobile phase is sodium hydroxide,
typically, the
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concentration of NaOH is from about 5 mM to 0.1 mM. Preferably, the
concentration of
NaOH is from about 2.5 mM to about 0.1 mM. More preferably, the concentration
of
NaOH is about 1 mM. If the isocratic mobile phase is a combination of ammonium
hydroxide and a volatile ammonium salt selected from the group consisting of
ammonium formate and ammonium acetate, the total concentration of the ammonium
hydroxide and the volatile ammonium salt selected from the group consisting of
ammonium formate and ammonium acetate is from about 5 mM to 0.1 mM.
Preferably,
the total concentration of the ammonium hydroxide and the volatile ammonium
acetate
is from about 2.5 mM to about 0.1 mM. More preferably, the total concentration
of the
ammonium hydroxide and the volatile ammonium salt selected from the group
consisting of ammonium formate and ammonium acetate is about 1 mM. Typically,
the
proportion of ammonium hydroxide and the volatile ammonium salt selected from
the
group consisting of ammonium formate and ammonium acetate is about 50:50.
[0065] In an alternative method to improve resolution, an evaporative light
scattering detector (ELSD) is employed employing altered elution conditions.
Typically,
in this method, the HPLC column is a silica gel column bonded to 018 compounds
and
finished with an endcapping procedure employing Lewis acid-Lewis base
chemistry
such as the YMC 018 column. Typically, elution is performed with a gradient of
95%
water/5`)/0 acetonitrile to 70% water/30% acetonitrile, returning to 95%
water/5`)/0
acetonitrile. Preferably, the time schedule for varying the eluant is as
follows: 0
minutes, 95% water/5`)/0 acetonitrile; 15 minutes, 95% water/5`)/0
acetonitrile; 15.1
minutes, 70% water/30% acetonitrile; 20 minutes, 70% water/30% acetonitrile;
20.1 to
35 minutes, 95% water/5`)/0 acetonitrile. Preferably, the HPLC method detects
a
monoepoxide degradation product of dianhydrogalactitol, a monoepoxide dimer,
and
dulcitol. More preferably, the HPLC method also detects a dimer of
dianhydrogalactitol
and condensed products.
[0066] Preferably, in this alternative of the method, the peaks resulting from
HPLC are analyzed by LC-MS.
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[0067] In another alternative method, as shown in Example 3, an Atlantis HPLC
column is employed. Typically, in this method, the column temperature is about
30 C.
Typically, in this method, the flow rate is about 0.5 mL/min. Typically, in
this method,
the injection volume is about 10 I_ to about 100 L. Typically, in this
method, an ELSD
detector is used. Typically, in this method, the ELSD detector is operated in
cooling
mode with the drift tube temperature of 35 C and gain 400, 2 pps, 45 PSI.
Typically, in
this method, Mobile Phase A and Mobile Phase B are employed, with Mobile Phase
A
being 0.05% formic acid in water and Mobile Phase B being 100% methanol.
Typically,
in this method, elution is performed from 0 minutes to 25 minutes with 100% of
0.05%
formic acid in water, from 25 minutes to 25.1 minutes with 90% of 0.05% formic
acid in
water and 10% of 100% methanol, from 25.1 minutes to 35 minutes with 10% of
0.05%
formic acid in water and 90% of 100% methanol, and from 35.1 minutes to 50
minutes
with 100% of 0.05% formic acid in water.
[0068] Typically, this alternative of the method further comprises the
preparation
of an external calibration standard curve for an impurity. The impurity can
be, but is not
limited to, an impurity selected from the group consisting of dulcitol, a
monoepoxide
degradation product of dianhydrogalactitol, and a dimer of
dianhydrogalactitol. In this
method, for an unknown impurity, the content of the unknown impurity can be
estimated
using a calibration standard curve established by chromatography of
dianhydrogalactitol
reference material.
[0069] In another alternative, as shown in Example 4, following the elution
sequence described above, namely where elution is performed from 0 minutes to
25
minutes with 100% of 0.05% formic acid in water, from 25 minutes to 25.1
minutes with
90% of 0.05% formic acid in water and 10% of 100% methanol, from 25.1 minutes
to 35
minutes with 10% of 0.05% formic acid in water and 90% of 100% methanol, and
from
35.1 minutes to 50 minutes with 100% of 0.05% formic acid in water, an
additional
elution sequence is performed as follows: from 0 minutes to 7.5 minutes with
100% of
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0.05% formic acid; from 7.5 minutes to 7.6 minutes with 97% of 0.05% formic
acid and
3% of methanol; and from 7.6 minutes to 20 minutes with 100% of 0.05% formic
acid.
In this alternative, typically the column temperature for HPLC is about 30 C,
the sample
temperature for HPLC is about 5 C, the flow rate for HPLC is about 0.5
mL/min, and
the injection volume is about 100 L. In this alternative, typically, for
ELSD, the gain is
about 400, the drift tube temperature is about 45 C, the gas pressure is about
35 PSI of
nitrogen, the nebulizer is set to cooling, the data rate is 2 points per
second, and the
Rayleigh factor is about 6Ø Typically, in this alternative, standards of
dulcitol at 0.005
to 0.1 mg/mL are employed to determine the sensitivity and linearity of the
system.
Typically, in this alternative, the retention time for dulcitol is about 6.4
minutes and the
retention time for dianhydrogalactitol is about 12.1 minutes. In this
alternative, the
amount and percentage of a dulcitol impurity can be determined from the
results of
HPLC and ELSD. Also, in this alternative, the amount and percentage of an
unknown
impurity other than dulcitol can be determined from the results of HPLC and
ELSD.
[0070] The invention is illustrated by the following Examples. These examples
are for illustrative purposes only, and are not intended to limit the
invention.
Examples
Example 1
HPLC Analysis of Dianhydrogalactitol Preparations Employing Isocratic Sodium
Hydroxide Elution
[0071] The procedure described in this Example is used for determining
dulcitol
and related impurities in a dianhydrogalactitol drug preparation by ion
exchange high
performance liquid chromatography with refractive index detection.
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[0072] In this procedure, the samples are prepared with dianhydrogalactitol at
a
target concentration of 5 mg/mL. Dulcitol, dianhydrogalactitol, and related
impurities are
separated using an anion exchange column (Hamilton RCX-10, 250 x 4.1 mm, 7
pm),
with 50 mM NaOH as isocratic mobile phase with refractive index detection.
Dulcitol
concentration is determined with an external reference standard and the
contents of
related substances are estimated using a DAG reference standard.
[0073] A suitable HPLC system and data acquisition system is an Agilent
Technologies 1200 Series HPLC system or equivalent equipped with the
following: Quat
pump, Model G1311A or equivalent; auto sampler, Model 1329A or equivalent; RID
detector, Model 1362A or equivalent; column temperature controller capable of
30 3
C; and degasser, Model G1322 or equivalent. The column is a Hamilton RCX anion
exchange column 250 x 4.1-mm, 7 pm, P/N 79440, or equivalent. Data acquisition
is
performed by a ChemStation and ChemStore Client/Server or an equivalent data
system.
[0074] The following chemicals are used. Water is Milli-Q water or deionized
water. Sodium hydroxide is standard purified grade. Dulcitol and DAG reference
standards are of purity > 98.0%.
[0075] For the mobile phase (50 mM NaOH), 2.0 g NaOH is dissolved in 1 liter
of water. The solution is filtered through an 0.45-pm filter. The mobile phase
can be
stored up to 1 month at room temperature. For the dulcitol reference stock
solution
(nominal 500 pg/mL), 25 mg of dulcitol reference standard is accurately
weighed into a
50-mL volumetric flask. The dulcitol is diluted to volume with deionized water
and
mixed well. The prepared stock solution can be stored up to 3 days at 2-8 C.
For the
DAG reference stock solution (nominal 500 pg/mL), 25 mg of DAG reference
standard
is accurately weighed into a 50-mL volumetric flask. The DAG is diluted to
volume with
deionized water and mixed well. The prepared stock solution can be stored up
to 3
days at 2-8 C. For the dulcitol-DAG standard solution (dulcitol 50 pg/mL +
DAG 50
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pg/mL; each at 1 /0 of 5 mg/mL DAG), 1.0 ml of dulcitol stock and 1.0 ml of
DAG stock
are each quantitatively transferred into a 10-mL volumetric flask, diluted to
volume with
water and mixed well.
[0076] For DAG sample preparation from an API sample (nominal 1 mg/mL),
about 25 mg of API sample of DAG is accurately weighed in a clean 25-mL
volumetric
flask. The DAG API sample is dissolved in approximately 5 mL of deionized
water,
diluted to volume with deionized water, and mixed. An aliquot of 1 to 2 mL of
the test
sample is transferred into an HPLC vial. Prepared samples can be stored for up
to 2
days at 2-8 C.
[0077] For DAG sample preparation (nominal 5 mg/mL) for an API sample,
about 50 mg of the API sample is accurately weighed into a clean 10-mL
volumetric
flask. The DAG API sample is dissolved in approximately 5 mL of water, diluted
to
volume with water, and mixed.
[0078] For DAG sample preparation from a lyophilized (40 mg/vial) sample, the
sample is removed from the refrigerator in which the sample is stored and the
seal
removed. A volume of water of 5.0 mL is quantitatively transferred and the
solution is
mixed to dissolve the DAG, yielding an 8 mg/mL solution. An aliquot of 1.0 g
of the
reconstituted solution is diluted to 8.0 g with deionized water and mixed. A
further
aliquot of 1 to 2 mL of the test sample is transferred into an HPLC vial.
Prepared
samples can be stored for up to 2 days at 2-8 C.
[0079] For DAG sample preparation (nominal 5 mg/mL) for the drug product
using lyophilized powder (40 mg/vial), the closure of the vial is cleaned and
removed.
The lyophilized vial is reconstituted with 8.0 mL water to yield a 5 mg/mL
solution. An
aliquot of 1 to 2 mL is transferred to an HPLC vial. Samples are prepared in
duplicate
(using two vials). Prepared samples can be stored at 2-8 C for up to 24
hours.
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[0080] For HPLC analysis, the HPLC system is turned on and the detector is
allowed to warm up for at least 20 minutes. If necessary, place the HPLC
mobile phase
prepared as described above into the appropriate solvent inlet. The solvent
line is
primed with the mobile phase. The system and the column are equilibrated with
HPLC
mobile phase at a flow rate of 1.5 mL/min for at least 30 minutes. A sample
analysis
sequence is created. Once system suitability has been confirmed, a water blank
is
injected followed by injections of the standards and then the samples. A
dulcitol-DAG
standard is inserted after every 10 injections of samples and then a last
bracketing
standard at the end of the run. A suitable sample analysis sequence is shown
in Table
1.
Table 1
Sample Analysis Sequence
Injectons
...............................................................................
...............................................................................
................................
Blank (100% water) 1
System Suitability Test, Dul-DAG Standard (50 6
pg/mL each)
Blank (100% water) 1
Test Samples (DAG drug substance and/or drug 2
product) (n
20)
- assay (duplicate preparations)
Bracketing Standard, Dul-DAG Standard (50 2
pg/mL each)
Blank (water) 1
[0081] The samples are analyzed using RID. As indicated above, a suitable
column is a Hamilton RCX ion exchange column (250 x 4.1 mm, 7 pm), P/N 79440
or
equivalent. The mobile phase is 50 mM NaOH in deionized water (isocratic
elution).
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The flow rate is 1.5 mL/min. The column temperature is 30 C. The injection
volume is
50 pL. Detection is by RID at 35 C. The run time is 8 minutes.
[0082] For analysis and integration of the chromatograms, the HPLC software is
used. The chromatograms for the blank, the samples, and the test standards are
reviewed and compared. Manual integration and assignment of some peaks may be
necessary. Integration parameters such as slope sensitivity, peak width, peak
height
threshold value for rejection, integration type of shoulder peak, baseline,
and split peak,
as well as other parameters, are adjusted to obtain appropriate integration
and values
for these parameters are recorded and applied to all samples and standards.
[0083] Suitability of the system is assessed as follows. The six replicated
injections of the dulcitol-DAG standard solution are evaluated using the
chromatographic performance requirements of Table 2.
Table 2
Chromatographic Performance Requirements
Dulcitol Retention time (RT): ¨ 2 min.
DAG Retention time (RT): ¨ 6 min.
Area Response variation (YoRSD: 10.0 A
Retention time variation (YoRSD: 2.0%
[0084] The dulcitol and DAG peak area in the bracketing standard solution
injections should be 80% to 120% of the average peak area of each in previous
SST
injections. In case one bracketing standard fails to meet the criterion, the
samples
analyzed after the final passing bracketing standard should be re-analyzed.
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[0085] In the analysis of the data, relative peak area = (peak area/total peak
area) x 100, where "peak area" is the individual peak area and "total peak
area" is the
sum of peak areas from all peaks.
[0086] Dulcitol concentration is calculated as indicated: Dulcitol (Cu, pg/mL)
=
Cs x mean sample peak area / mean dulcitol peak area of Dul-DAG standard
injections,
where Cs is dulcitol concentration in pg/mL.
[0087] Dulcitol content (wt %) in DAG drug substance or drug product is
calculated as indicated: Dulcitol wt (:)/0 = Cu (pg/mL) /1000 / SC (mg/mL) x
100%, where
Cu is dulcitol concentration (pg/mL) calculated as above, and SC is sample
concentration (mg/mL) as prepared for drug substance or drug product. If
dulcitol is
present, the weight percent of dulcitol is reported if equal to or greater
than 0.05%; it is
reported to the nearest 0.01%.
[0088] If an unknown or previously unidentified impurity other than dulcitol
is
present in the DAG preparation, its concentration is calculated as follows:
Unknown
impurity concentration (pg/mL) = Cs x mean sample peak area / mean DAG peak
area
of Dul-DAG standard injections. If present, the unknown impurity weight
percent is
calculated as follows: Cu (pg/mL) /1000 / SC (mg/mL) x 100%, where Cu =
unknown
concentration (pg/mL) calculated as above, and SC = sample concentration
(mg/mL)
as prepared in [0077] for drug substance or [0079] for drug product. Each
unknown
impurity, if present, is reported if equal to or greater than 0.05%; it is
reported to the
nearest 0.01%.
[0089] The assay results in weight percent are calculated for each sample and
for the mean of duplicate samples.
Example 2
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HPLC Analysis Employing Evaporative Light Scattering Detector Using Gradient
of
Water/Acetonitrile
[0090] To improve resolution of impurities, another method of HPLC analysis
was employed using an evaporative light scattering detector (ELSD) with a
gradient of
water/acetonitrile as detailed below.
[0091] Due to the limitations of the refractive index (RI) detector, the
HPLC/RI
method does not have sufficient specificity to obtain reliable impurity
profile data, which
pose the risks of exposure of patients to unacceptable levels of impurities
that are
unknown or are incompletely characterized. To address this concern, a more
sensitive
detector, such as the evaporative light scattering detector (ELSD)
manufactured by
Agilent, is used in conjunction with HPLC system for determination of
impurities found in
dianhydrogalactitol drug substance or drug product.
[0092] For example, a DAG sample was analyzed by HPLC/ELSD method using
a YMC 018 column with the gradient shown in Table 3:
Table 3
Time (miri) % water
aceton true
0 95 5
15 95 5
15.1 70 30
20 70 30
20.1 to 35 95 5
[0093] As shown in the chromatogram (Figure 3), dulcitol was eluted at
retention
time of 4.5 minutes. The peaks eluted right after dulcitol were identified to
be mono-
epoxide related compounds, as supported by LC-MS data summarized in Table 4.
The
peak observed at 11.46 minutes is possibly DAG dimer and the peak eluted at
22.6
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minutes contributed multiple peaks in LC-MS with m/z of 271, 357, 417, 512,
and other
peaks, which might be condensed products (Figure 4). These data are consistent
with
the impurity profile expected by previous studies. As expected, the
monoepoxide and
dulcitol are two major degradation products obtained by this method.
Table 4
RT (min) % Area Base peak m/z* m/z Comments
4.55 1.22 [M+Na] = 205.1 182 dulcitol
328 Mono-epoxide dimer
4.92 2.68 [M+Naf = 351.1
187 Mono-epoxide Na adduct
5.21 2.31 [M+Na] = 187.1 164 Mono-epoxide
6..81 91 35 = 169.1 146 DAG
11.46 0.22 [M+Na] = 289.1 266 DAG dimer
22.61 2.22 271, 357, 417, 521 Condensed products
Example 3
HPLC Analysis with Formic Acid in Water/Methanol Gradient to Improve
Resolution of
Mono-Epoxide Peaks
[0094] To improve the resolution of mono-epoxide peaks, a new method was
developed. This new method employed the following parameters: The column was
Atlantis 018, 250 x 4.6 mm, 5 m. The column temperature was 30 C. The flow
rate
was 0.5 mL/min. The injection volume was 100 L. The ELSD detector was
operated
in cooling mode with the drift tube temperature of 35 C and gain 400, 2 pps,
45 PSI.
Mobile Phase A was 0.05% formic acid in water. Mobile Phase B was 100%
methanol.
The gradient was shown in Table 5:
Table 5
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Time, min %A %B
0 100 0
25 90 10
25.1 - 35 10 90
35.1 ¨ 50 100 0
[0095] Better resolution of the early eluting impurities has been observed
(refer
to chromatogram of DAG sample below in Figure 5). Dulcitol labeled peak 2 was
eluted
at retention time of 6.26 minutes or relative retention time (RRT) of 0.59.
Dianhydrogalactitol was eluted at 10.86 minutes.
[0096] Since ELSD response is not linear, an external calibration standard
curve
is required for a known impurity, such as dulcitol, to determine the impurity
content in a
dianhydrogalactitol sample tested. For an unknown impurity contained in a
sample of
dianhydrogalactitol, the unknown impurity content can be estimated using a
calibration
standard curve established by chromatography of dianhydrogalactitol reference
material.
Example 4
Further Improved Method for Detection or Determination of Impurities Employing
Dual-
Gradient HPLC Elution
[0097] A further improved analytical method for the detection or determination
of
impurities in dianhydrogalactitol employs HPLC and ELSD with dual-gradient
elution in
HPLC. This method is described below.
[0098] In this analytical method, the following materials and equipment are
used:
an Atlantis 018, 250 x 4.6-mm, 5- m HPLC column; a quaternary or binary HPLC
pump; an Evaporative Light Scattering Detector (ELSD); an integrator or
computer-
based analytical system; a calibrated analytical balance; and Class A
volumetric flasks
and pipettes. The following reagents and standards are used: a dulcitol
reference
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standard as described above; HPLC grade water; HPLC grade or equivalent formic
acid
(FA); HPLC grade or equivalent acetonitrile (ACN); and HPLC grade or
equivalent
methanol (Me0H).
[0099] For solutions that are employed in the method, the volume may be scaled
to suit the needs of the analysis. It is important that all mobile phases are
filtered. The
sintered glass in the filtration apparatus may be a source of buffers that may
interfere
with sensitivity in the ELSD. All filtration apparatus should be rinsed
thoroughly with
Milli-Q grade water. To perform this, approximately 500 mL of Milli-Q grade
water is
filtered through the filtration apparatus. The water is discarded and the
mobile phase is
then filtered.
[0100] Test solution preparations are to be made in a fume hood using
appropriate PPE (gloves, lab coat, and safety glasses). Test solution
preparations are
to be stored in a fume hood for disposal and are to be labelled appropriately.
[0101] Mobile Phase A is prepared by pipetting 0.5 mL of formic acid into 1000
mL of water and mixing well. The Mobile Phase A is filtered and degassed.
[0102] Mobile Phase B is Me0H. The Mobile Phase B is filtered and degassed.
[0103] Diluent A is water. Diluent B is prepared by mixing 20 mL of ACN with
180 mL of water and mixing well.
[0104] The standard and sample solution preparation is described below. The
blank solution is water. The dulcitol stock solution is prepared by accurately
transferring
100 mg of dulcitol reference standard to a 20-mL volumetric flask. About 15 mL
of
Diluent B is added and sonicated to dissolve. The solution is allowed to cool
down to
room temperature and diluted to volume with Diluent B and mixed well (5
mg/mL). The
following standard solutions are prepared: 0.2, 0.1, 0.08, 0.05, 0.03, 0.01
and 0.005
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mg/mL (system sensitivity solution). A 4.0% standard solution is prepared by
pipetting
2.0 mL of dulcitol stock solution into a 50-mL volumetric flask. The solution
is diluted to
volume with water and mixed well (0.2 mg/mL). A 2.0% standard solution is
prepared
by pipetting 5.0 mL of 4.0% standard solution into a 10-mL volumetric flask.
The
solution is diluted to volume with water and mixed well (0.10 mg/mL). A 1.6%
standard
solution is prepared by pipetting 4.0 mL of 4.0% standard solution into a 10-
mL
volumetric flask. The solution is diluted to volume with water and mixed well
(0.08
mg/mL). A 1.0% standard solution is prepared by pipetting 2.5 mL of 4.0%
standard
solution into a 10-mL volumetric flask. The solution is diluted to volume with
water and
mixed well (0.05 mg/mL). An 0.60% standard solution is prepared by pipetting
3.0 mL
of 4.0% standard solution into a 20-mL volumetric flask. The solution is
diluted to
volume with water and mixed well (0.30 mg/mL). An 0.20% standard solution is
prepared by pipetting 1.0 mL of 4.0% standard solution into a 20-mL volumetric
flask.
The solution is diluted to volume with water and mixed well (0.01 mg/mL). An
0.10%
standard solution (system sensitivity solution) is prepared by pipetting 5.0
mL of 0.20%
standard solution into a 10-mL volumetric flask. The solution is diluted to
volume with
water and mixed well (0.005 mg/mL).
[0105] Test sample working solutions are to be prepared in duplicate (A and
B).
Sample solutions must be prepared just before analysis. Sample injections must
be
performed within 15 minutes of sample solution preparation. Sample dilution
may be
required, in some cases, to quantitate any impurities that are overloaded.
[0106] For sample preparation, approximately 50 mg of test sample is
accurately
transferred into a 10-mL volumetric flask. The test sample is dissolved in
water and
diluted to volume and mixed well (5 mg/mL).
[0107] The HPLC operating conditions are as follows: The column is the
Atlantis 018 250 x 4.6-mm, 5- m HPLC column. Mobile Phase A is 0.05% FA in
water.
Mobile Phase B is Me0H. Gradients A and B are described below in Table 6. The
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column temperature is 30 C. The sample temperature is 5 C. The flow rate is
0.5
mL/min. The injection volume is 100 L. The run time is 50 minutes for
Gradient A and
20 minutes for Gradient B.
Table 6
Gradient A Time, %A %B
mins
0 100 0
25 90 10
25.1 10 90
35 10 90
35.1 100 0
50 100 0
Gradient B Time, %A %B
mins
0 100 0
7.5 97 3
7.6 100 0
20 100 0
[0108] The ELSD operating conditions are as follows: The gain is 400. The
drift
tube temperature is 45 C. The gas pressure (nitrogen) is 35 PSI. The
nebulizer is set
to cooling. The data rate is 2 points per second. The Rayleigh factor, set
directly in the
detector, is 6Ø
[0109] The injection sequence is shown in Table 7. Blank injections are to be
repeated until a stable and reproducible baseline is obtained.
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Table 7
Solution # of Injections Gradient
Blank Solution 1* A
0.1% Standard Solution (System Sensitivity) 1
0.20% Standard Solution 5
0.60% Standard Solution 1
1.0% Standard Solution 1
1.6% Standard Solution 1
2.0% Standard Solution 1
0.20% Standard Solution Check 1
Test Sample Solution Prep A 1 A
Test Sample Solution Prep B 1 A
0.20% Standard Solution Check 1
[0110] Additional test sample injections may be added as required. No more
than 6 test sample solution injections are to be performed before repeating
the 0.20%
standard solution check injection.
[0111] For evaluation, all peaks should be integrated associated with any
unknown impurities detected. If baseline noise is an issue, make sure that the
waste is
properly drained from the detector. It may be necessary to check that there is
no liquid
accumulated inside the tubing draining the waste from the ELSD. The tubing
position
should be corrected as needed. If the waste is being properly drained and the
baseline
noise is an issue, the detector may be cleaned. If required, the following
cleaning
method may be used prior to analysis. For cleaning, the HPLC conditions are as
follows: The column is to be removed from the instrument and a union is to be
used.
The mobile phase is 100% H20 (isocratic 100%). The flow rate is 1.0 mL per
minute.
The column temperature is ambient temperature. The run time is 60 minutes. The
ELSD operating conditions are as follows: The gain is 50. The drift tube
temperature is
100 C. The gas pressure (nitrogen) is 50 PSI. The nebulizer is set to heating
at 75%.
[0112] Typical retention times are shown in Table 8. In Table 8, "DAG" is
dianhydrogalactitol. DAG in the test sample is not quantitated in this method.
DAG is
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observed as a wide peak due to the concentration of DAG required. Retention
time for
DAG in sample solution is approximately between 10 and 13 minutes.
Table 8
Component RT, min RRT
Dulcitol 6.4 0.53
*DAG 12.1* 1.00
[0113] Figure 6 is an example chromatogram of a blank solution.
[0114] Figure 7 is an example chromatogram of 0.10% standard solution
(system sensitivity solution).
[0115] Figure 8 is an example chromatogram of a test solution.
[0116] System suitability requirements are as follows. For blank solution
injection, no interfering peaks should be observed at the retention time of
the dulcitol
peak or of any known impurities. A stable and reproducible baseline should be
observed; continue to inject the blank solution until this condition is met.
For system
sensitivity, the dulcitol peak subsequent to the injection of the 0.10%
standard solution
should be observed. The signal-to-noise ratio for the dulcitol peak should be
reported.
If contamination in the mobile phase is suspected (i.e., baseline noise is
greater than
1.0 LSU) or the dulcitol peak is not observed, the mobile phase should be re-
prepared
or the clean-up procedure described above should be performed. The USP tailing
factor for the dulcitol peak for the first and last injections of the 0.20%
standard solution
is no more than 2Ø For precision, the % RSD for the log of peak area in the
five
injections is calculated. The % RSD must be no more than 15%.
[0117] For calculations, all peaks that are not attributed to the blank should
be
integrated. The logarithm of the response versus the logarithm of the
concentration for
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the 0.10% through the 2.0% standard solutions for dulcitol (including the 5
precision
injections of the 0.20% standard) is plotted. The correlation coefficient (r)
must be no
less than 0.98 for the linearity curve. The slope and intercept are determined
from the
curve. The linearity curve for dulcitol is used to determine the concentration
in mg/mL
of unknowns and dulcitol impurities in the sample.
[0118] For determination of individual unknown impurities in the sample, the
slope and intercept from the linearity curve for dulcitol as described above
are used.
Using the Log [area response] of the Unknown, the Log [concentration] of the
unknown
is determined as follows using Equation (1):
Log [Unknown concentration] = Log [Unknown responsel-Intercept
Slope
(1).
[0119] The amount of unknown impurity (in mg/mL) is determined as follows
using Equation (2):
Unknown concentration (mg/mL) = 1 oLog[Unknown concentration]
(2).
[0120] The percentage of each unknown impurity is determined as follows using
Equation (3):
(:)/0 Unknown = Unknown concentration (mg/mL) x 100
Spl. Conc. (mg/mL)
(3).
[0121] Alternatively, quantitation using dulcitol standards may be formed
using
log-log linear function in Empower (Waters Corp.)
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[0122] Similar equations, specifically Equations (4)-(6), are used for the
determination of the dulcitol impurity in the sample.
[0123] Using the log [area response] for dulcitol, the log [concentration] of
dulcitol is determined using Equation (4):
Log [Dulcitol concentration] = Log [Dulcitol responsel-Intercept
Slope
(4).
[0124] The concentration of dulcitol impurity (in mg/mL) is determined using
Equation (5):
Dulcitol concentration (mg/mL) = 1 oLog[Dulcitol concentration]
(5).
[0125] The percentage of dulcitol impurity is determined using Equation (6):
A Dulcitol = Dulcitol concentration (mg/mL) x 100
Spl. Conc. (mg/mL)
(3).
ADVANTAGES OF THE INVENTION
[0126] The present invention provides an improved analytical method for the
detection and quantitation of impurities present in dianhydrogalactitol
preparations,
including dulcitol and unknown impurities, as well as methods for isolation
and
identification of unknown impurities present in dianhydrogalactitol
preparations. The
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methods of the present invention allow the large-scale preparation of
dianhydrogalactitol
of high purity suitable for pharmaceutical use and reduce the possibility of
significant
side effects caused by the presence of impurities in dianhydrogalactitol
preparations
intended for pharmaceutical use.
[0127] Methods according to the present invention possess industrial
applicability for analysis of dianhydrogalactitol preparations and
determination and
quantitation of impurities in dianhydrogalactitol preparations.
[0128] With respect to ranges of values, the invention encompasses each
intervening value between the upper and lower limits of the range to at least
a tenth of
the lower limit's unit, unless the context clearly indicates otherwise.
Moreover, the
invention encompasses any other stated intervening values and ranges including
either
or both of the upper and lower limits of the range, unless specifically
excluded from the
stated range.
[0129] Unless defined otherwise, the meanings of all technical and scientific
terms used herein are those commonly understood by one of ordinary skill in
the art to
which this invention belongs. One of ordinary skill in the art will also
appreciate that any
methods and materials similar or equivalent to those described herein can also
be used
to practice or test this invention.
[0130] The publications and patents discussed herein are provided solely for
their disclosure prior to the filing date of the present application. Nothing
herein is to be
construed as an admission that the present invention is not entitled to
antedate such
publication by virtue of prior invention. Further the dates of publication
provided may be
different from the actual publication dates which may need to be independently
confirmed.
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[0131] All the publications cited are incorporated herein by reference in
their
entireties, including all published patents, patent applications, and
literature references,
as well as those publications that have been incorporated in those published
documents. However, to the extent that any publication incorporated herein by
reference refers to information to be published, applicants do not admit that
any such
information published after the filing date of this application to be prior
art.
[0132] As used in this specification and in the appended claims, the singular
forms include the plural forms. For example the terms "a," "an," and "the"
include plural
references unless the content clearly dictates otherwise. Additionally, the
term "at least"
preceding a series of elements is to be understood as referring to every
element in the
series. The inventions illustratively described herein can 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 future shown and described or any portion thereof, and 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
inventions herein disclosed can be resorted by those skilled in the art, and
that such
modifications and variations are considered to be within the scope of the
inventions
disclosed herein. The inventions have been described broadly and generically
herein.
Each of the narrower species and subgeneric groupings falling within the scope
of the
generic disclosure also form part of these inventions. This includes the
generic
description of each invention with a proviso or negative limitation removing
any subject
matter from the genus, regardless of whether or not the excised materials
specifically
resided therein. In addition, where features or aspects of an invention are
described in
terms of the Markush group, those schooled in the art will recognize that the
invention is
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also thereby described in terms of any individual member or subgroup of
members of
the Markush group. It is also to be understood that the above description is
intended to
be illustrative and not restrictive. Many embodiments will be apparent to
those of in the
art upon reviewing the above description. The scope of the invention should
therefore,
be determined not with reference to the above description, but should instead
be
determined with reference to the appended claims, along with the full scope of
equivalents to which such claims are entitled. Those skilled in the art will
recognize, or
will be able to ascertain using no more than routine experimentation, many
equivalents
to the specific embodiments of the invention described. Such equivalents are
intended
to be encompassed by the following claims.
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