Field of the invention The present invention relates to a new HPLC method for the analysis of the drug substance tiotropium and related substances. In particular, the present invention relates to a new HPLC method for the analysis of the salt tiotropium bromide 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 phosphate salt, a dihydrogen phosphate salt, a phosphoric acid, or a mixture thereof. A third method comprises the detection and optional quantification of methyl-di-2-thienyl glycolate. The present invention also relates to tiotropium and associated pharmaceutical compositions from which samples have been analysed by the methods of the invention and/ or which are substantially free of specific impurities.

Background art

In order to secure marketing approval for a pharmaceutical product, a manufacturer must submit detailed evidence to the appropriate regulatory authorities to prove that the product is suitable for release onto the market. It is, therefore, necessary to satisfy regulatory authorities that the product is acceptable for administration to humans and that the particular pharmaceutical composition, which is to be marketed, is sufficiently free from impurities at the time of release and that it has acceptable storage stability (shelf life). Therefore, applications to regulatory authorities for die approval of drug substances must include analytical data which demonstrate that impurities in the active pharmaceutical ingredient (API), at the time of manufacture and during storage, are absent or are present only in acceptable levels. The likely impurities in APIs and pharmaceutical compositions include residual quantities of synthetic precursors (intermediates), by-products which arise during synthesis of the API, residual solvents, isomers of the API (e.g. geometrical isomers, diastereomers or enantiomers), contaminants which are 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 degradants of the API, for instance formed by hydrolysis or oxidation. The health authorities have very stringent standards and manufacturers must demonstrate that their product is relatively free from impurities or within acceptable limits and that these standards are reproducible for each batch of pharmaceutical product that is produced. The tests that are required to demonstrate that the API or pharmaceutical compositions are safe and effective include purity assay, related substances, content uniformity and dissolution tests. The purity assay test determines the purity of the test product when compared to a standard of a known purity, while the related substances test is used to quantify all the impurities present in the product. The content uniformity test ensures that batches of product like a tablet contain a uniform amount of API and the dissolution test ensures that each batch of product has a consistent dissolution and release of the API.

The technique of choice for the analysis of APIs or pharmaceutical compositions (e.g. the tablet or capsule) is usually High Performance Liquid Chromatography (HPLC) coupled with a UV- Visible detector. The API and the impurities present, if any, are separated on the HPLC stationary phase and they can be detected and quantified using their response obtained from the UV- Visible detector.

HPLC is a chromatographic separation technique in which high-pressure pumps force the substance or mixture being analysed together with a 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. In isocratic mode, the mobile phase composition is constant throughout. A gradient HPLC mode is carried out by a gradual change over a period of time in the percentage of the two or more solvents making up the mobile phase. The change in solvent is controlled by a mixer which mixes the solvents to produce the mobile phase prior to its passing through the column. If a 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 elutes out of the column sooner. Depending upon the strength of interactions, the various constituents of the analyte appear at the end of the separating column at different times, known as retention times, where they can be detected and quantified by means of a suitable detector, such as a UV- Visible detector.

Tiottopium bromide (I), chemically known as (la,2p,4p,5a,7 )-7-[(hydroxy-di-2- tMenylacetyl)oxy]-9,9-dimemyl-3-oxa-9-azordatricyclo[3.3.1.0 ^2,4 ] nonane bromide, is an anticholinergic bronchodilator used in the management of chronic obstructive pulmonary disease (COPD). Tiotropium bromide is marketed as capsules for inhalation with the brand name Spiriva ^® .

Several HPLC methods have been reported in the literature for the estimation of tiotropium bromide in biological fluids and pharmaceutical formulations, but none of these methods have been primarily developed for the detection and quantitation of impurities in tiotropium bromide (see, for example: L. Ding et al, J. Chrom. Sci., vol. 46, pages 445-449, 2008; J. Wang et al, Rapid Communications in Mass Spectrometry, vol. 21 (11), pages 1755- 1758, 2007). Additionally, a monograph on drug substance tiotropium bromide monohydrate was published in the Indian Pharmacopeia, vol. 3, page 1812, 2007.

All these current HPLC methods are not suitable for the detection and estimation of total impurities, which include unknown impurities, that are present in tiotropium bromide samples, in particular samples synthesized by the process disclosed in commonly owned patent application WO 2009/087419 Al.

Therefore, the HPLC methods reported in the prior art are not particularly convenient or suitable for analysing tiotropium, particularly with respect to related substances.

Consequently, although several HPLC methods have been reported in the prior art for the analysis of tiotropium bromide 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 present inventors have culminated in the development and validation of a new, efficient, reproducible and simple HPLC method for the analysis of tiotropium, particularly with respect to the related substances formed during the synthetic process.

Object of the invention

It is, therefore, an object of the present invention to provide a new, accurate and sensitive HPLC method for the detection and quantitation of all intermediates and related substances that are formed and may remain in the batches of tiotropium whilst avoiding the typical problems associated with the prior art methods.

A particular object of the invention is to provide a new, accurate and sensitive HPLC method for the detection and quantitation of all intermediates and related substances that are formed and may remain in the batches of tiotropium bromide synthesized by the process disclosed in commonly owned patent application WO 2009/087419 Al.

Summary of the invention The term "tiotropium" as used herein throughout the description and claims means tiotropium and/ or any salt, solvate, hydrate, anhydrate, tautomer or isomer thereof. The present invention is particularly useful for the analysis of tiotropium bromide. A first aspect of the present invention provides a HPLC method for analysing tiotropium or a salt thereof, wherein the mobile phase comprises two or more liquids and the relative concentration of the liquids is varied to a predetermined gradient. Preferably the mobile phase comprises a first liquid A which is aqueous based, such as water or an aqueous solution of a buffer.

Preferably the buffer is selected from an acid, an organic salt, an inorganic salt, an organic base or a mixture thereof.

Preferably the buffer is a phosphate salt, a dihydrogen phosphate salt, an acetate salt, a trifluoroacetate salt, a formate salt, acetic acid, trifluoroacetic acid, formic acid, a phosphoric acid such as orthophosphoric acid, or a mixture thereof. Most preferably the buffer is a phosphate salt, a dihydrogen phosphate salt, a phosphoric acid such as orthophosphoric acid, or a mixture thereof.

In a particularly preferred embodiment of the first aspect of the present invention, the buffer is a mixture of a phosphate salt or a dihydrogen phosphate salt, with a phosphoric acid such as orthophosphoric acid. Most preferably the buffer is a mixture of a dihydrogen phosphate salt with orthophosphoric acid.

Where the buffer is or comprises a salt, preferably the counter cation is an inorganic counter cation such as an alkali metal or alkali earth metal cation. More preferably the counter cation is an alkali metal cation such as Li ⁺ , Na ⁺ or K ⁺ . Most preferably the counter cation is K ⁺ .

The buffer can be present at a concentration of 0.001 to 0.2 M, preferably at a concentration of 0.005 to 0.1 M, more preferably at a concentration of 0.005 to 0.05 M, and most preferably at a concentration of about 0.01M.

Preferably the buffer comprises potassium dihydrogen phosphate, optionally mixed with orthophosphoric acid, wherein the potassium dihydrogen phosphate is present at a concentration of 0.005 to 0.05 M. Most preferably the buffer is potassium dihydrogen phosphate present at a concentration of about 0.01 M, preferably mixed with orthophosphoric acid.

Preferably the pH of the buffer solution is approximately 1 to 7. More preferably the pH of the buffer solution is approximately 2 to 6. More preferably still the pH of the buffer solution is approximately 2 to 4. Most preferably the pH of the buffer solution is about pH 3.

Preferably the method of the first aspect of the present invention is carried out at a column temperature between approximately 15 to 40°C.

The mobile phase preferably comprises a second liquid B which is or comprises an organic solvent, preferably selected from an alkyl alcohol, such as methanol, ethanol, propanol or iso-propanol, or acetonitrile or a mixture thereof.

In one embodiment of the first aspect of the present invention, the second liquid B comprises or is a polar protic organic solvent such as acetic acid, methanol, ethanol, n- propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol, or a mixture thereof. Preferably the polar protic organic solvent is an alcohol such as a Q-Q alcohol. More preferably the alcohol is an alkyl alcohol. More preferably still the alcohol is a C _r C ₄ alkyl alcohol such as methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol. Most preferably the second liquid B is methanol.

For the purposes of the present invention, an "alkyl" group is defined as a monovalent saturated hydrocarbon, which may be straight-chained or branched, or be or include cyclic groups. An alkyl group may optionally include one or more heteroatoms N, O or S in its carbon skeleton. Examples of alkyl groups are methyl, ethyl, /7-propyl, i-propyl, /z-butyl, i- butyl, /-butyl and «-pentyl groups. Preferably an alkyl group is straight-chained or branched and does not include any heteroatoms in its carbon skeleton. Preferably an alkyl group is a C _j -C ₁₂ alkyl group, which is defined as an alkyl group containing from 1 to 12 carbon atoms. More preferably an alkyl group is a Q-Q alkyl group, which is defined as an alkyl group containing from 1 to 6 carbon atoms. In another embodiment of the first aspect of the present invention, the second liquid B is substantially water miscible.

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 Δχ _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 _Y , is from 0.40 to 0.45, or from 0.70 to 0.75; in both cases Δχ _γ = 0.05. Preferably the magnitude of Δχ _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. In one embodiment of the first aspect of the present invention, the mobile phase contains less than 10%, less than 5% or less than 1% acetonitrile by volume. In one embodiment, the mobile phase contains no acetonitrile.

In another embodiment of the first aspect of the present invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any organic dipolar aprotic solvent by volume. In one embodiment, d e mobile phase contains no organic dipolar aprotic solvent.

A preferred embodiment of the first aspect of the present invention is when the first liquid A is an aqueous solution of a dihydrogen phosphate salt mixed with or hophosphoric acid and the second liquid B is methanol.

A particularly preferred embodiment of the first aspect of the present invention is when the first liquid A is 0.01 M potassium dihydrogen phosphate mixed with orfhophosphoric acid and die second liquid B is methanol.

The method of the first aspect of the present invention comprises a gradient programming so that the relative concentration of die liquids A and B by volume is typically varied to a gradient between 100 % A : 0 % B to 0 % A : 100 % B over a period of 10 to 180 minutes. Preferably the gradient is between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 120 minutes, more preferably 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 60 minutes.

Alternatively, the HPLC method of the first aspect of the present invention may comprise a gradient programming so that the relative concentration of the liquids A and B by volume starts at a first ratio, then is varied to a first gradient over a first period of time, to arrive at a second ratio, then is varied to a second gradient over a second period of time, to arrive at a third ratio, then optionally is varied to a third gradient over a third period of time, to arrive at a fourth ratio.

The first ratio may be 75-95 % A : 5-25 % B. Preferably the first ratio is 80-90 % A : 10-20 % B. Most preferably the first ratio is about 85 % A : 15 % B.

The first period of time may be from 0 to 60 minutes. Preferably the first period of time is from 5 to 30 minutes. Most preferably the first period of time is about 15 minutes.

The second ratio may be 60-80 % A : 20-40 % B. Preferably the second ratio is 65-75 % A : 25-35 % B. Most preferably the second ratio is about 70 % A : 30 % B.

The second period of time may be from 0 to 60 minutes. Preferably the second period of time is from 2 to 20 minutes. Most preferably the second period of time is about 10 minutes.

The third ratio may be 15-35 % A : 65-85 % B. Preferably the third ratio is 20-30 % A : 70- 80 % B. Most preferably the third ratio is about 25 % A : 75 % B.

The third period of time may be from 0 to 60 minutes. Preferably the third period of time is from 1 to 15 minutes. Most preferably the third period of time is about 5 minutes.

The fourth ratio may be 0-20 % A : 80-100 % B. Preferably the fourth ratio is 5-15 % A 85-95 % B. Most preferably the fourth ratio is about 10 % A : 90 % B. 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 still a mobile phase flow rate of between 0.5 and 1.5 ml/min is used, most preferably a mobile phase flow rate of about 1 ml/ min is used.

In one embodiment of the first aspect of the present invention, the stationary phase used is a gel, preferably a silica gel.

In another embodiment, the stationary phase used is chiral and/or the mobile phase further comprises a chiral selector.

Preferably the stationary phase used in the first aspect of the present invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, arninopropyl silica gel or an aUtyl-diol silica gel. Particularly suitable stationary phases include octadecylsilyl silica gel or octylsilyl silica gel. A particularly preferred stationary phase comprises an Inertsil ODS 3V (250 mm x 4.6 mm), 5μηι column.

Preferably the stationary phase has a particle size of between 0.1 and ΙΟΟμηι, or between 0.5 and 25μιτι, or between 1 and ΙΟμηι, or between 4.5 and 6μηι. More preferably the stationary phase has a particle size of about 5μπι.

Preferably the stationary phase has a pore size of between 10 and ΙΟΟθΑ, or between 25 and 500A, or between 50 and 20θΑ. More preferably the stationary phase has a pore size of between 75 and 125A, or between 90 and HOA. Most preferably the stationary phase has a pore size or about lOOA.

In one embodiment of the first aspect of the present 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 between 200mm and 280mm in length. Most 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 1mm and 10mm in internal diameter. More preferably the chromatography is carried out in a column about 4.6mm in internal diameter.

A particularly preferred method according to the first aspect of the present invention is when the first liquid A is 0.01 M potassium dihydrogen phosphate mixed with orthophosphoric acid and the second liquid B is methanol and the gradient is as follows:

The eluent may be analysed by a detector such as a UV and/ or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector. In one embodiment of the first aspect of the present invention the HPLC method detects and optionally quantifies in a single run one or more impurities selected from:

scopine-di-2-thienyl glycolate; and

mediyl-di-2-thienyl glycolate. Preferably the HPLC method detects and optionally quantifies in a single run both scopine- di-2-thienyl glycolate and methyl-di-2-thienyl glycolate.

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

scopine-di-2-thienyl glycolate; and methyl-di-2-thienyl glycokte.

In one embodiment of the first aspect of the present invention, scopine-di-2-thienyl glycolate and/ or methyl-di-2-thienyl glycolate is used as internal or external reference marker, or as internal or external reference standard.

In another embodiment of the first aspect of the present invention, the HPLC method is used for the analysis of tiotropium or a salt thereof that is suitable for use in a pharmaceutical composition.

Preferably the HPLC method is used for the analysis of tiotropium or a salt thereof that has not entered the human or animal body. Preferably the tiotropium or the salt thereof that is analysed is not in contact with a human or animal bodily fluid such as plasma. Preferably the tiotropium or the salt thereof that is analysed is not in solution.

In another embodiment of the first aspect of the present invention, the HPLC method is used for the analysis of a pharmaceutical composition comprising tiotropium or a salt thereof. In yet another embodiment of the first aspect of the present invention, the HPLC method is used for the analysis of a substance comprising at least 5% tiotropium or a salt thereof by weight. Preferably the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% tiotropium or a salt thereof by weight. Most preferably the substance comprises at least 95% tiotropium or a salt thereof by weight.

In yet another embodiment of the first aspect of the present invention, the HPLC method is used for the analysis of a substance comprising tiotropium or a salt thereof as the only active pharmaceutical ingredient. A second aspect of the present invention provides a chromatographic method for analysing tiotropium or a salt thereof, wherein the mobile phase comprises a phosphate salt, a dihydrogen phosphate salt, a phosphoric acid such as orthophosphoric acid, or a mixture thereof. In one embodiment of the second aspect of the present invention, the mobile phase comprises a mixture of a phosphate salt or a dihydrogen phosphate salt, with a phosphoric acid such as orthophosphoric acid. Most preferably d e mobile phase comprises a mixture of a dihydrogen phosphate salt with orthophosphoric acid.

Where the mobile phase comprises a phosphate salt or a dihydrogen phosphate salt, preferably the counter cation is an inorganic counter cation such as an alkali metal or alkali earth metal cation. More preferably the counter cation is an alkali metal cation such as Li ⁺ , Na ⁺ or K ⁺ . Most preferably the counter cation is K ⁺ .

Preferably the mobile phase further comprises water. More preferably the mobile phase comprises an aqueous solution of the phosphate salt, dihydrogen phosphate salt, phosphoric acid, or mixture thereof. More preferably the mobile phase comprises an aqueous solution of a mixture of a phosphate salt or a dihydrogen phosphate salt, with a phosphoric acid such as orthophosphoric acid. Most preferably the mobile phase comprises an aqueous solution of a mixture of a dihydrogen phosphate salt with orthophosphoric acid. The phosphate salt, dihydrogen phosphate salt, phosphoric acid, or mixture thereof can be present at a concentration of 0.001 to 0.2 M, preferably at a concentration of 0.005 to 0.1 M, more preferably at a concentration of 0.005 to 0.05 M, and most preferably at a concentration of about 0.01M. Preferably the mobile phase comprises potassium dihydrogen phosphate, optionally mixed with orthophosphoric acid, wherein the potassium dihydrogen phosphate is present at a concentration of 0.005 to 0.05 M. Most preferably the potassium dihydrogen phosphate is present at a concentration of about 0.01 M, preferably mixed with orthophosphoric acid. Preferably the pH of the aqueous solution is approximately 1 to 7. More preferably the pH of the aqueous solution is approximately 2 to 6. More preferably still the pH of the aqueous solution is approximately 2 to 4. Most preferably the pH of the aqueous solution is about pH 3. In one embodiment of the second aspect of the present invention, the mobile phase comprises two or more liquids, including a first liquid A and a second liquid B, wherein at least one of said liquids comprises the phosphate salt, dihydrogen phosphate salt, phosphoric acid, or mixture thereof. Preferably the first liquid A is an aqueous solution of the phosphate salt, dihydrogen phosphate salt, phosphoric acid, or mixture thereof.

The second liquid B preferably comprises or is an organic solvent, preferably selected from an alkyl alcohol, such as methanol, ethanol, propanol or iso-propanol, or acetonitrile or a mixture thereof.

In one embodiment of the second aspect of the present invention, the second liquid B comprises or is a polar protic organic solvent such as acetic acid, methanol, ethanol, n- propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol, or a mixture thereof. Preferably the polar protic organic solvent is an alcohol such as a Q-Q alcohol. More preferably the alcohol is an alkyl alcohol. More preferably still the alcohol is a C _r C ₄ alkyl alcohol such as methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol. Most preferably the second liquid B is methanol. In another embodiment of the second aspect of the present invention, die second liquid B is substantially water miscible.

In one embodiment of the second aspect of the present invention, the mobile phase contains less than 10%, less than 5% or less than 1% acetonitrile by volume. In one embodiment, the mobile phase contains no acetonitrile.

In another embodiment of the second aspect of the present invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any organic dipolar aprotic solvent by volume. In one embodiment, the mobile phase contains no organic dipolar aprotic solvent. A preferred embodiment of the second aspect of the present invention is when the first liquid A is an aqueous solution of a dihydrogen phosphate salt mixed with orthophosphoric acid and the second liquid B is methanol. A particularly preferred embodiment of the second aspect of the present invention is when the first liquid A is 0.01 M potassium dihydrogen phosphate mixed with orthophosphoric acid and the second liquid B is methanol.

In one embodiment of the second aspect of the present invention, the chromatographic method is a liquid chromatographic method such as a HPLC, LC-MS or LC-MS/MS method; preferably the chromatographic method is a HPLC method.

The chromatographic method may be an isocratic method, preferably such that the relative concentration of the liquids A and B by volume 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 still the relative concentration of the liquids A and B by volume is about 55 % A : 45 % B.

Alternately, the relative concentration of the liquids in the mobile phase may be varied to a predetermined gradient. Typically, the relative concentration of the liquids A and B by volume is varied to a gradient between 100 % A : 0 % B to 0 % A : 100 % B over a period of 10 to 180 minutes. Preferably the gradient is between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 120 minutes, more preferably 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 60 minutes.

Alternatively, the chromatographic method of the second aspect of the present invention may comprise a gradient programming so that the relative concentration of the liquids A and B by volume starts at a first ratio, then is varied to a first gradient over a first period of time, to arrive at a second ratio, then is varied to a second gradient over a second period of time, to arrive at a third ratio, then optionally is varied to a third gradient over a third period of time, to arrive at a fourth ratio. The first ratio may be 75-95 % A : 5-25 % B. Preferably the first ratio is 80-90 % A : 10-20 % B. Most preferably the first ratio is about 85 % A : 15 % B.

The first period of time may be from 0 to 60 minutes. Preferably the first period of time is from 5 to 30 minutes. Most preferably the first period of time is about 15 minutes.

The second ratio may be 60-80 % A : 20-40 % B. Preferably the second ratio is 65-75 % A : 25-35 % B. Most preferably the second ratio is about 70 % A : 30 % B. The second period of time may be from 0 to 60 minutes. Preferably the second period of time is from 2 to 20 minutes. Most preferably the second period of time is about 10 minutes.

The third ratio may be 15-35 % A : 65-85 % B. Preferably the third ratio is 20-30 % A : 70- 80 % B. Most preferably the third ratio is about 25 % A : 75 % B.

The third period of time may be from 0 to 60 minutes. Preferably the third period of time is from 1 to 15 minutes. Most preferably the third period of time is about 5 minutes. The fourth ratio may be 0-20 % A : 80-100 % B. Preferably the fourth ratio is 5-15 % A : 85-95 % B. Most preferably the fourth ratio is about 10 % A : 90 % B.

A particularly preferred method according to the second aspect of the present invention is when the first liquid A is 0.01 M potassium dihydrogen phosphate mixed with orthophosphoric acid and the second liquid B is methanol and the gradient is as follows:

Time (min) % A (by volume) % B (by volume)

0 85 15

15 70 30

25 25 75

30 10 90

40 10 90

45 85 15 Preferably the method of the second aspect of the present invention is carried out at a column temperature between approximately 15 to 40°C. 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 still a mobile phase flow rate of between 0.5 and 1.5 ml/min is used, most preferably a mobile phase flow rate of about 1 ml/ min is used. In one embodiment of the second aspect of the present invention, the stationary phase used is a gel, preferably a silica gel.

In another embodiment, the stationary phase used is chiral and/or the mobile phase further comprises a chiral selector.

Preferably the stationary phase used in the second aspect of the present 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 an Inertsil ODS 3V (250 mm x 4.6 mm), 5μηι column.

Preferably the stationary phase has a particle size of between 0.1 and ΙΟΟμη , or between 0.5 and 25μηι, or between 1 and ΙΟμηι, or between 4.5 and 6μιη. More preferably the stationary phase has a particle size of about 5 ιτι.

Preferably the stationary phase has a pore size of between 10 and ΙΟΟθΑ, or between 25 and 500A, or between 50 and 200A. More preferably the stationary phase has a pore size of between 75 and 125A, or between 90 and llOA. Most preferably the stationary phase has a pore size of about ΙΟθΑ.

In one embodiment of the second aspect of the present 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 between 200mm and 280mm in length. Most 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 1mm 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 and/ or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.

In one embodiment of the second aspect of the present invention the chromatographic method detects and optionally quantifies in a single run one or more impurities selected from:

scopine-di-2-thienyl glycolate; and

methyl-di-2-thienyl glycolate.

Preferably the chromatographic method detects and optionally quantifies in a single run both scopine-di-2-thienyl glycolate and methyl-di-2-thienyl glycolate.

In a preferred embodiment the chromatographic method according to the second aspect of the present invention efficiently detects and quantifies in a single run all impurities including those selected from the following compounds:

scopine-di-2-thienyl glycolate; and

methyl-di-2-thienyl glycolate.

In one embodiment of the second aspect of the present invention, scopine-di-2-tl ienyl glycolate and/or methyl-di-2-thienyl glycolate is used as internal or external reference marker, or as internal or external reference standard. In another embodiment of the second aspect of the present invention, the chromatographic method is used for the analysis of tiotropium or a salt thereof that is suitable for use in a pharmaceutical composition. Preferably the chromatographic method is used for the analysis of tiotropium or a salt thereof that has not entered the human or animal body. Preferably the tiotropium or the salt thereof that is analysed is not in contact with a human or animal bodily fluid such as plasma. Preferably the tiotropium or the salt thereof that is analysed is not in solution. In another embodiment of the second aspect of the present invention, the chromatographic method is used for the analysis of a pharmaceutical composition comprising tiotropium or a salt thereof.

In yet another embodiment of the second aspect of the present invention, the chromatographic method is used for the analysis of a substance comprising at least 5% tiotropium or a salt thereof by weight. Preferably the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% tiotropium or a salt thereof by weight. Most preferably the substance comprises at least 95% tiotropium or a salt thereof by weight.

In yet another embodiment of the second aspect of the present invention, the chromatographic method is used for the analysis of a substance comprising tiotropium or a salt thereof as the only active pharmaceutical ingredient. A third aspect of the present invention provides a method for analysing a substance, comprising the detection and optional quantification of methyl-di-2-thienyl glycolate.

Preferably the method of the third aspect of the present invention further comprises the detection and optional quantification of tiotropium or a salt thereof and/or scopine-di-2- thienyl glycolate.

Preferably the method of the third aspect of the present invention comprises the detection and optional quantification of all three of: tiotropium or a salt thereof;

scopine-di-2-thienyl glycolate; and

mediyl-di-2-thienyl glycolate. In one embodiment of the third aspect of the present invention, the substance is an active pharmaceutical ingredient. Preferably the substance is tiotropium, optionally in the form of a salt, solvate, hydrate or anhydrate. Most preferably the tiotropium is the bromide salt, preferably in monohydrate or anhydrous form. Preferably the tiotropium analysed is for use in a pharmaceutical composition.

Preferably the substance that is analysed has not entered the human or animal body. Preferably the substance that is analysed is not in contact with a human or animal bodily fluid such as plasma. Preferably the substance that is analysed is not in solution. In another embodiment of the third aspect of the present invention, the method is a method of analysing a pharmaceutical composition comprising tiotropium or a salt thereof.

In yet another embodiment of the third aspect of the present invention, the substance comprises at least 5% tiotropium or a salt thereof by weight. Preferably the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% tiotropium or a salt thereof by weight. Most preferably die substance comprises at least 95% tiotropium or a salt thereof by weight.

In yet another embodiment of the third aspect of the present invention, the substance comprises tiotropium or a salt thereof as the only active pharmaceutical ingredient.

In a preferred embodiment of the third aspect of the present invention, the method is a chromatographic method, preferably wherein 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 selected from an acid, an organic salt, an inorganic salt, an organic base or a mixture thereof.

Preferably the buffer is a phosphate salt, a dihydrogen phosphate salt, an acetate salt, a trifluoroacetate salt, a formate salt, acetic acid, trifiuoroacetic acid, formic acid, a phosphoric acid such as orthophosphoric acid, or a mixture thereof. Most preferably the buffer is a phosphate salt, a dihydrogen phosphate salt, a phosphoric acid such as orthophosphoric acid, or a mixture thereof. In a particularly preferred embodiment of the third aspect of the present invention, the buffer is a mixture of a phosphate salt or a dihydrogen phosphate salt, with a phosphoric acid such as orthophosphoric acid. Most preferably the buffer is a mixture of a dihydrogen phosphate salt with orthophosphoric acid. Where the buffer is or comprises a salt, preferably the counter cation is an inorganic counter cation such as an alkali metal or alkali earth metal cation. More preferably the counter cation is an alkali metal cation such as Li ⁺ , Na ⁺ or K ⁺ . Most preferably the counter cation is K ⁺ . The buffer can be present at a concentration of 0.001 to 0.2 M, preferably at a concentration of 0.005 to 0.1 M, more preferably at a concentration of 0.005 to 0.05 M, and most preferably at a concentration of about 0.01M.

Preferably the buffer comprises potassium dihydrogen phosphate, optionally mixed with orthophosphoric acid, wherein the potassium dihydrogen phosphate is present at a concentration of 0.005 to 0.05 M. Most preferably the buffer is potassium dihydrogen phosphate present at a concentration of about 0.01 M, preferably mixed with orthophosphoric acid. Preferably the pH of the buffer solution is approximately 1 to 7. More preferably the pH of the buffer solution is approximately 2 to 6. More preferably still the pH of the buffer solution is approximately 2 to 4. Most preferably the pH of the buffer solution is about pH 3. The second liquid B preferably comprises or is an organic solvent, preferably selected from an alkyl alcohol, such as methanol, ethanol, propanol or iso-propanol, or acetonitrile or a mixture thereof.

In one embodiment of the third aspect of the present invention, the second liquid B comprises or is a polar protic organic solvent such as acetic acid, methanol, ethanol, n- propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol, or a mixture thereof. Preferably the polar protic organic solvent is an alcohol such as a C _r C ₆ alcohol. More preferably the alcohol is an alkyl alcohol. More preferably still the alcohol is a Q-C ₄ alkyl alcohol such as methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol. Most preferably the second liquid B is methanol.

In another embodiment of the third aspect of the present invention, the second liquid B is substantially water miscible.

In one embodiment of the third aspect of the present invention, the mobile phase contains less than 10%, less than 5% or less than 1% acetonitrile by volume. In one embodiment, the mobile phase contains no acetonitrile.

In another embodiment of the third aspect of the present invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any organic dipolar aprotic solvent by volume. In one embodiment, die mobile phase contains no organic dipolar aprotic solvent.

A preferred embodiment of the third aspect of the present invention is when the first liquid A is an aqueous solution of a dihydrogen phosphate salt mixed with orthophosphoric acid and the second liquid B is methanol. A particularly preferred embodiment of the third aspect of the present invention is when the first liquid A is 0.01 M potassium dihydrogen phosphate mixed with orthophosphoric acid and the second liquid B is methanol. In one embodiment of the third aspect of the present invention, the chromatographic method is a liquid chromatographic method such as a HPLC, LC-MS or LC-MS/MS method; preferably the chromatographic method is a HPLC method. Preferably in any chromatographic method of the third aspect of the present invention, said method detects and optionally quantifies in a single run methyl-di-2-thienyl glycolate. More preferably said method also detects and optionally quantifies in the same run tiotropium or a salt thereof and/ or scopine-di-2-thienyl glycolate. Most preferably said method detects and optionally quantifies in a single run all three of: tiotropium or a salt thereof;

scopine-di-2-thienyl glycolate; and

methyl-di-2-thienyl glycolate. In one embodiment of the third aspect of the present invention, tiotropium or a salt thereof, scopine-di-2-thienyl glycolate and/or methyl-di-2-thienyl glycolate is used as internal or external reference marker, or as internal or external reference standard.

The chromatographic method may be an isocratic method, preferably such that the relative concentration of the liquids A and B by volume 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 still the relative concentration of the liquids A and B by volume is about 55 % A : 45 % B. Alternately, the relative concentration of the liquids in the mobile phase may be varied to a predetermined gradient. Typically, the relative concentration of the liquids A and B by volume is varied to a gradient between 100 % A : 0 % B to 0 % A : 100 % B over a period of 10 to 180 minutes. Preferably the gradient is between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 120 minutes, more preferably 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 60 minutes.

Alternatively, the chromatographic method of the third aspect of the present invention may comprise a gradient programming so that the relative concentration of the liquids A and B by volume starts at a first ratio, then is varied to a first gradient over a first period of time, to arrive at a second ratio, then is varied to a second gradient over a second period of time, to arrive at a third ratio, then optionally is varied to a third gradient over a third period of time, to arrive at a fourth ratio.

The first ratio may be 75-95 % A : 5-25 % B. Preferably the first ratio is 80-90 % A : 10-20 % B. Most preferably the first ratio is about 85 % A : 15 % B.

The first period of time may be from 0 to 60 minutes. Preferably the first period of time is from 5 to 30 minutes. Most preferably the first period of time is about 15 minutes.

The second ratio may be 60-80 % A : 20-40 % B. Preferably the second ratio is 65-75 % A : 25-35 % B. Most preferably the second ratio is about 70 % A : 30 % B. The second period of time may be from 0 to 60 minutes. Preferably the second period of time is from 2 to 20 minutes. Most preferably the second period of time is about 10 minutes.

The third ratio may be 15-35 % A : 65-85 % B. Preferably the third ratio is 20-30 % A : 70- 80 % B. Most preferably the third ratio is about 25 % A : 75 % B.

The third period of time may be from 0 to 60 minutes. Preferably the third period of time is from 1 to 15 minutes. Most preferably the third period of time is about 5 minutes. The fourth ratio may be 0-20 % A : 80-100 % B. Preferably the fourth ratio is 5-15 % A : 85-95 % B. Most preferably the fourth ratio is about 10 % A : 90 % B.

A particularly preferred method according to the third aspect of the present invention is when the first liquid A is 0.01 M potassium dihydrogen phosphate mixed with orfhophosphoric acid and the second liquid B is methanol and the gradient is as follows: Time (min) % A (by volume) % B (by volume)

0 85 15

15 70 30

25 25 75

30 10 90

40 10 90

45 85 15

55 85 15

Preferably the method of the third aspect of the present invention is carried out at a column temperature between approximately 15 to 40°C.

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 still a mobile phase flow rate of between 0.5 and 1.5 ml/min is used, most preferably a mobile phase flow rate of about 1 ml/ min is used.

In one embodiment of the third aspect of the present invention, the stationary phase used is a gel, preferably a silica gel.

In another embodiment, the stationary phase used is chiral and/or the mobile phase further comprises a chiral selector.

Preferably the stationary phase used in the third aspect of the present 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 an Inertsil ODS 3V (250 mm x 4.6 mm), 5μηι column.

Preferably the stationary phase has a particle size of between 0.1 and ΙΟΟμηι, or between 0.5 and 25μιη, or between 1 and ΙΟμηι, or between 4.5 and 6μπι. More preferably the stationary phase has a particle size of about 5μπι. Preferably the stationary phase has a pore size of between 10 and ΙΟΟθΑ, or between 25 and 500A, or between 50 and 20θΑ. More preferably the stationary phase has a pore size of between 75 and 125A, or between 90 and HOA. Most preferably the stationary phase has a pore size of about ΙΟθΑ.

In one embodiment of the third aspect of the present 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 between 200mm and 280mm in length. Most 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 1mm 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 and/ or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.

A fourth aspect of the present invention provides a process for preparing a batch of a substance, said process comprising the steps of:

(i) providing a source quantity of the substance;

(ii) removing a sample from said source quantity and subjecting said sample to a method according to any of the first to third aspects of the present invention; and

(iii) retaining some or all of the remainder of said source quantity to give the batch of the substance.

In one embodiment of the fourth aspect of the present invention, the substance comprises or is an active pharmaceutical ingredient. Preferably the substance comprises or is tiotropium, optionally in the form of a salt, solvate, hydrate or anhydrate. More preferably the tiotropium is the bromide salt, preferably in monohydrate or anhydrous form. Preferably the substance is for use in a pharmaceutical composition. In another embodiment of the fourth aspect of the present invention, the substance comprises or is a pharmaceutical composition. Preferably the pharmaceutical composition comprises tiotropium, optionally in the form of a salt, solvate, hydrate or anhydrate. More preferably the tiotropium is the bromide salt, preferably in monohydrate or anhydrous form. Preferably the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

Preferably the substance of the fourth aspect of the present invention has not entered the human or animal body. Preferably the substance is not in contact with a human or animal bodily fluid such as plasma. Preferably the substance is not in solution.

In another embodiment of the fourth aspect of the present invention, the substance comprises at least 5% tiotropium or a salt thereof by weight. Preferably the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% tiotropium or a salt thereof by weight. Most preferably the substance comprises at least 95% tiotropium or a salt thereof by weight.

In yet another embodiment of the fourth aspect of the present invention, the substance comprises tiotropium or a salt thereof as the only active pharmaceutical ingredient.

A fifth aspect of the present invention provides a batch of tiotropium or a salt thereof which has been prepared by a process according to the fourth aspect of the present invention. Preferably the tiotropium or the salt thereof is substantially free of scopine-di-2- thienyl glycokte and/ or metnyl-di-2-thienyl glycolate.

Tiotropium or a salt thereof is "substantially free" of a compound, if it comprises less than about 5% of that compound, preferably less than about 3%, preferably less than about 2%, preferably less than about 1%, preferably less than about 0.5%, preferably less than about 0.1%, preferably less than about 0.05%, preferably as measured by HPLC.

A sixth aspect of the present invention provides a process for preparing a pharmaceutical composition, said process comprising the step of combining one or more pharmaceutically acceptable excipients with part or all of a batch of tiotropium or a salt thereof which has been prepared by a process according to the fourth aspect of the present invention.

A seventh aspect of the present invention provides a pharmaceutical composition prepared by a process according to the sixth aspect of the present invention.

An eighth aspect of die present invention provides a batch of one or more pharmaceutical compositions which have been prepared by a process according to the fourth aspect of the present invention, wherein the pharmaceutical composition(s) comprise tiotropium or a salt thereof. Preferably the pharmaceutical composition(s) also comprise one or more pharmaceutically acceptable excipients.

For the avoidance of doubt, insofar as is practicable any embodiment of a given aspect of die 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 present invention

The present invention can be used to analyse tiotropium and/ or its salts, in particular the bromide salt, as an API or when prepared as a pharmaceutical composition. The pharmaceutical compositions that can be analysed by the present 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, inhalation or infusion routes.

The term "impurities" or "related substances" as used herein throughout die 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 tiotropium, particularly with respect to the related substances formed in the synthesis of tiotropium bromide and/ or other salts prepared by the process disclosed in commonly owned WO 2009/087419 Al.

However, the present 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. Identification of all impurities in a single run is particularly advantageous and cost saving in a commercial environment. The present invention is also advantageous as the method is selective, linear and precise for the analysis of related substances in tiotropium and/or its salts. In addition, the present invention is highly sensitive and allows detection and quantification of related substances in tiotropium and/ or its salts at levels much lower than acceptance limits specified by health authorities.

In addition, the method of the present invention can be used to easily detect and quantify all degradation impurities formed on storage of samples of tiotropium. This was established by carrying out forced degradation studies as per ICH Q1A (R2) Guidelines and validated as per ICH Q2C (Rl) Guidelines covering the parameters Specificity, Linearity and Range, Precision (Reproducibility), Limit of Detection (LOD), Limit of Quantitation (LOQ) 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, dihydrogen phosphate, acetate, formate and mixtures thereof. Alternatively the buffer can be an organic salt such as die ammonium salt of acetate or formate and mixtures thereof. Alternatively the buffer can be a mineral acid or a carboxylic acid, such as acetic acid, trifluoroacetic acid, a phosphoric acid or orthophosphoric acid. Alternatively the buffer can be an organic base such as cuethylarnine, n-propylamine or triethylamine. Preferably the buffer comprises 0.01 M potassium dihydrogen phosphate mixed with orthophosphoric acid. The second liquid B is an organic solvent such as an alcohol, preferably a Q to Q alkyl alcohol like methanol, ethanol, propanol, butanol or iso-propanol or mixtures thereof. Alternatively, the organic solvent(s) may be tetrahydrofuran, ethyl acetate or acetonitrile or any suitable organic solvent(s). Most preferably the organic solvent is methanol. Preferably the stationary phase used in the method of the present invention is selected from octadecylsilyl silica gel (RP-18) or octylsilyl silica gel (R.P-8).

An internal standard reference compound may be used in the method of the present 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 present invention extensively to show that they are reproducible, precise and linear with respect to concentration. 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 present invention is illustrated but in no way limited by the following example.

Example

Experimental conditions:

Column: Inertsil ODS 3V (250 mm x 4.6 mm), 5μ, ΙΟθΑ pore size;

Flow rate: 1 ml/min;

Detection: 235 nm;

Sample concentration: 1000 ppm;

Autosampler temperature: 10°C; Column oven temperature: 25°C;

Diluent: [0.01 M potassium dihydrogen phosphate, pH 3.0 with orthophosphoric acid] - methanol (50:50 v/v);

The sample of tiotropium bromide (anhydrous) is initially dissolved in a small volume of the diluent; the sample solution is then injected into the column which is run using the mobile phase outlined below;

First Liquid A: 0.01 M potassium dihydrogen phosphate, pH 3.0 with orthophosphoric acid;

Second Liquid B: methanol;

Mobile phase: First Liquid A - Second Liquid B gradient; the gradient program is described below, with the program between 40 and 55 minutes being used to equilibrate and prepare the column for the next run:

Retention times (RT), Relative retention times (RRT), Limit of Detection (LOD) and Limit of Quantitation (LOQ) obtained for the impurities and tiotropium are given in Table 1.

% LOD and LOQ values are with respect to sample concentration of 1000 ppm.

Table 1 It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the invention. Various modifications and embodiments can be made without departing from the scope and spirit of the invention, which is defined by the following claims only.