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Novel Chromatography Methods

Field of the invention

The present invention relates to novel HPLC methods for the analysis of the API formoterol and related substances. In a first method the mobile phase comprises two or more liquids, and the relative concentration of the liquids is varied to a predetermined gradient. In a second method the mobile phase comprises a first liquid A comprising an aqueous solution of ammonium acetate and a second liquid B comprising a dipolar aprotic solvent. In a third method the mobile phase comprises a first liquid A comprising an aqueous solution of ammonium acetate with a concentration of 0.001 to 0.025M, and a second liquid B. The present invention also relates to a method for analysing a substance, comprising the detection and optional quantification of one or more specific impurities.

Background of the invention

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 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 (intermediates) 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

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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.

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 is usually High Performance Liquid Chromatography (HPLC) combined with a UV- Visible detector. 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 UV- 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.

HPLC analysis may be performed in isocratic or gradient mode. An isocratic HPLC separation is one which is carried out under a constant mobile phase composition. A gradient HPLC separation is characterized by a gradual change over time in the percentage of the two or more solvents making up the mobile phase. The change in solvent often is

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controlled by a mixing device which mixes the solvents to produce the HPLC mobile phase just prior to its movement through the column.

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 and quantified by means of a suitable detector.

Formoterol, the common chemical name for N-[2-hydroxy-5-[l-hydroxy-2-[[2-(4- meώoxyphenyl)-l-metiιylemyl]amino]ethyl]phenyl]formamide, is a long acting beta agonist and is useful for the treatment of a range of respiratory disorders such as asthma and chronic obstructive pulmonary disease (COPD). Formoterol is currently marketed as the dihydrate form of the fumarate salt of the racemate of the enantiomers which have the RR (I) and SS configuration.

(I)

Methods for the preparation of formoterol are known in the art and these methods include those disclosed in US patent 3994974.

Several methods have been published in the literature to analyse formoterol but these various methods have been primarily developed for the detection and determination of formoterol in biological fluids (see, for example, the methods disclosed by J. Campestrini et al. in J. Chromatography B, 704, pages 221-229, 1997; and by D.K. Nadarassan et al. in J. Chromatography B, 850, pages 31-37, 2007). Alternative HPLC methods have been

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published which are more suited to the analysis of formoterol in bulk drug products (see, for example, S.O. Akapo & M. Asif in J. Pharm. Biomed. Anal., 33, pages 935-945, 2003) or in an aerosol formulation (see K.H. Assi et al. in J. Pharm. Biomed. Anal., 41, pages 325- 328, 2006).

Additionally, an official monograph on formoterol fumarate dihydrate appeared in European Pharmacopoeia 5.0 (volume 2, pages 1632-1634, 2005), but two different HPLC methods have to be employed to detect and/or measure all the named impurities or related substances.

However, all these current methods are not suitable for the detection and quantitation of all synthetic intermediates and other related substances that are present in a formoterol sample, particularly a formoterol fumarate dihydrate sample synthesized by the route disclosed in US patent 3994974. Current methods are also deficient in estimating the total impurities in formoterol fumarate dihydrate.

Therefore, the HPLC methods reported in the prior art are not particularly convenient or suitable for analysing formoterol as a bulk drug substance, particularly with respect to related substances.

Consequently, although several HPLC methods have been disclosed for the analysis of formoterol 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 lead to the development and validation of a novel, efficient, reproducible and simple HPLC method to analyse formoterol, particularly with respect to the related substances formed during the synthesis.

Object of the invention

It is an object of the present invention to provide a novel, alternative method for analysing formoterol, its impurities and related substances, preferably whilst avoiding the typical prior art problems associated with the prior art methods.

It is another object of the present invention to provide a novel, accurate and sensitive HPLC method for the quantification of intermediates that are formed and may remain in batches of formoterol fumarate dihydrate, especially when synthesized by the process disclosed in US patent 3994974.

Summary of the invention

The term "formoterol" as used herein throughout the description and claims means formoterol and/or any salt, solvate, isomer, diastereomer or enantiomer thereof. Preferably formoterol is used in the dihydrate form of the fumarate salt of the racemate of the enantiomers which have the RR and SS configuration.

A first aspect of the current invention provides a HPLC method for analysing formoterol, wherein the mobile phase comprises two or more liquids, including a first liquid A and a second liquid B, and the relative concentration of the liquids is varied to a predetermined gradient. In a preferred embodiment, there is provided a HPLC method for the analysis of formoterol and related substances, wherein the mobile phase comprises two or more liquids, the relative concentration of the liquids is varied to a predetermined gradient and the stationary phase used is reverse phase.

Preferably, the first liquid A is aqueous based, such as water or an aqueous solution of a buffer.

Preferably, the buffer is an acid or an organic salt or an inorganic salt.

Typically, the buffer is a phosphate salt, an acetate salt, a formate salt or trifluoroacetic acid. Most preferably, the buffer is an ammonium salt, such as ammonium acetate.

The buffer can be present at a concentration of 0.001 to 0.1 M, preferably at a concentration of 0.001 to 0.01 M, more preferably at a concentration of 0.005 to 0.01 M, more preferably at a concentration of approximately 0.007 M.

Preferably the buffer is ammonium acetate present at a concentration of 0.001 to 0.01 M. Most preferably, the buffer is ammonium acetate present at a concentration of approximately 0.007 M.

Preferably, the pH of the buffer is approximately 2 to 6, more preferably the pH is between 3.8 and 5.8, more preferably the pH of the buffer is about 4.8.

Typically, the method of the first aspect of the current invention is carried out at a temperature between approximately 15 to 40°C.

The second liquid B is preferably an organic solvent, such as methanol, ethanol, acetonitrile, propanol or isopropanol or a mixture thereof.

In one embodiment of the first aspect of the current invention the second liquid B is a substantially water miscible solvent.

As used herein, the term "substantially miscible" in relation to two liquids X and Y means that when mixed together at 20°C and 1 atmosphere pressure, X and Y form a single phase between two mole fractions of Y, x _Y1 and x _Y2 , wherein the magnitude of δx _γ (= x _Y2 — x _Y1 ) is at least 0.05. For example, X and Y may form a single phase where the mole fraction of Y, x _γ , is from 0.40 to 0.45, or from 0.70 to 0.75; in both cases δx _γ = 0.05. Preferably, the magnitude of δx _γ is at least 0.10, more preferably at least 0.25, more preferably at least 0.50, more preferably at least 0.75, more preferably at least 0.90, even more preferably at least 0.95. Most preferably the term "substantially miscible" in relation to two liquids X and Y means that when mixed together at 20°C and 1 atmosphere pressure, X and Y form a single phase when mixed together in any proportion.

Preferably the second liquid B is a polar protic solvent such as acetic acid, methanol, ethanol, n-propanol or isopropanol, or a dipolar aprotic solvent such as acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF. More preferably where the second liquid B is a dipolar aprotic solvent, it is selected from acetone, acetonitrile, dimethoxyethane or 1,4-dioxane. Most preferably, the second liquid B is acetonitrile.

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A preferred embodiment of the first aspect of the current invention is when the first liquid A is an aqueous solution of ammonium acetate and the second liquid B is acetonitrile.

Preferably a mobile phase flow rate of between 0.01 and 10 ml/min is used, more preferably a mobile phase flow rate of between 0.1 and 4 ml/min is used, more preferably a mobile phase flow rate of about 1 ml/min is used.

Typically, the first aspect of the current invention comprises a gradient programming so that the relative concentration of the liquids A and B are varied to a gradient between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 10 to 180 minutes. Preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 120 minutes. More preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 60 minutes.

Alternatively, the first aspect of the current invention may comprise a gradient programming so that the relative concentration of the liquids A and B are varied to a gradient from about 95%A : 5%B, or from about 90%A : 10%B, or from about 85%A : 15%B, to about 5%A : 95%B, or to about 10%A : 90%B, or to about 15%A : 85%B. The variation in gradient may typically take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably over 30 to 60 minutes.

A particularly preferred embodiment of the first aspect of the current invention is when the first liquid A is an aqueous solution of 0.007 M ammonium acetate and the second liquid B is acetonitrile.

A particularly preferred method according to the first aspect of the current invention is when the first liquid A is an aqueous solution of 0.007 M ammonium acetate and the second liquid B is acetonitrile and the gradient is as follows:

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In one embodiment of the first aspect of the current invention, the stationary phase is chiral. In another embodiment, the mobile phase further comprises a chiral selector.

Preferably, the stationary phase used in the fkst aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel. Particularly suitable stationary phases include octadecylsilyl silica gel or octylsilyl silica gel. A particularly preferred stationary phase comprises a YMC Pack pro Cl 8 (250 mm x 4.6 mm), 5μ column, preferably with a 12nm pore size.

Preferably the stationary phase has a particle size of between 0.1 and lOOμm, or between 0.5 and 25μm, or between 1 and lOμm. More preferably the stationary phase has a particle size of about 5μm.

Preferably the stationary phase has a pore size of between 1 and lOOnm, or between 2 and 40nm, or between 5 and 15nm. More preferably the stationary phase has a pore size of about 12nm.

In one embodiment of the first aspect of the current invention, the chromatography is carried out in a column between 10mm and 5000mm in length, or in a column between 50mm and 1000mm in length, or between 100mm and 500mm in length. More preferably the chromatography is carried out in a column about 250mm in length.

The chromatography may be carried out in a column between 0.01mm and 100mm in internal diameter, or between 0.1mm and 50mm in internal diameter, or between lmm and 10mm in internal diameter. More preferably the chromatography is carried out in a column about 4.6mm in internal diameter.

The eluent may be analysed by a detector such as a UV or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.

In a pteferred embodiment of the first aspect of the current invention, the formoterol is in the form of formoterol fumarate dihydrate.

In one embodiment of the first aspect of the current invention the HPLC method detects and optionally quantifies in a single run one or more of the following impurities:

N-Benzyl-N-(1 -methyl-2-p-methoxyphenylethyl) amine;

4-Benzyloxy-3-nitto-α-[N-benzyl-N-(l-meώyl-2-p-methoxyp henylethyl)amino] acetophenone;

4-Benzyloxy-3-rύtro-α-[N-benzyl-N-(l-methyl-2-p-niethox yphenylemyl)aminomethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-mtro-α-[N-benzyl-N-(l-methyl-2-p-memoxyphe nylemyl)aminomethyl] benzyl alcohol diastereomer-II;

4-Benzyloxy-3-amino-α-|TSf-benzyl-N-(l-rnethyl-2-p-medio xyphenylethyl)arninoniethyl] benzyl alcohol diastereomer-I; 4-Benzyloxy-3-amino-α-|N-benzyl-N-(l-meλyl-2-p-memoxypheny lemyl)aminomethyl] benzyl alcohol diastereomer-II; and/ or

4-Benzyloxy-3-formylamino-α-[N-benzyl-N-(l-methyl-2-p-rn ethoxyphenylethyl) aminomethyl] benzyl alcohol.

In a preferred embodiment the HPLC method according to the current invention efficiently detects and quantifies in a single run all impurities including those selected from the following compounds:

N-Benzyl-N-(1 -methyl-2-p-methoxyphenylethyl) amine;

4-Benzyloxy-3-nitro-α-pSI-benzyl-N-(l-methyl-2-p-methoxy phenylethyl)amino] acetophenone;

4-Benzyloxy-3-nitro-α-[N-benzyl-N-(l-methyl-2-p-methoxyp henylemyl)aminomethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-nitro-α-[N-benzyl-N-(l-rnethyl-2-p-πietho xyphenylethyl)arninoniethyl] benzyl alcohol diastereomer-II; 4-Benzyloxy-3-attτino-α-[N-benzyl-N-(l-memyl-2-p-methoxyph enylethyl)aminomethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-amino-α-pSJ-benzyl-N-(l-methyl-2-p-methoxy phenyle1±iyl)aminomethyl] benzyl alcohol diastereomer-II;

4-Benzyloxy-3-foiωiylamiαo-α-[N-benzyl-N-(l-methyl-2-p -methoxyphenyletliyl) aminomethyl] benzyl alcohol.

A second aspect of the current invention provides a HPLC method for analysing formoterol, wherein the mobile phase comprises two or more liquids, including a first liquid A comprising an aqueous solution of ammonium acetate and a second liquid B comprising a dipolar aprotic solvent.

Preferably the aqueous solution of ammonium acetate has a concentration of 0.001 to 0.1 M, more preferably the aqueous solution of ammonium acetate has a concentration of 0.001 to 0.01 M, or of 0.005 to 0.01 M. More preferably the aqueous solution of ammonium acetate has a concentration of approximately 0.007 M.

Preferably, the pH of the aqueous solution is approximately 2 to 6, more preferably the pH is between 3.8 and 5.8, more preferably the pH of the aqueous solution is about 4.8.

In one embodiment of the second aspect of the current invention the second liquid B is a substantially water miscible solvent.

Preferably the second liquid B is selected from acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF. More preferably the second liquid B is selected from acetone, acetonitrile, dimethoxyethane or 1,4-dioxane. Most preferably the second liquid B is acetonitrile.

Preferably a mobile phase flow rate of between 0.01 and 10 ml/min is used, more preferably a mobile phase flow rate of between 0.1 and 4 ml/min is used, more preferably a mobile phase flow rate of about 1 ml/min is used.

In one embodiment of the second aspect of the current invention the HPLC method is an isocratic method, preferably such that the relative concentration of the liquids A and B is set between 99.5%A : 0.5%B and 0.5%A : 99.5%B, or between 90%A : 10%B and 10%A :

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90%B, more preferably between 75%A : 25%B and 25%A : 75%B. More preferably the relative concentration of the liquids A and B is about 40%A : 60%B.

In an alternate embodiment of the second aspect of the current invention the relative concentration of the liquids of the mobile phase is varied to a predetermined gradient. Typically, a gradient programming is used so that the relative concentration of the liquids A and B are varied to a gradient between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 10 to 180 minutes. Preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 120 minutes. More preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 60 minutes. Alternatively, a gradient programming may be used so that the relative concentration of the liquids A and B are varied to a gradient from about 95%A : 5%B, or from about 90%A : 10%B, or from about 85%A : 15%B, to about 5%A : 95%B, or to about 10%A : 90%B, or to about 15%A : 85%B. The variation in gradient may typically take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably over 30 to 60 minutes.

A particularly preferred method according to the second aspect of the current invention is when the first liquid A is an aqueous solution of 0.007 M ammonium acetate and the second liquid B is acetonitrile and the gradient is as follows:

In one embodiment of the second aspect of the current invention, the stationary phase is chiral. In another embodiment, the mobile phase further comprises a chiral selector.

Preferably, the stationary phase used in the second aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel,

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cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel. Particularly suitable stationary phases include octadecylsilyl silica gel or octylsilyl silica gel. A particularly preferred stationary phase comprises a YMC Pack pro Cl 8 (250 mm x 4.6 mm), 5μ column, preferably with a 12nm pore size.

Preferably the stationary phase has a particle size of between 0.1 and lOOμm, or between 0.5 and 25μm, or between 1 and lOμm. More preferably the stationary phase has a particle size of about 5μm.

Preferably the stationary phase has a pore size of between 1 and lOOnm, or between 2 and 40nm, or between 5 and 15nm. More preferably the stationary phase has a pore size of about 12nm.

Typically, the method of the second aspect of the current invention is carried out at a temperature between approximately 15 to 40°C.

In one embodiment of the second aspect of the current invention, the chromatography is carried out in a column between 10mm and 5000mm in length, or in a column between 50mm and 1000mm in length, or between 100mm and 500mm in length. More preferably the chromatography is carried out in a column about 250mm in length.

The chromatography may be carried out in a column between 0.01mm and 100mm in internal diameter, or between 0.1mm and 50mm in internal diameter, or between lmm and 10mm in internal diameter. More preferably the chromatography is carried out in a column about 4.6mm in internal diameter.

The eluent may be analysed by a detector such as a UV or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.

In a preferred embodiment of the second aspect of the current invention, the formoterol is in the form of formoterol fumarate dihydrate.

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In one embodiment of the second aspect of the current invention the HPLC method detects and optionally quantifies in a single run one or more of the following impurities:

N-Benzyl-N-(1 -methyl-2-p-methoxyphenylethyl) amine;

4-Benzyloxy-3-nitro-α-pSI-benzyl-N-(l-methyl-2-p-methoxy phenylethyl)amino] acetophenone;

4-Benzyloxy-3-nitro-α-(N-benzyl-N-(l-methyl-2-p-rnedioxy phenylemyl)aminomethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-nitro-α-|N-benzyl-N-(l-rnetJiyl-2-p-methox yphenylemyl)aminornethyl] benzyl alcohol diastereomer-II; 4-Benzyloxy-3-amino-α-|N-benzyl-N-(l-methyl-2-p-rnethoxyphe nylethyl)arninornethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-arnino-α-[N-benzyl-N-(l-mediyl-2-p-methoxy phenylethyl)arninomethyl] benzyl alcohol diastereomer-II; and/or

4-Benzyloxy-3-formylarnino-α-[N-benzyl-N-(l-rnethyl-2-p- methoxyphenylethyl) aminomethyl] benzyl alcohol.

A third aspect of the current invention provides a HPLC method for analysing formoterol, wherein the mobile phase comprises two or more liquids, including a first liquid A comprising an aqueous solution of ammonium acetate with a concentration of 0.001 to 0.025 M, and a second liquid B.

Preferably the aqueous solution of ammonium acetate has a concentration of 0.001 to 0.01 M, more preferably the aqueous solution of ammonium acetate has a concentration of 0.005 to 0.01 M. More preferably the aqueous solution of ammonium acetate has a concentration of approximately 0.007 M.

Preferably, the pH of the aqueous solution is approximately 2 to 6, more preferably the pH is between 3.8 and 5.8, more preferably the pH of the aqueous solution is about 4.8.

The second liquid B of the third aspect of the current invention is preferably an organic solvent, such as methanol, ethanol, acetonitrile, n-propanol or isopropanol or a mixture thereof.

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In one embodiment of the third aspect of the current invention the second liquid B is a substantially water miscible solvent.

Preferably the second liquid B is a polar protic solvent such as acetic acid, methanol, ethanol, n-propanol or isopropanol, or a dipolar aprotic solvent such as acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF. More preferably where the second liquid B is a dipolar aprotic solvent, it is selected from acetone, acetonitrile, dimethoxyethane or 1,4-dioxane. Most preferably, the second liquid B is acetonitrile.

Preferably a mobile phase flow rate of between 0.01 and 10 ml/min is used, more preferably a mobile phase flow rate of between 0.1 and 4 ml/min is used, more preferably a mobile phase flow rate of about 1 ml/min is used.

In one embodiment of the third aspect of the current invention the HPLC method is an isocratic method, preferably such that the relative concentration of the liquids A and B is set between 99.5%A : 0.5%B and 0.5%A : 99.5%B, or between 90%A : 10%B and 10%A : 90%B, more preferably between 75%A : 25%B and 25%A : 75%B. More preferably the relative concentration of the liquids A and B is about 40%A : 60%B.

In an alternate embodiment of the third aspect of the current invention the relative concentration of the liquids of the mobile phase is varied to a predetermined gradient. Typically, a gradient programming is used so that the relative concentration of the liquids A and B are varied to a gradient between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 10 to 180 minutes. Preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 120 minutes. More preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 60 minutes. Alternatively, a gradient programming may be used so that the relative concentration of the liquids A and B are varied to a gradient from about 95%A : 5%B, or from about 90%A : 10%B, or from about 85%A : 15%B, to about 5%A : 95%B, or to about 10%A : 90%B, or to about 15%A : 85%B. The variation in gradient may typically take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably over 30 to 60 minutes.

A particularly preferred method according to the third aspect of the current invention is when the first liquid A is an aqueous solution of 0.007 M ammonium acetate and the second liquid B is acetonitrile and the gradient is as follows:

In one embodiment of the third aspect of the current invention, the stationary phase is chiral. In another embodiment, the mobile phase further comprises a chiral selector.

Preferably, the stationary phase used in the third aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel. Particularly suitable stationary phases include octadecylsilyl silica gel or octylsilyl silica gel. A particularly preferred stationary phase comprises a YMC Pack pro Cl 8 (250 mm x 4.6 mm), 5μ column, preferably with a 12nm pore size.

Preferably the stationary phase has a particle size of between 0.1 and lOOμm, or between 0.5 and 25μm, or between 1 and lOμm. More preferably the stationary phase has a particle size of about 5μm.

Preferably the stationary phase has a pore size of between 1 and lOOnm, or between 2 and 40nm, or between 5 and 15nm. More preferably the stationary phase has a pore size of about 12nm.

Typically, the method of the third aspect of the current invention is carried out at a temperature between approximately 15 to 4O ⁰ C.

In one embodiment of the third aspect of the current invention, the chromatography is carried out in a column between 10mm and 5000mm in length, or in a column between 50mm and 1000mm in length, or between 100mm and 500mm in length. More preferably the chromatography is carried out in a column about 250mm in length.

The chromatography may be carried out in a column between 0.01mm and 100mm in internal diameter, or between 0.1mm and 50mm in internal diameter, or between lmm and 10mm in internal diameter. More preferably the chromatography is carried out in a column about 4.6mm in internal diameter.

The eluent may be analysed by a detector such as a UV or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.

In a preferred embodiment of the third aspect of the current invention, the formoterol is in the form of formoterol fumarate dihydrate.

In one embodiment of the third aspect of the current invention the HPLC method detects and optionally quantifies in a single run one or more of the following impurities:

N-Benzyl-N-(1 -methyl-2-p-methoxyphenylethyl) amine;

4-Benzyloxy-3-mtto-α-[N-benzyl-N-(l-mediyl-2-p-methoxyph enyleώyl)amino] acetophenone;

4-Benzyloxy-3-nitro-α-[N-benzyl-N-(l-memyl-2-p-me1±ιox yphenylethyl)aminomethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-nitro-α-[N-benzyl-N-(l-methyl-2-p-m.eώoxy phenylethyl)aminoniethyl] benzyl alcohol diastereomer-II;

4-Benzyloxy-3-arnino-α-|N-benzyl-N-(l-metiiyl-2-p-methox yphenylethyl)aminomethyl] benzyl alcohol diastereomer-I; 4-Benzyloxy-3-amino-α-[N-benzyl-N-(l-niethyl-2-p-nietiioxyp henyletiιyl)arninornethyl] benzyl alcohol diastereomer-II; and/or

4-Benzyloxy-3-formylamino-α-[N-benzyl-N-(l -methyl-2-p-methoxyphenylethyl) aminotnethyl] benzyl alcohol.

A fourth aspect of the current invention provides a method for analysing a substance, comprising the detection and optional quantification of one or more impurities selected from:

N-Benzyl-N-(1 -methyl-2-p-methoxyphenylethyl) amine;

4-Benzyloxy-3-rdtro-α-|N-benzyl-N-(l-methyl-2-p-methoxyp henylethyl)arnino] acetophenone; 4-Benzyloxy-3-mtto-α-pSI-benzyl-N-(l-niethyl-2-p-memoxyphen ylethyl)aminornethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-mtro-α-(N-benzyl-N-(l-rnediyl-2-p-methoxyp henylethyl)aminornethyl] benzyl alcohol diastereomer-II;

4-Benzyloxy-3-amino-α-[N-benzyl-N-(l-ttiethyl-2-p-methox yphenylethyl)aminoniethyl] benzyl alcohol diastereomer-I;

4-Benzyloxy-3-ammo-α-(N-benzyl-N-(l-niethyl-2-p-inethoxy phenylethyl)aminomethyl] benzyl alcohol diastereomer-II; and/or

4-Benzyloxy-3-formylamino-α-[N-benzyl-N-(l -methyl-2-p-methoxyphenylethyl) aminomethyl] benzyl alcohol.

In one embodiment of the fourth aspect of the current invention, the substance is an active pharmaceutical ingredient. Preferably the substance is formoterol, most preferably in the form of formoterol fumarate dihydrate.

In another embodiment of the fourth aspect of the current invention, the substance comprises less than 25 wt.% of the one or more impurities. Preferably, the substance comprises less than 10 wt.%, less than 5 wt.% or less than 2 wt.% of the one or more impurities. More preferably the substance comprises less than 1 wt.%, or less than 0.5 wt.% of the one or more impurities.

In another embodiment of the fourth aspect of the current invention, the method comprises the use of HLPC, preferably such that the mobile phase comprises two or more liquids, including a first liquid A and a second liquid B.

Preferably, the first liquid A is aqueous based, such as water or an aqueous solution of a buffer.

Preferably, the buffer is an acid or an organic salt or an inorganic salt.

Typically, the buffer is a phosphate salt, an acetate salt, a formate salt or trifluoroacetic acid. Most preferably, the buffer is an ammonium salt, such as ammonium acetate.

The buffer can be present at a concentration of 0.001 to 0.1 M, preferably at a concentration of 0.001 to 0.01 M, more preferably at a concentration of 0.005 to 0.01 M, most preferably at a concentration of approximately 0.007 M.

Preferably, the pH of the buffer is approximately 2 to 6, more preferably the pH is between 3.8 and 5.8, more preferably the pH of the buffer is about 4.8.

The second liquid B is preferably an organic solvent, such as methanol, ethanol, acetonitrile, n-propanol or isopropanol or a mixture thereof.

Preferably the second liquid B is a substantially water miscible solvent.

Preferably the second liquid B is a polar protic solvent such as acetic acid, methanol, ethanol, n-propanol or isopropanol, or a dipolar aprotic solvent such as acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF. More preferably where the second liquid B is a dipolar aprotic solvent, it is selected from acetone, acetonitrile, dimethoxyethane or 1,4-dioxane. Most preferably, the second liquid B is acetonitrile.

Preferably the first liquid A is an aqueous solution of ammonium acetate and the second liquid B is acetonitrile.

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Preferably a mobile phase flow rate of between 0.01 and 10 ml/min is used, more preferably a mobile phase flow rate of between 0.1 and 4 ml/min is used, more preferably a mobile phase flow rate of about 1 ml/min is used.

Preferably the method is an isocratic HPLC method, preferably such that the relative concentration of the liquids A and B is set between 99.5%A : 0.5%B and 0.5%A : 99.5%B, or between 90%A : 10%B and 10%A : 90%B, more preferably between 75%A : 25%B and 25%A : 75%B. More preferably the relative concentration of the liquids A and B is about 40%A : 60%B.

Alternatively, the relative concentration of the liquids of the mobile phase is varied to a predetermined gradient. Typically, a gradient programming is used so that the relative concentration of the liquids A and B are varied to a gradient between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 10 to 180 minutes. Preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 120 minutes. More preferably, the gradient is between 99.5%A : 0.5%B to 0.5%A : 99.5%B over 30 to 60 minutes. Alternatively, a gradient programming may be used so that the relative concentration of the liquids A and B are varied to a gradient from about 95%A : 5%B, or from about 90%A : 10%B, or from about 85%A : 15%B, to about 5%A : 95%B, or to about 10%A : 90%B, or to about 15%A : 85%B. The variation in gradient may typically take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably over 30 to 60 minutes.

A particularly preferred method of the fourth aspect of the current invention is when the first liquid A is an aqueous solution of 0.007 M ammonium acetate and the second liquid B is acetonitrile and the gradient is as follows:

—

In one embodiment of the fourth aspect of the current invention, the stationary phase is chiral. In another embodiment, the mobile phase further comprises a chiral selector.

Preferably, the stationary phase used in the fourth aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel. Particularly suitable stationary phases include octadecylsilyl silica gel or octylsilyl silica gel. A particularly preferred stationary phase comprises a YMC Pack pro C18 (250 mm x 4.6 mm), 5μ column, preferably with a 12nm pore size.

Preferably the stationary phase has a particle size of between 0.1 and lOOμm, or between 0.5 and 25μm, or between 1 and lOμm. More preferably the stationary phase has a particle size of about 5μm.

Preferably the stationary phase has a pore size of between 1 and lOOnm, or between 2 and 40nm, or between 5 and 15nm. More preferably the stationary phase has a pore size of about 12nm.

Typically, the method of the fourth aspect of the current invention is carried out at a temperature between approximately 15 to 40°C.

In one embodiment of the fourth aspect of the current invention, the chromatography is carried out in a column between 10mm and 5000mm in length, or in a column between 50mm and 1000mm in length, or between 100mm and 500mm in length. More preferably the chromatography is carried out in a column about 250mm in length.

The chromatography may be carried out in a column between 0.01mm and 100mm in internal diameter, or between 0.1mm and 50mm in internal diameter, or between lmm and 10mm in internal diameter. More preferably the chromatography is carried out in a column about 4.6mm in internal diameter.

The eluent may be analysed by a detector such as a UV or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.

For the avoidance of doubt, insofar as is practicable any embodiment of a given aspect of the present invention may occur in combination with any other embodiment of the same aspect of the present invention. In addition, insofar as is practicable it is to be understood that any preferred or optional embodiment of any aspect of the present invention should also be considered as a preferred or optional embodiment of any other aspect of the present invention.

Detailed description of the invention

The current invention can be used to analyse formoterol API or formoterol when prepared as a pharmaceutical composition, preferably in the form of formoterol fumarate dihydrate.

The pharmaceutical compositions that can be analysed by the current 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.

The term "impurities" or "related substances" as used herein throughout the specification can mean either impurities formed in the manufacture of the API or the pharmaceutical composition and/ or formed by degradation of the API or in the pharmaceutical composition on storage.

As discussed above, the HPLC methods reported in the prior art are not suitable for analysing formoterol, particularly with respect to the related substances formed in the synthesis of formoterol fumarate dihydrate synthesized using the scheme described in US patent 3994974. A reason for the difficulties encountered in the prior art could be the large polarity differences between the related substances and formoterol fumarate dihydrate.

However, a preferred embodiment of the current invention solves this problem and efficiently detects and quantifies, in a single run, all impurities and intermediates formed in this particular synthetic process. The present invention is advantageous as the gradient method allows the elution of all polar to non-polar impurities.

The current invention is also advantageous as the method is selective, linear, precise, accurate and robust for the analysis of related substances in formoterol fumarate dihydrate. In addition, the current invention is highly sensitive and allows detection and quantification of related substances in formoterol fumarate dihydrate at levels much lower than acceptance limits specified by health authorities.

In addition, the method of the current invention can be used to easily detect and quantify all degradation impurities formed on storage of samples of formoterol. This was established by carrying out forced degradation studies as per ICH QlA Guidelines and validated as per ICH Q2A Guidelines covering the parameters Specificity, Linearity and Range, Precision (Repeatability, Reproducibility and Intermediate Precision), Accuracy, Limit of Detection (LOD), Limit of Quantitation (LOQ), Robustness and System Suitability.

The buffer optionally used in the first liquid A can be an inorganic salt such as sodium, potassium, calcium, magnesium, lithium or aluminium salts of phosphate, acetate or formate and mixtures thereof. Alternatively the buffer can be an organic salt such as the ammonium salt of phosphate, acetate or formate and mixtures thereof. Alternatively the buffer can be a mineral acid or a carboxylic acid, such as acetic acid or trifluoroacetic acid. Preferably the first liquid A is a 0.007 M aqueous solution of ammonium acetate. Ammonium acetate is particularly advantageous to use as the buffer as it is compatible if mass spectrometry is needed as the detector (LC-MS).

The organic solvent(s) used as the second liquid B can be lower alkyl alcohols, such as methanol, ethanol, propanol, butanol or isopropanol or mixtures thereof. Alternatively, the organic solvent(s) may be tetrahydrofuran or acetonitrile or any suitable organic solvent(s). Preferably the organic solvent is acetonitrile.

Preferably the stationary phase used in the method of the current invention is selected from octadecylsilyl silica gel (RP-18) or octylsilyl silica gel (RP-8).

An internal standard reference compound may be used in the method of the current invention if required. Alternatively the concentration of the components analysed may be determined by comparison with one or more external reference compounds.

The inventors have tested the methods of the current invention extensively to show that they are reproducible, accurate, precise, linear with respect to concentration, and robust.

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 formoterol.

The present invention is illustrated but in no way limited by the following example.

Example

Experimental conditions:

Column: YMC Pack pro Cl 8 (250 mm x 4.6 mm), 5μ, 12nm pore size; Flow rate: 1 ml/min;

Detection: 214 nm;

Sample concentration: 2000 ppm;

Diluent: Water-Acetonitrile (1:1 v/v);

Mobile phase: 0.007 M aqueous ammonium acetate (A) - acetonitrile (B) gradient. The gradient program is described below:

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Retention Times (RT), Relative Retention Times (RRT), Limit of Detection (LOD), and Limit of Quantitation (LOQ) obtained for all the intermediates and formoterol fumarate dihydrate are summarized in Table 1.

Table 1: Retention Times (RT), Relative Retention Times (RRT), Limit of Detection (LOD), and Limit of Quantitation (LOQ)