Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Novel Assay Methods
Technical Field
The present invention relates to a novel assay for the analysis of memantine
and related
compounds.
Background Art
In order to secure marketing approval for a pharmaceutical product, a
manufacturer must
submit detailed evidence to the appropriate regulatory authorities to show
that the product
is suitable for release on to the market. The regulatory authority must be
satisfied, inter
alia, that the product is acceptable for administration to humans and that the
particular
pharmaceutical composition which is to be marketed is free from impurities at
the time of
release and that it has an acceptable storage stability (shelf-life).
Submissions made to regulatory authorities therefore must include analytical
data which
demonstrate that impurities are absent from the active pharmaceutical
ingredient (API) at
the time of manufacture, or are present only at a negligible level, and that
the shelf-life of
the pharmaceutical composition is acceptable.
Potential impurities in APIs and pharmaceutical compositions include residual
amounts of
synthetic precursors to the API, by-products which arise during synthesis of
the active
agent, residual solvent, isomers of the active agent, contaminants which were
present in
materials used in the synthesis of the API or in the preparation of the
pharmaceutical
composition, and unidentified adventitious substances. Other impurities which
may appear
on storage include substances resulting from degradation of the active agent,
for instance
by oxidation or hydrolysis.
The health authorities have very stringent standards and manufacturers must
demonstrate
that their product is relatively free from impurities (within certain agreed
limits) and that
this standard is reproducible for each batch of pharmaceutical product that is
produced.
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The tests that are required to satisfy the relevant health authorities that
the API and
pharmaceutical compositions are safe and effective include a purity assay,
content
uniformity test, dissolution testing and related substances test. The purity
assay determines
the purity of the test product (analyte) when compared to a standard of a
known purity,
while the related substances test is used to quantify all of the impurities
present in the
product. The content uniformity test ensures that batches of product (e.g. a
tablet) contain
a uniform amount of API and the dissolution testing ensures that each batch of
product
has a consistent dissolution and release of the API.
When developing these methods for the analysis of either the API or the
pharmaceutical
composition (e.g. the tablet or capsule), the technique of choice would
usually be High
Performance I.iquid Chromatography (HPLC) combined with a UV-Visible
Spectrometer.
The API and any impurities that are present in the mixture are separated on
the HPLC
stationary phase and they can be quantified by detection and measurement via
the W-
Visible spectrometer.
HPLC is a chromatographic separating technique in which high-pressure pumps
force the
substance or mixture being analysed (analyte) together with a liquid solvent -
the mobile
phase (also referred to as the eluent) - through a separating column
containing the
stationary phase. If a constituent substance interacts strongly with the
stationary phase, it
remains in the column for a relatively long time, whereas a substance that
does not interact
with the stationary phase as strongly leaves the column sooner. Depending on
the strength
of the interactions, the various constituents of the analyte appear at the end
of the
separating column at different times - retention times - where they can be
identified by
means of a suitable detector.
This method works well for compounds that have a chromophore, that is to say
compounds that absorb energy in the UV-Visible range of the electromagnetic
spectrum.
However, for other compounds which do not contain a chromophore, an
alternative
detection method must be found. This sometimes involves derivatising the
subject
compound with a reagent such that the resultant analyte contains a chromophore
which
can be detected using a UV-Visible detector.
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An alternative detection method is to replace the UV-Visible detector with a
fluorescence
detector. However, this technique requires the analyte to have a fluorophore
but, as
compounds which fluoresce are very rare, the subject compound must usually be
derivatised with a chemical moiety which does fluoresce. This again involves
derivatising
the analyte, this time with a reagent to produce a fluorescent compound.
Derivatisation of
the analyte is both time consuming and difficult to develop, particularly as
some analytes
will not have a functional group that is readily reacted with a derivatising
reagent. In
addition, some related substances may lose, on degradation, the functional
group which can
be derivatised and consequently these related substances will not be detected.
Another
drawback of fluorescent derivatisation is that fluorescence detectors are not
as common in
the analytical laboratory of a pharmaceutical company as a UV-Visible
detector.
Gas chromatography (GC) is regularly used by pharmaceutical companies to
quantify
residual solvents in both pharmaceutical compositions and APIs. The method
generally
involves dissolving the test sample in a quantity of one or more solvents,
injecting the
sample and volatilisation of the solvents using a headspace technique. The
analytes are
typically detected using a flame ionisation detector (FID) technique. These
FID detectors
can be used for almost all pharmaceutical products, as they are able to detect
any analyte
which contains a C-C or C-H bond. However, in order to analyse compounds using
gas
chromatography, the compounds must have a low enough vapour pressure to allow
them
to be volatilised prior to interaction with the chromatographic column.
Gas chromatography is a highly efficient, sensitive method, which is used to
analyse
complex mixtures of substances. It is a chromatographic technique that uses
gas as the
mobile phase and the gas is routed through a column of a defined diameter
containing the
stationary phase, a bighly viscous liquid. Most capillary columns are
generally made of
fused silica, have an internal diameter of between 0.1 and 0.5 mm and are
between 5 and 60
m long. The inner column wall is coated with the stationary phase, whereby the
film is 0.1
to 5 m thick. Hydrogen, helium or nitrogen is usually used as the carrier
gas, whereby
hydrogen offers significant advantages in terms of separating efficiency and
analytical
speed.
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One basic prerequisite for gas chromatography is that the substance can be
vaporised
without decomposing - assuming that the substance is not available in gaseous
form.
Substances with inadequate volatility can be derivatised selectively to
produce more volatile
substances, such as trimethylsilyl derivatives. The sample can be introduced
into the
capillary column by directly injecting a liquid sample or by extracting from
the gas space of
a sample vial. The latter method, referred to as headspace gas chromatography,
is a special
technique which offers a means of detecting low concentrations of highly
volatile
substances in liquid or solid samples.
An alternative detection method is to use mass spectrometry to detect the
compounds
separated by either gas chromatography (GC-MS) or liquid chromatography (LC-
MS).
However, this method requires an expensive mass spectrometer which is not
convenient
for many analytical laboratories.
Memantine is the common chemical name for 3,5-
dimethyltricyclo[3.3.1.13'7]decan-l-amine
or 1-amino-3,5-dimethyladamantane. It is a moderate affinity N-methyl-D-
aspartate
(NMDA) receptor antagonist.
Bormann et al discloses in US patent 5061703 that memantine hydrochloride is
useful for
the prevention and treatment of cerebral ischemia after apoplexy, open-heart
surgery,
subarachnoidal hemorrhage, transient cerebro-ischemic attacks, perinatal
asphyxia, anoxia,
hypoglycemia, apnoea and Alzheimer's disease. In addition, a method of using
memantine
for reducing non-ischemic NMDA receptor mediated neuronal degeneration in a
mammal
is disclosed in US patent 5614560.
Memantine hydrochloride, represented by Formula I, is currently approved and
marketed
for the prevention and treatment of moderate to severe Alzheimer's disease.
NH2 HCl
4 CH3 (1)
CH3
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Methods for the preparation of memantine are known in the art and these
methods include
those disclosed in US patent 4122193 and by K Gerzon et al in J. Med. Chem.,
vol. 6, pages
760-763, 1963.
Memantine is very unusual as a pharmaceutical compound, because the molecule
does not
contain a chromophore. As discussed above, this means that the usual choice of
HPLC
with UV-Visible detector is not feasible as a method for analysing memantine
and its
impurities. Memantine also contains very few functional groups, which means
that
derivatisation to form a compound containing a suitable chromophore or
fluorophore
would be difficult in addition to the general disadvantages of such a method.
Several methods have been published in the literature to analyse memantine,
but these
various methods are all encumbered with the problems described above as they
involve
derivatisation and/or specialist detection equipment.
Examples of more recent publications disclosing these methods include the
following:
HPLC methods using fluorescent reagents to derivatise memantine in articles by
T-H Duh
et al in J. Chromatography A, vol. 987, pages 205-209, 2003, and J.
Chromatography A, vol.
1088, pages 175-181, 2005. An HPLC method using derivatisation and UV
detection by C
Shuangjin in J. Pharm. & Biomed. Analysis (in press). A liquid chromatography-
mass
spectrometry (LC-MS) method by MJ Koeberle et al in J. Chromatography B, vol.
787,
pages 313-322, 2003. A specific liquid chromatography-tandem mass spectrometry
(LC-
MS/MS) method by AA Almeida et al in J. Chromatography B, vol. 848, pages 311-
316,
2007. An electrophoretic method by N Reichova et al in Electrophoresis, vol.
23, pages
259-262, 2002.
Consequently, although several methods have been disclosed for the analysis of
memantine
and its impurities, there is still a need for an alternative method which
avoids the problems
associated with the known methods as discussed above.
Studies by the inventors have surprisingly shown that memantine is volatile
enough to be
used in GC without derivatisation and without decomposition. This enables
analysis of the
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product using GC and a sample of memantine can be injected directly onto the
column
without the need for the headspace sampling described above. It is also
possible to
quantify the organic impurities of inemantine using this GC method. The
inventors have
also developed novel, efficient and reproducible methods of sample preparation
for the GC
method.
Object of the Invention
It is therefore an object of the present invention to provide a novel,
alternative method for
analysing memantine and its impurities whilst avoiding the typical prior art
problems
associated with derivatisation and the use of specialist detection methods.
Summary of the Invention
Therefore, a first aspect of the present invention provides a method for
analysing
memantine, or a pharmaceutically acceptable salt thereof, and its impurities
using gas
chromatography. The method preferably involves a test sample of memantine
being
injected directly onto the GC column without headspace sampling. The method
preferably
uses flame ionisation detection. Memantine is preferably in the form of the
free base, the
hydrochloride salt or another acid addition salt.
The method of the first aspect of the invention can be used to analyse
memantine API or
memantine when prepared as a pharmaceutical composition. Preferably the
memantine is
prepared as a pharmaceutical composition.
The pharmaceutical compositions that can be analysed by the method of the
first aspect of
the invention include solid and liquid compositions and optionally comprise
one or more
pharmaceutically acceptable carriers or excipients. Solid form compositions
include
powders, tablets, pills, capsules, cachets, suppositories, and dispersible
granules. Liquid
compositions include solutions or suspensions which can be administered by
oral,
injectable or infusion routes.
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The term "impurities" as used herein throughout the specification can mean
either
impurities formed in the manufacture of the API or the pharmaceutical
composition
and/or impurities (or related substances) formed by degradation in the
pharmaceutical
composition on storage.
As described above, memantine is usually present as a hydrochloride salt for
pharmaceutical use. This introduces an additional problem for analysis by gas
chromatography, because the salt is very acidic and can corrode the column
used for
separation. Therefore, the salt must typically be neutralised before analysis
to form
memantine free base. A typical sample preparation technique in such cases
would be to
neutralise the memantine hydrochloride, present as the API or when present in
a
pharmaceutical composition, by reaction with an aqueous solution of an alkali,
such as
sodium hydroxide. The memantine free base thus formed and the corresponding
impurities are then extracted using an organic solvent such as chloroform.
However, this extraction technique can be very unreliable for several reasons
including
variable extraction rates into the organic layer and incomplete separation of
the aqueous
and organic solvent layers. The inventors have found that the quantity of
memantine that
is recovered following this type of extraction process is not consistently
reproducible and
this leads to additional problems in quantitation of the related substances.
Consequently a second aspect of the invention affords a method for analysing
memantine
hydrochloride, or another pharmaceutically acceptable acid addition salt
thereof, and its
impurities using gas chromatography, wherein a test sample is prepared by
mixing
memantine hydrochloride or another pharmaceutically acceptable acid addition
salt thereof,
or a pharmaceutical composition comprising memantine hydrochloride or another
pharmaceutically acceptable acid addition salt thereof, with one or more
solvent(s) and a
non-aqueous alkaline reagent, wherein the solvent(s) is capable of dissolving
the memantine
and the alkaline reagent.
The method of the second aspect of the invention preferably involves the test
sample of
memantine being injected directly onto the GC column without headspace
sampling. The
method preferably uses flame ionisation detection.
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The method of the second aspect of the invention can be used to analyse
memantine API
or memantine when prepared as a pharmaceutical composition. Preferably the
memantine
is prepared as a pharmaceutical composition.
The pharmaceutical compositions that can be analysed by the method of the
second aspect
of the invention include solid and liquid compositions and optionally comprise
one or
more pharmaceutically acceptable carriers or excipients. Solid form
compositions include
powders, tablets, pills, capsules, cachets, suppositories, and dispersible
granules. Liquid
compositions include solutions or suspensions which can be administered by
oral,
injectable or infusion routes.
Alkaline reagents that can be used in the method of the second aspect of the
invention
include inorganic alkaline reagents such as sodium carbonate; sodium
bicarbonate;
potassium carbonate; potassium bicarbonate; calcium carbonate; calcium
bicarbonate;
magnesium carbonate; magnesium bicarbonate; sodium hydroxide; potassium
hydroxide;
calcium hydroxide; lithium hydroxide; ammonium hydroxide; aluminium hydroxide;
magnesium oxide; magnesium hydroxide; magnesium aluminium hydroxide; magnesium
aluminium silicate; phosphate salts (e.g. sodium, potassium or calcium dibasic
phosphate,
tribasic calcium phosphate or trisodium phosphate); and mixtures thereof. The
alkaline
reagent is preferably an alkali metal hydroxide such as sodium hydroxide or
potassium
hydroxide. Most preferably, the alkaline reagent is potassium hydroxide.
The organic solvent(s) used in the method of the second aspect of the
invention are
preferably lower alkyl alcohols, such as methanol, ethanol, propanol, butanol
or
isopropanol or mixtures thereof. Alternatively, the organic solvent(s) may be
tetrahydrofuran, ethyl acetate or acetonitrile or any suitable organic
solvent(s) which allow
complete dissolution of the alkaline reagent and the liberated memantine free
base.
Preferably the organic solvent is methanol or ethanol and, most preferably, it
is methanol.
If required, the test sample solution formed can be separated from any
residual solids from
the remaining pharmaceutical composition by standard methods in the art such
as filtration
or centrifugation. Preferably, the test sample solution is separated by
centrifugation.
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A standard reference compound is a sample of an analyte that has a well
established purity
and a solution of the standard is usually prepared in parallel to the sample
solution and
used as a so called "external reference standard". The purity of the major
compound or its
impurities can then be calculated based on their response relative to the
standard.
However, this method has been found to be unsuitable for the analysis of
memantine due
to the poor reproducibility of the sample preparation process.
An internal reference standard is a known amount of a compound which is added
to the
sample solution prior to analysis. In most cases, a cheap and simple
hydrocarbon with a
similar structure to the analyte would be chosen. This ensures that it has a
similar retention
time to the analyte and that highly pure standards can be obtained.
The inventors have developed an alternative technique using an internal
reference standard,
which does not require extraction of an aqueous layer with an organic solvent
such as
chloroform.
The methods that the inventors have developed use amantadine hydrochloride as
the
internal reference standard. The inventors have found that, surprisingly,
amantadine and
its salts behave in a similar way to memantine and its salts during the sample
preparation
process and that the ratio of memantine : amantadine that is recovered from
the sample
preparation process matches the ratio of inemantine hydrochloride : amantadine
hydrochloride that was present in the samples prior to the sample preparation
process.
Using this approach, the problems with sample extraction are completely
overcome to
afford a simple, reliable and cost effective method for the analysis of
memantine, or a
pharmaceutically acceptable salt thereof, and its related substances using gas
chromatography.
The inventors have tested the methods extensively to show that they are
reproducible,
accurate, precise, linear with respect to concentration, and robust. The
limits of detection
and quantification have also been determined.
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The first and second aspects of the invention may include the use of an
internal reference
standard. Preferably, the internal standard reference compound is amantadine
or
rimantadine. Most preferably, the internal standard reference compound is
amantadine.
A third aspect of the invention provides a method for the analysis of
inemantine
hydrochloride and its impurities using gas chromatography, comprising:
(a) preparing a solution of an internal standard reference compound in a first
organic
solvent;
(b) mixing the internal standard reference compound solution with memantine
hydrochloride or a pharmaceutical composition comprising memantine
hydrochloride, a
second organic solvent, and a non-aqueous alkaline reagent to liberate
memantine free base
in solution; and
(c) analysing the supernatant solution.
The first and second organic solvents used in the method of the third aspect
of the
invention may or may not be the same and are preferably lower alkyl alcohols,
such as
methanol, ethanol, propanol, butanol or isopropanol. Alternatively, the first
and second
organic solvents may be tetrahydrofuran, ethyl acetate or acetonitrile or any
suitable organic
solvent which allows complete dissolution of the alkaline reagent and the
liberated
memantine free base. Preferably the organic solvent is methanol or ethanol
and, most
preferably, it is methanol.
A suitable alkaline reagent for use in the method of the third aspect of the
invention can be
an inorganic alkaline reagent such as sodium carbonate; sodium bicarbonate;
potassium
carbonate; potassium bicarbonate; calcium carbonate; calcium bicarbonate;
magnesium
carbonate; magnesium bicarbonate; sodium hydroxide; potassium hydroxide;
calcium
hydroxide; lithium hydroxide; ammonium hydroxide; aluminium hydroxide;
magnesium
oxide; magnesium hydroxide; magnesium aluminium hydroxide; magnesium aluminium
silicate; phosphate salts (e.g. sodium, potassium or calcium dibasic
phosphate, tribasic
calcium phosphate or trisodium phosphate); and mixtures thereof. Preferably,
the alkaline
reagent is a hydroxide salt such as sodium hydroxide or potassium hydroxide.
Most
preferably, the alkaline reagent is potassium hydroxide.
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Usually the alkaline reagent is added to the solution in step (b).
Alternatively, the alkaline
reagent can be added to the solution in step (a) instead of in step (b).
Preferably, the internal standard reference compound is amantadine or
rimantadine. Most
preferably, the internal standard reference compound is amantadine.
If required, the supernatant solution in step (c) can be separated from any
residual solids
from the pharmaceutical composition by standard methods in the art such as
filtration or
centrifugation. Preferably, the supernatant solution is separated by
centrifugation.
The term "non-aqueous" as used in the second and third aspects of the
invention means
that there is not enough water present to form a distinct aqueous layer which
separates
from the organic solvent layer. Accordingly, the term "non-aqueous alkaline
reagent"
means that, even though the alkaline reagent may be soluble in water, the
alkaline reagent is
dissolved in one or more organic solvents, since there is not enough water
present to form
a distinct aqueous layer which separates from the organic solvent layer.
The second and third aspects of the invention involve methods of analysing
memantine
free base using a monophasic solvent extraction. Consequently an
organic/aqueous
biphasic solvent extraction is avoided and no separation of an aqueous and
organic layer is
required, thus avoiding the problems associated with the prior art processes
described
above.
However, if a biphasic solvent extraction is preferred, the inventors have
found that the use
of chlorinated solvents such as chloroform is not very suitable for sample
preparation,
since results are not reproducible, and since the chloroform layer is below
the aqueous layer
and is consequently more difficult to separate for assay purposes. The
inventors have
surprisingly found that an aqueous alkaline/organic solvent biphasic
extraction works very
well, when lower alkyl hydrocarbon organic solvents such as pentane, hexane,
heptane and
octane or aromatic hydrocarbons such as benzene or toluene are used. The
inventors have
tested this method extensively to show that it is reproducible, accurate,
precise, linear with
respect to concentration, and robust.
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Consequently a fourth aspect of the invention provides a method for the
analysis of
memantine hydrochloride, or another pharmaceutically acceptable acid addition
salt
thereof, and its impurities using gas chromatography, comprising mixing
memantine
hydrochloride or another pharmaceutically acceptable acid addition salt
thereof, or a
pharmaceutical composition comprising memantine hydrochloride or another
pharmaceutically acceptable acid addition salt thereof, with one or more
organic solvent(s)
and an aqueous alkaline reagent to liberate memantine free base in solution in
the organic
solvent(s), separating the biphasic mixture to afford an organic solvent
layer, and analysing
the organic solvent solution.
The fourth aspect of the invention may involve the use of an internal standard
reference
compound. Preferably, the internal standard reference compound is amantadine
or
rimantadine. Most preferably, the internal standard reference compound is
amantadine.
The organic solvent(s) used in the fourth aspect of the invention is
preferably a
hydrocarbon solvent selected from one or more of the following solvents:
pentane, hexane,
heptane, octane, benzene or toluene. Preferably the hydrocarbon solvent is n-
hexane.
A suitable atkaline reagent for use in the method of the fourth aspect of the
invention can
be an inorganic alkaline reagent such as sodium carbonate; sodium bicarbonate;
potassium
carbonate; potassium bicarbonate; calcium carbonate; calcium bicarbonate;
magnesium
carbonate; magnesium bicarbonate; sodium hydroxide; potassium hydroxide;
calcium
hydroxide; lithium hydroxide; ammonium hydroxide; aluminium hydroxide;
magnesium
oxide; magnesium hydroxide; magnesium aluminium hydroxide; magnesium aluminium
silicate; phosphate salts (e.g. sodium, potassium or calcium dibasic
phosphate, tribasic
calcium phosphate or trisodium phosphate); and mixtures thereof. Preferably,
the alkaline
reagent is a hydroxide salt such as sodium hydroxide or potassium hydroxide.
Most
preferably, the alkaline reagent is potassium hydroxide.
If required, the organic solvent solution can be separated from any residual
solids from the
pharmaceutical composition by standard methods in the art such as filtration
or
centrifugation. Preferably, the organic solvent solution is separated by
centrifugation.
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The term "aqueous" as used in the fourth aspect of the invention means that
there is
enough water present to form a distinct aqueous layer which separates from the
organic
solvent layer. Accordingly, the term "aqueous alkaline reagent" means that at
least some of
the alkaline reagent is dissolved in the aqueous layer.
Alternatively to the hydrochloride salt, the four aspects of the invention may
be used to
analyse other acid addition salts of inemantine such as the hydrobromide,
hydroiodide,
maleate, mesylate, besylate, tosylate, oxalate, acetate, propionate or formate
salts. However,
the inventive method is preferably used to analyse the hydrochloride salt of
inemantine.
The pharmaceutical compositions that can be analysed by all four aspects of
the invention
include solid and liquid compositions which optionally comprise one or more
pharmaceutically acceptable carriers or excipients. Solid form compositions
include
powders, tablets, pills, capsules, cachets, suppositories, and dispersible
granules. Typical
excipients used in the solid dosage forms comprise additives conventional in
the dosage
form in question. Tableting aids commonly used in tablet formulation can be
used and
reference is made to the extensive literature on the subject, see in
particular Fiedler's
"Lexikon der Hilfsstoffe", 4th edition, ECV Aulendorf, 1996, which is
incorporated herein
in its entirety by reference. Typical excipients include but are not limited
to fillers, binders,
disintegrants, glidants, lubricants, stabilising agents, diluents, surfactants
and the like.
Consequently, the pharmaceutical composition to be analysed typically
comprises: one or
more fillers, such as microcrystalline cellulose, lactose, lactose
monohydrate, sugars,
starches, modified starch, mannitol, sorbitol and other polyols, dextrin,
dextran or
maltodextrin; one or more binders, such as lactose, starches, modified starch,
maize starch,
dextrin, dextran, maltodextrin, microcrystalline cellulose (e.g. Avicel ),
sugars, polyethylene
glycols, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethyl
cellulose,
hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, gelatin,
acacia gum,
tragacanth, polyvinylpyrrolidone or crospovidone; one or more disintegrants,
such as
croscarmellose sodium, cross-linked polyvinylpyrrolidone, crospovidone, cross-
linked
carboxymethyl starch, starches, microcrystalline cellulose, polyacrylin
potassium; one or
more different glidants or lubricants, such as magnesium stearate, calcium
stearate, zinc
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stearate, calcium behenate, sodium stearyl fumarate, talc, magnesium
trisilicate, stearic acid,
palmitic acid, carnauba wax or silicon dioxide (e.g. colloidal silicon
dioxide).
If required, the pharmaceutical compositions to be analysed may also include
surfactants
and other conventional excipients. Typical surfactants that may be used are
ionic
surfactants, such as sodium lauryl sulphate, or non-ionic surfactants, such as
different
poloxamers (polyoxyethylene and polyoxypropylene copolymers), natural or
synthesized
lecithins, esters of sorbitan and fatty acids (such as Spano ), esters of
polyoxyethylene-
sorbitan and fatty acids (such as Tween ), polyoxyethylated hydrogenated
castor oil (such
as Cremophor~, polyoxyethylene stearates (such as Brij~, dimethylpolysiloxane,
or any
combination of the above mentioned surfactants.
If the solid pharmaceutical composition is in the form of coated tablets, the
coating may be
prepared from at least one film-former, such as hydroxypropyl methyl
cellulose,
hydroxypropyl cellulose or methacrylate polymers, which optionally may contain
at least
one plasticizer, such as polyethylene glycols, dibutyl sebacate, triethyl
citrate, and other
pharmaceutical auxiliary substances conventional for film coatings, such as
pigments, fillers
and others.
One or more of these excipients can be selected and used by the skilled person
having
regard to the particular desired properties of the solid oral dosage form by
routine
experimentation and without any undue burden.
The absolute amounts of each excipient and the amounts relative to other
additives is
similarly dependent on the desired properties of the solid oral dosage form
and may also be
chosen by the skilled person by routine experimentation without undue burden.
For
example, the solid oral dosage form may be chosen to exhibit accelerated
and/or delayed
release of the active agent with or without quantitative control of the
release of active
agent.
In powders, the carrier is a finely divided solid that is in a mixture with
the finely divided
API. In tablets, the API is mixed with the carrier having the necessary
binding properties
in suitable proportions and compacted in the shape and size desired.
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I.iquid form preparations include solutions, suspensions and emulsions, for
example water
or water propylene glycol solutions. For parenteral injection, liquid
preparations can be
formulated in solution in aqueous polyethylene glycol solution.
Aqueous solutions suitable for oral use can be prepared by dissolving the API
in water and
adding suitable colorants, flavours, stabilizing and thickening agents as
desired.
The quantity of active component in a unit dose preparation may be varied or
adjusted
from 0.5 mg to 50 mg, preferably 2 mg to 30 mg, and most preferably 5 mg to 20
mg. A
daily dose range of about 5 mg to about 20 mg is preferred. The composition
can, if
desired, also contain other compatible therapeutic agents.
While the present invention has been described in terms of its specific
embodiments,
certain modifications and equivalents will be apparent to those skilled in the
art and are
intended to be included within the scope of the present invention.
The methods of the invention disclosed herein can also be used for the
analysis of
compounds with similar chemical structures and/or similar chemical or physical
properties
to memantine, e.g. rimantadine and pharmaceutically acceptable salts thereof.
The present invention is illustrated but in no way limited by the following
examples.
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Examples
Example 1: Content Uniformity Test
GC SYSTEM: Agilent 6890 or equivalent
INLET LINER: Agilent Split/Splitless Liner Part No. 5183-4711
COLUMN: ZB-1, 30 m length, 0.32 mm internal diameter,
1.0 m film thickness
PRE-COLUMN: de-activated fused silica tubing, 5 m length,
0.32 mm internal diameter
CARRIER GAS FLOW: Helium, 2.5 mL/min (constant flow)
INLET: Temperature: 340 C
Split Injection: Split Ratio: 20:1
Gas Saver: ON On Time: 2.0 min
Flow: 20.0
OVEN TEMPERATURE: Initial Temperature 140 C
Initial Time 1 min
Rate 1 10 C/min
Temperature 1 200 C
Time 1 0 min
Rate 2 40 C/min
Temperature 2 340 C
Time 2 1 min
DETECTOR: Flame Ionization Detector (FID) at 340 C
Hydrogen 30 mL/min
Air 350 mL/min
Helium (makeup) 22.5 mL/min
INJECTION VOLUME: 1 L
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INJECTOR: 5 pre-washes with Diluent
post-washes with Methanol
5 sample washes
5 injector pumps
5
DILUENT: 0.005M Methanolic Potassium Hydroxide
RUN TIME: 12 minutes
Internal Standard Working Solution (ISTD),
Accurately weigh about 100 mg of Amantadine HC1 (C10H17N=HC1) and transfer
into a
1000 mL volumetric flask. Add about 600 mL of Diluent and sonicate for about 2
minutes
or until dissolved. Let cool to room temperature, dilute to volume with
Diluent and mix
well. (Conc. ;:z~ 100 gg/mL of Amantadine HCl).
Standard Working Solution (prepare in duplicate)
Accurately weigh about 25.0 mg of Memantine HC1 RS (C12H21N=HC1) and transfer
into a
250 mL volumetric flask. Add about 150 mL of ISTD and sonicate for about 2
minutes or
until dissolved. Let cool to room temperature, dilute to volume with ISTD and
mix well.
(Conc. ,z~ 100 g/mL of Memantine HC1 and ;t~ 100 g/mL of Amantadine HC1).
Sample Working Solution
Transfer one tablet into a 100 mL volumetric flask. Add 70 mL of ISTD. Shake
mechanically for 20 minutes or until the tablet is disintegrated completely.
Sonicate for 10
minutes with occasional swirling. Allow to stand at room temperature for 10
minutes.
Dilute to volume with ISTD. Mix well. Centrifuge a portion at 3000 rpm for 5
minutes.
Take supernatant for analysis.
(Conc. ;zz~ 100 g/mL of Memantine HCl and ;zz~ 100 g/mL of Amantadine HCI).
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Example 2: Dissolution Testing
GC SYSTEM: Agilent 6890 or equivalent
INLET LINER: Agilent Split/Splitless Liner Part No. 5183-4711
COLUMN: ZB-1, 30 m length, 0.32 mm internal diameter,
1.0 m film thickness
CARRIER GAS FLOW: Helium, 2.5 mL/min (constant flow)
INLET: Temperature: 250 C
Split Injection: Split Ratio: 10:1
Gas Saver: OFF
OVEN TEMPERATURE: Initial Temperature 140 C
Initial Time 1 min
Rate 1 10 C/min
Temperature 1 200 C
Time 1 0 min
Rate 2 40 C/min
Temperature 2 300 C
Time 2 2 min
DETECTOR: Flame Ionization Detector (FID) at 300 C
Hydrogen 40 mL/min
Air 400 mL/min
Helium (makeup) 22.5 mL/min
INJECTION VOLUME: 5 L
INJECTOR: 5 pre-washes with 0.01M NaOH in Ethanol*
5 post-washes with Ethanol
5 sample washes
5 injector pumps
*0.01M Ethanolic NaOH: 100 L of 1.OM aqueous NaOH diluted to 10 mL with
anhydrous ethanol
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RUN TIME: 12 minutes
DISSOLUTION EOUIPMENT AND PARAMETERS
MEDIUM: 0.1N HCl with NaCI*
VOLUME: 900 mL
METHOD: Apparatus I (Basket)
SPEED: 100 rpm
RUN TIME: 30 minutes
TEMPERATURE: 37 C 0.5 C
MEDIUM*: Weigh accurately 12 g of Sodium Chloride (NaCl) and dissolve in
5 L of water. Add 40 mL of Hydrochloric Acid (HCl). Dilute to
6 L with water. Mix well. Adjust pH to 1.2 0.05 with HCI.
INTERNAL STANDARD PREPARATION (ISTD),
Stock Internal Standard Solution
Weigh 27.8 mg of Amantadine HCl (C10H17N=HC1) and transfer into a 50 mL
volumetric
flask. Add about 40 mL of Medium. Sonicate for 2 minutes or until dissolved.
Dilute to
volume with Medium. Mix well.
Internal Standard Intermediate Solution
Transfer 30.0 mL of Stock Internal Standard Solution into a 100 mL volumetric
flask. Add
5.0 mL of 1.ON NaOH Solution. Swirl to mix. Add 20.0 mL of n-Hexane. Stopper
tightly. Shake mechanically for 5 minutes. Allow to stand for 10 minutes.
Internal Standard Working Solution (IS=
Transfer 5.0 mL of upper layer from Internal Standard Intermediate Solution
into a 250
mL volumetric flask. Dilute to volume with n-Hexane. Mix well.
(Conc. z 16.7 g/mL of Amantadine HCl).
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Standard Stock Solution
Accurately weigh about 22.2 mg of Memantine HCl RS (C12H21N=HC1) and transfer
into a
100 mL volumetric flask. Add about 30 mL of Medium and sonicate for about 2
minutes
or until dissolved. Dilute to volume with Medium and mix well.
(Conc. ;t~ 222 g/mL of Memantine HCl).
For 10mg Tablets:
Pipette 5.0 mL of Standard Stock Solution into a 100 mL volumetric flask.
Dilute to
volume with Medium and mix. (Conc. ;z~ 11.1 g/mL of Memantine HCl).
For 5mg Tablets:
Pipette 5.0 mL of Standard Stock Solution into a 200 mL volumetric flask.
Dilute to
volume with Medium and rnix. (Conc. ;t; 5.56 g/mL of Memantine HCl).
For 10mg Tablets:
Pipette 6.0 mL of Standard Stock Solution into a 25 mL volumetric flask. Add
1.0 mL of
1.ON NaOH Solution. Mix by swirling. Add 4.0 mL of ISTD. Stopper tightly.
Shake
mechanically for 5 minutes. Allow to stand for 10 minutes. Using a disposable
glass
pipette, carefully transfer the upper layer into a GC vial.
(Conc. ;t~ 16.7 g/mL of Memantine HCl and ;z:~ 16.7 g/mL of Amantadine HCl).
For 5mg Tablets:
Pipette 6.0 mL of Standard Stock Solution into a 25 mL volumetric flask. Add
1.0 mL of
1.ON NaOH Solution. Mix by swirling. Add 4.0 mL of ISTD. Stopper tightly.
Shake
mechanically for 5 minutes. Allow to stand for 10 minutes. Using a disposable
glass
pipette, carefully transfer the upper layer into a GC vial.
(Conc. P~ 8.33 g/mL of Memantine HCl and ;z:~ 16.7 g/mL of Amantadine HCl).
Pipette 6.0 mL of Medium into a 25 mL volumetric flask. Add 1.0 mL of 1.ON
NaOH
Solution. Mix by swirling. Add 4.0 mL of ISTD. Stopper tightly. Shake
mechanically for
5 minutes. Allow to stand for 10 minutes. Using a disposable glass pipette,
carefully
transfer the upper layer into a GC vial. (Conc. ;-- 16.7 g/mL of Amantadine
HCl).
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Sample Stock Solution
Place 900 mL of Medium into each of six dissolution vessels and equilibrate
the Medium to
37 C 0.5 C. Preset the baskets speed to 100 rpm. Place one tablet into each
of the dry
baskets. Lower the baskets into the Medium and start rotating the baskets.
After 30
minutes, withdraw 10 mL sample from each vessel using probes from a zone
midway
between the surface of the Medium and the top of the rotating basket, not less
than 1 cm
from the vessel wall. Filter the solutions immediately.
For 10mg Tablets:
Pipette 6.0 mL of Sample Stock Solution into a 25 mL volumetric flask. Add 1.0
mL of
1.ON NaOH Solution. Mix by swirling. Add 4.0 mL of ISTD. Stopper tightly.
Shake
mechanically for 5 minutes. Allow to stand for 10 minutes. Using a disposable
glass
pipette, carefully transfer the upper layer into a GC vial.
(Conc. - 16.7 g/mL of Memantine HC1 and,& 16.7 g/mL of Amantadine HC1).
For 5mg Tablets:
Pipette 6.0 mL of Sample Stock Solution into a 25 mL volumetric flask. Add 1.0
mL of
1.ON NaOH Solution. Mix by swirling. Add 4.0 mL of ISTD. Stopper tightly.
Shake
mechanically for 5 minutes. Allow to stand for 10 minutes. Using a disposable
glass
pipette, carefully transfer the upper layer into a GC vial.
(Conc. - 8.33 g/mL of Memantine HCI and ;:t~ 16.7 g/mL of Amantadine HCl).
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Example 3: Related Substances Test
GC SYSTEM: Agilent 6890 or equivalent
INLET LINER: Agilent Split/Splitless Liner Part No. 5183-4711
COLUMN: ZB-1, 60 m length, 0.32 mm internal diameter,
1.0 m film thickness
CARRIER GAS FLOW: Helium, 2.5 mL/min (constant flow)
INLET: Temperature: 250 C
Split Injection: Split Ratio: 20:1
Gas Saver: ON On Time: 2.0 min
Flow: 20.0
OVEN TEMPERATURE: Initial Temperature 140 C
Initial Time 1 min
Rate 1 2 C/min
Temperature 1 200 C
Time 1 0 min
Rate 2 30 C/min
Temperature 2 300 C
Time2 5min
DETECTOR: Flame Ionization Detector (FID) at 250 C
Hydrogen 40 mL/min
Air 400 mL/min
Helium (makeup) 22.5 mL/min
INJECTION VOLUME: 5 L
INJECTOR: 5 pre-washes with 0.01M Ethanolic NaOH*
5 post-washes with Ethanol
5 sample washes
5 injector pumps
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*0.01M Ethanolic NaOH: 100 L of 1.OM aqueous NaOH diluted to 10 mL with
anhydrous ethanol
RUN TIME: 40 minutes
Standard Stock Solution:
Accurately weigh about 5.0 mg of Memantine HC1 RS (C12H21N=HC1) and transfer
into a
100 mL volumetric flask. Add about 60 mL of H20, and sonicate for 30 seconds
if
necessary to clissolve. Dilute to volume with H20 and mix well.
(Conc. ;z~ 50 g/mL of Memantine HCI).
Standard Intermediate Solution:
Pipette 4.0 mL of Standard Stock Solution into a 100 mL volumetric flask.
Dilute to
volume with H20 and mix well. (Conc. ;t~ 2 g/mL of Memantine HCl).
Standard Working Solution:
Pipette 5.0 mL of Standard Intermediate Solution into a 25 mL volumetric
flask. Add 1.0
mL of 6M NaOH Solution and 5.0 mL of n-Hexane. Ensure the flask is capped
tightly
immediately after adding the n-Hexane to prevent any leaking and/or
evaporation. Shake
the flask by hand for 5 minutes. Let stand for 5 minutes to allow the layers
to separate.
Transfer a portion of the upper organic layer to a GC vial and cap
immediately.
(Conc. ;z~ 2 g/mL of Memantine HC1).
Sample Working Solution:
Accurately weigh 20 tablets and determine average tablet weight. Finely powder
the tablets
using mortar and pestle. Accurately weigh tablet powder equivalent to 20.0 mg
of
Memantine HCl and transfer into a 40 mL polycarbonate centrifuge tube with
Nalgene
screw cap. Add 10.0 mL of H20 and sonicate for 2 minutes with occasional
shaking. Add
2.0 mL of 6M NaOH Solution and 10.0 mL of n-Hexane. Ensure the tube is capped
tightly immediately after adding the n-Hexane to prevent any leaking and/or
evaporation.
Shake the tube by hand for 5 minutes. Let stand for 10 minutes to allow the
layers to
separate. Transfer a portion of the upper organic layer to a GC vial and cap
immediately.
(Conc. ;zt~ 2 mg/mL of Memantine HCI).
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Example 4: Purity Assay
GC SYSTEM: Agilent 6890 or equivalent
INLET LINER: Agilent Split/Splitless Liner Part No. 5183-4711
COLUMN: ZB-1, 30 m length, 0.32 mm internal diameter,
1.0 m flm tbickness
PRE-COLUMN: de-activated fused silica tubing, 5 m length,
0.32 mm internal diameter
CARRIER GAS FLOW: Helium, 2.5 mL/min (constant flow)
INLET: Temperature: 340 C
Split Injection: Split Ratio: 20:1
Gas Saver: ON On Time: 2.0 min
Flow: 20.0
OVEN TEMPERATURE: Initial Temperature 140 C
Initial Time 1 min
Rate 1 10 C/min
Temperature 1 200 C
Time 1 0 min
Rate 2 40 C/min
Temperature 2 340 C
Time 2 1 min
DETECTOR: Flame Ionization Detector (FID) at 340 C
Hydrogen 30 mL/min
Air 350 mL/min
Helium (makeup) 22.5 mL/min
INJECTION VOLUME: 1 L
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INJECTOR: 5 pre-washes with Diluent
post-washes with Methanol
5 sample washes
5 injector pumps
S
DILUENT: 0.005M Methanolic Potassium Hydroxide*
*Dilute 10 mL of 0.5M Methanolic Potassium Hydroxide to 1000 mL with Methanol
RUN TIME: 12 minutes
Internal Standard Working Solution (ISTD~
Accurately weigh about 125 mg of Amantadine HCl (C10H17N=HC1) and transfer
into a
1000 mL volumetric flask. Add about 600 mL of Diluent and sonicate for about 2
minutes
or until dissolved. Let cool to room temperature, dilute to volume with
Diluent and mix
well. (Conc. z 125 g/mL of Amantadine HCl).
Standard Working Solution (prepare in duplicate)
Accurately weigh about 25.0 mg of Memantine HCl RS (C12H21N=HCl) and transfer
into a
200 mL volumetric flask. Add about 150 mL of ISTD and sonicate for about 2
minutes or
until dissolved. Let cool to room temperature, dilute to volume with ISTD and
mix well.
(Conc. ;z~ 125 g/mL of Memantine HCl and,& 125 g/mL of Amantadine HCl).
Sample Working Solution (prepare in duplicate)
Accurately weigh 20 tablets and determine average tablet weight. Finely powder
the tablets
using mortar and pestle. Accurately weigh tablet powder equivalent to 25.0 mg
of
Memantine HCl and transfer into a 200 mL volumetric flask. Add about 100 mL of
ISTD.
Shake mechanically for 10 minutes. Sonicate for 5 minutes with occasional
swirling. Allow
to stand at room temperature for 10 minutes. Dilute to volume with ISTD and
mix well.
Centrifuge a portion at 3000 rpm for 5 minutes. Transfer portion of clear
supernatant into
GC vial. (Conc. ge 125 g/mL of Memantine HCl and ;z~ 125 g/mL of Amantadine
HCl).
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Inject Blank Solution (ISTD) and ensure the baseline is clean and stable.
Inject Standard Working Solution 1 five times.
Calculate the relative standard deviation (RSD) of peak area ratio of
Memantine to ISTD.
The RSD is NMT 2.0%. Calculate the tailing factor (T) and column efficiency
(N) for
Memantine peak. T is within 0.8 and 1.2, and N is NLT 100000. The resolution
(R)
between ISTD and Memantine is NLT 3.5.
Inject Standard Working Solution 2, Blank Solution (ISTD), and Sample Working
Solutions
once.
Inject Standard Working Solution 1 once after every six samples.
Inject Standard Working Solution 1 once each at the end of the run.
