WO2011095803A1

WO2011095803A1 - Hplc method for analyzing frovatriptan

Info

Publication number: WO2011095803A1
Application number: PCT/GB2011/050168
Authority: WO
Inventors: Erra Koteswara Satya Vijayakumar; Vinayak Vele; Sandesh Parab; Avinash Nikam; Maitreyee Samel
Original assignee: Generics [Uk] Limited; Mylan India Private Limited
Priority date: 2010-02-02
Filing date: 2011-02-01
Publication date: 2011-08-11

Abstract

The present invention relates to a new HPLC method for the analysis of the drug substance frovatriptan and related substances. In particular, the present invention relates to a new HPLC method for the analysis of the drug substance frovatriptan monosuccinate monohydrate 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 other methods the stationary phase is reverse phase and the mobile phase comprises an alcohol and/or an alkylamine. In yet another method the mobile phase comprises a formate salt. A final method comprises the detection and optional quantification of specific impurities. The present invention also relates to frovatriptan 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.

Description

HPLC METHOD FOR ANALYZING PROVATRIPTAN

Field of the invention The present invention relates to a new HPLC method for the analysis of the drug substance frovatriptan and related substances. In particular, the present invention relates to a new HPLC method for the analysis of the drug substance frovatriptan monosuccinate monohydrate and related substances. In a first method the mobile phase comprises two or more liquids and the relative concentration of die liquids is varied to a predetermined gradient. In other methods the stationary phase is reverse phase and the mobile phase comprises an alcohol and/or an aUtylamine. In yet another method the mobile phase comprises a formate salt. A final method comprises the detection and optional quantification of specific impurities. The present invention also relates to frovatriptan and associated pharmaceutical compositions from which samples have been analysed by the methods of die 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 the 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 ate 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 Mice 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 malting 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.

Frovatriptan, chemically known as (+)-R-3-methykmino-6-carboxamido-l, 2,3,4- tetrahydrocarbazole, is an orally active 5-hydroxytriptamine (5-HT) receptor agonist and is used for the management of migraine symptoms. Frovatriptan is marketed as its monosuccinate monohydrate salt (I) (commonly referred to as frovatriptan succinate monohydrate) with the brand name Frova^®.

Several HPLC methods have been reported in the literature for the estimation of frovatriptan in biological fluids and pharmaceutical formulations, but none of these methods have been primarily developed for the detection and quantitation of all impurities in the drug substance frovatriptan monosuccinate monohydrate. Examples of these publications are: HPLC Methods for Recently Approved Pharmaceuticals by G. Lunn, Willey Interscience, pages 286-287, 2005; L. Laugher et al, Chromatographia, vol. 52, pages S113-S119, 2000. Additionally, chiral HPLC methods have been reported, to analyse the enantiomeric purity of frovatriptan, in M. Khan et al, J. Chromatography B, vol. 846 (1-2), pages 119-123, 2007.

However, the current HPLC methods are not suitable for the detection and estimation of total impurities, especially with respect to unknown impurities that are present in a frovatriptan or frovatriptan monosuccinate monohydrate sample, particularly samples synthesized by the process disclosed in co-pending Indian patent application IN 657/KOL/2009 and the corresponding international patent application WO 2010/122343.

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

Consequently, although several HPLC methods have been reported in the prior art for the analysis of frovatriptan 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 frovatriptan, 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 frovatriptan 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 frovatriptan synthesized by the process disclosed in co-pending Indian patent application IN 657/KOL/2009 and the corresponding international patent application WO 2010/122343.

Summary of the invention The term "frovatriptan" as used herein throughout the description and claims means frovatriptan and/or any salt, solvate, hydrate, anhydrate, tautomer, isomer or enantiomer thereof. The present invention is particularly useful for the analysis of frovatriptan monosuccinate monohydrate. A first aspect of the present invention provides a HPLC method for analysing frovatriptan or a salt thereof, wherein the mobile phase comprises two or more Hquids 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 (such as a c aUcylamine or a triaUiylamine) or a mixture thereof.

Typically, the buffer is a phosphate salt, an acetate salt, a formate salt, a trifluoroacetate salt, a phosphoric acid, acetic acid, formic acid, trifluoroacetic acid, an aU^lamine such as cHemylamine, n-propylamine or ttiemylamine, or a mixture thereof. More typically, the buffer is a phosphate salt, an acetate salt, a formate salt, acetic acid, trifluoroacetic acid, diemylamine, n-propylamine, ttiemylamine or a mixture thereof.

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, /?-propyl, z-propyl, »-butyl, i- butyl, /-butyl and /z-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_rC₁₂ alkyl group, which is defined as an alkyl group containing from 1 to 12 carbon atoms. More preferably an alkyl group is a C C₆ alkyl group, which is defined as an alkyl group containing from 1 to 6 carbon atoms.

In one embodiment of the first aspect of the present invention, the buffer comprises a carboxylate salt and/ or an organic base. Preferably the carboxylate salt is an acetate salt, a formate salt or a trifluoroacetate salt. More preferably the carboxylate salt is a formate salt such as ammonium formate. Preferably the organic base is an amine such as an alkylamine. The alkylamine may be selected from a monoalkylamine such as emylamine, n-propylamine or isopropylamine, a dialkylamine such as diemylamine, N-etiiyl-N-methylamine or N- emyl-N-n-propylamine, a cycKc-alkylamine such as piperidine, pyrrolidine or morpholine, or a tiialfylamine such as toe ylamine, dnsopropylemylamine or dimemylemylamine. Preferably the all^ amine is a monoalkylamine such as emylamine, n-propylarnine or isopropylamine. More preferably the alfylamine is n-propylamine.

For the purposes of the present invention, an '^U^lamine" preferably contains from 1 to 20 carbon atoms, more preferably contains from 2 to 12 carbon atoms and most preferably contains from 3 to 8 carbon atoms.

Where the buffer is or comprises a salt, preferably the counter cation is an ammonium cation.

Most preferably the buffer is a mixture of a formate salt, such as ammonium formate, and a base, more preferably an organic base, such as n-propylamine.

The salt in 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 0.01 to 0.1 M, and most preferably at a concentration of about 0.02 M; and/ or the base in the buffer can be present at a concentration of 0.001 to 0.2 % v/v, preferably at a concentration of 0.005 to 0.2 % v/v, more preferably at a concentration of 0.01 to 0.2 % v/v, and most preferably at a concentration of about 0.05 % v/v.

Preferably the buffer is a mixture of ammonium formate present at a concentration of 0.01 to 0.1 M and n-propylamine at a concentration of 0.01 % to 0.2 % v/v. Most preferably the buffer is a mixture of ammonium formate present at a concentration of about 0.02 M and n-propylamine at a concentration of about 0.1 % v/v.

Preferably the pH of the buffer solution is approximately 7.5 to 10. More preferably the pH of the buffer solution is approximately 8.5 to 9.5. Most preferably the pH of the buffer- solution is approximately 9.2.

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 can comprise a second liquid B which is or comprises an organic solvent, preferably selected from an alcohol, preferably 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 C_rC₆ alcohol. More preferably the alcohol is an alkyl alcohol. More preferably still the alcohol is a C_rC₄ 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 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, the 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 buffer comprising a formate salt mixed with n-propylamine, 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 0.02 M ammonium formate containing 0.1 % v/v n-propylamine and the second liquid B is methanol.

The method of the first aspect of the present invention preferably comprises a gradient programming so that the relative concentration of the 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 70 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.

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

The first period of time may be from 0 to 120 minutes. Preferably the first period of time is from 30 to 90 minutes. Most preferably the first period of time is about 50 minutes. The second ratio may be 10-30 % A : 70-90 % B. Preferably the second ratio is 15-25 % A : 75-85 % B. Most preferably the second ratio is about 20 % A : 80 % 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. In other embodiments however the stationary phase used is non-chiral.

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, 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 Waters XTerra RP18 (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 25μη , or between 1 and ΙΟμηι, or between 4.5 and 6μηι. More preferably the stationary phase has a particle size of about 5μηι.

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 100 and 175A, or between 125 and 15θΑ. Most preferably the stationary phase has a pore size or about 137A.

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 lengtli. 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 0.02 M ammonium formate containing 0.1 % v/v n-propylamine 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:

(+)-3-amino-6-carboxarnido-l ,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxamido-3-N-memylarrimo-l,2,3,4-tetrahydiOcarbazole. (+)-3-Arrimo-6-carboxamido-l,2,3,4-tetrahydrocarbazole has the structure of compound (VII) in Scheme 7 on page 17 of WO 2010/122343, whilst (+)-3-N-benzyl-6-carboxamido- 3-N-memylamino-l,2,3,4-tetrahydrocarbazole has the structure of compound (VIII) in the same scheme. Preferably the HPLC method detects and optionally quantifies in a single run both (+)-3- amino-6-carboxamido-l ,2,3,4-tetrahydrocarbazole and (+)-3-N-benzyl-6-carboxamido-3- N-memylamino-l,2,3,4-tetrahydrocarbazole. 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:

(+)-3-arrrino-6-carboxamido- ,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxamido-3-N-memylamino-l,2,3,4-tetrahydrocarbazole.

In one embodiment of the first aspect of the present invention, (+)-3-amino-6- carboxamido-l,2,3,4-tetrahydrocarbazole and/ or (+)-3-N-benzyl-6-carboxamido-3-N- methylamino-l,2,3,4-tetrahydrocarbazole 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, frovatriptan and/ or R- 3-emylamino-6-carboxamido-l,2,3,4-tetrahydrocarbazole is used as internal or external reference marker, or as internal or external reference standard.

Preferably the method of the first aspect of the present invention is for analysing frovatriptan monosuccinate. More preferably the method of the first aspect of the present invention is for analysing frovatriptan monosuccinate monohydrate.

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

Preferably the HPLC method is used for the analysis of frovatriptan or a salt thereof that has not entered the human or animal body. Preferably the frovatriptan or the salt thereof that is analysed is not in contact with a human or animal bodily fluid such as whole blood or plasma. Preferably the frovatriptan 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 frovatriptan 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% frovatriptan 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% frovatriptan or a salt thereof by weight. Most preferably the substance comprises at least 95% frovatriptan 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 frovatriptan or a salt thereof as the only active pharmaceutical ingredient.

A second aspect of the present invention provides a chromatographic method for analysing frovatriptan or a salt thereof, wherein the stationary phase is reverse phase and the mobile phase comprises an alcohol. Preferably the alcohol is substantially water miscible.

In one embodiment of the second aspect of the present invention, the alcohol is a C₁-C₆ alcohol. Preferably the alcohol is an alkyl alcohol. More preferably the alcohol is a C_rC₄ alkyl alcohol such as methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol. Most preferably the alcohol is methanol.

In a preferred 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 alcohol. Preferably the second liquid B is the alcohol.

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 (such as a diaUtylamine or a trialliylamine) or a mixture thereof. Typically, the buffer is a phosphate salt, an acetate salt, a formate salt, a trifluoroacetate salt, a phosphoric acid, acetic acid, formic acid, trifluoroacetic acid, an alfylarnine such as diethylamine, n-propylamine or triethylamine, or a mixture thereof. More typically, the buffer is a phosphate salt, an acetate salt, a formate salt, acetic acid, trifluoroacetic acid, diethylamine, n-propylamine, triethylamine or a mixture thereof.

In one embodiment of the second aspect of the present invention, the buffer comprises a carboxylate salt and/ or an organic base. Preferably the carboxylate salt is an acetate salt, a formate salt or a trifluoroacetate salt. More preferably the carboxylate salt is a formate salt such as ammonium formate. Preferably the organic base is an amine such as an aUcylamine. The aUtylamine may be selected from a monoalliylamine such as ethylamine, n-propylamine or isopropylamine, a cualkylamine such as ckethylamine, N-eAyl-N-memylamine or N- ethyl-N -n-propylamine, a cyclic-alkylamine such as piperidine, pyrrolidine or morpholine, or a ttiaU^lamine such as triethylamine, dusopropyletJiylamine or dimelitylethylamine. Preferably the aUiylamine is a monoalkylamine such as ethylamine, n-propylamine or isopropylamine. More preferably the alfylamine is n-propylamine. Where the buffer is or comprises a salt, preferably the counter cation is an ammonium cation.

The salt in 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 0.01 to 0.1 M, and most preferably at a concentration of about 0.02 M; and/ or the base in die buffer can be present at a concentration of 0.001 to 0.2 % v/v, preferably at a concentration of 0.005 to 0.2 % v/v, more preferably at a concentration of 0.01 to 0.2 % v/v, and most preferably at a concentration of about 0.05 % v/v.

Preferably the buffer is a mixture of ammonium formate present at a concentration of 0.01 to 0.1 M and n-propylamine at a concentration of 0.01 % to 0.2 % v/v. Most preferably the buffer is a mixture of ammonium formate present at a concentration of about 0.02 M and n-propylamine at a concentration of about 0.1 % v/v. Preferably the pH of the buffer solution is approximately 7.5 to 10. More preferably the pH of the buffer solution is approximately 8.5 to 9.5. Most preferably the pH of the buffer solution is approximately 9.2. 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% acetonittile 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 buffer comprising a formate salt mixed with n- propylamine, 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 0.02 M ammonium formate containing 0.1 % v/v n-propylamine 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 50 % A : 50 % 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 70 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.

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

The first period of time may be from 0 to 120 minutes. Preferably the first period of time is from 30 to 90 minutes. Most preferably the first period of time is about 50 minutes.

The second ratio may be 10-30 % A : 70-90 % B. Preferably the second ratio is 15-25 % A : 75-85 % B. Most preferably the second ratio is about 20 % A : 80 % B. A particularly preferred method according to the second aspect of the present invention is when the first liquid A is 0.02 M ammonium formate containing 0.1 % v/v n-propylamine and the second liquid B is methanol and the gradient is as follows:

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 reverse phase stationary phase used is a gel, preferably a silica gel.

Preferably the reverse phase stationary phase used in the second aspect of the present invention is selected from an octadecylsilyl silica gel, an octylsilyl silica gel, a phenylalkyl silica gel, a cyanopropyl silica gel, an 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 Waters XTerra RP18 (250 mm x 4.6 mm), 5μηι column. Preferably the stationary phase has a particle size of between 0.1 and ΙΟΟμιη, or between 0.5 and 25μηι, or between 1 and ΙΟμιη, or between 4.5 and 6μιη. More preferably the stationary phase has a particle size of about 5 ιη.

Preferably the stationary phase has a pore size of between 10 and ΙΟΟθΑ, or between 25 and 50θΑ, or between 50 and 200A. More preferably the stationary phase has a pore size of between 100 and 175A, or between 125 and 15θΑ. Most preferably the stationary phase has a pore size of about 137A.

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:

(+)-3-amino-6-carboxamido-l ,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxaiTddo-3-N-methylamino-l,2,3,4-tetrahydrocarbazole.

Preferably the chromatographic method detects and optionally quantifies in a single run both (+)-3-ammo-6-carboxamido-l,2,3,4-tetrahydrocarbazole and (+)-3-N-benzyl-6- carboxamido-3-N-memykrnino-l,2,3,4-tetrahydrocarbazole.

In a preferred embodiment die 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:

(+)-3-amino-6-carboxamido-l ,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxamido-3-N-methylamino-l,2,3,4-tetrahydrocarbazole.

In one embodiment of the second aspect of the present invention, (+)-3-amino-6- carboxamido-l,2,3,4-tetrahydrocarbazole and/ or (+)-3-N-benzyl-6-carboxamido-3-N- memylamino-l,2,3,4-tetrahydrocarbazole 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, frovatriptan and/or R-3-ethylamino-6-carboxamido-l,2,3,4-tetrahydrocarbazole is used as internal or external reference marker, or as internal or external reference standard. Preferably the method of the second aspect of the present invention is for analysing frovatriptan monosuccinate. More preferably the method of the second aspect of the present invention is for analysing frovatriptan monosuccinate monohydrate.

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

Preferably the chromatographic method is used for die analysis of frovatriptan or a salt thereof that has not entered the human or animal body. Preferably the frovatriptan or the salt thereof that is analysed is not in contact with a human or animal bodily fluid such as whole blood or plasma. Preferably the frovatriptan 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 frovatriptan 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% frovatriptan 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% frovatriptan or a salt thereof by weight. Most preferably the substance comprises at least 95% frovatriptan 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 frovatriptan or a salt thereof as the only active pharmaceutical ingredient. A third aspect of the present invention provides a chromatographic method for analysing frovatriptan or a salt thereof, wherein the mobile phase comprises a formate salt.

The counter cation to the formate salt is preferably an ammonium cation, i.e. the formate salt is preferably ammonium formate.

Preferably die mobile phase comprises an aqueous solution of the formate salt. The formate salt is typically 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 0.01 to 0.1 M, and most preferably at a concentration of about 0.02 M.

In one embodiment of the third aspect of the present invention, the aqueous solution further comprises a base, more preferably an organic base. Preferably the organic base is an amine such as an aU^lamine. The all^½mine may be selected from a monoaUcylamine such as ethylamine, n-propylamine or isopropylamine, a diaUtylamine such as diethylamine, N- ethyl-N-methylamine or N-ethyl-N-n-propylamine, a cycHc-aUcylamine such as piperidine, pyrrolidine or morpholine, or a trialfylamine such as triethylamine, dHsopropylethylamine or dimethylethylamine. Preferably the alfylamine is a monoaUiylamine such as eti ylamine, n-propylamine or isopropylamine. More preferably the ancylamine is n-propylamine.

The base in the aqueous solution can be present at a concentration of 0.001 to 0.2 % v/v, preferably at a concentration of 0.005 to 0.2 % v/v, more preferably at a concentration of 0.01 to 0.2 % v/v, and most preferably at a concentration of about 0.05 % v/v. Preferably the pH of the aqueous solution is approximately 7.5 to 10. More preferably the pH of the aqueous solution is approximately 8.5 to 9.5. Most preferably the pH of the aqueous solution is approximately 9.2.

In one embodiment of the third 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 formate salt. Preferably the first liquid A is an aqueous solution of the formate salt. More preferably die first liquid A is an aqueous solution of a mixture of the formate salt and a base. Most preferably the first liquid A is an aqueous solution of a mixture of the formate salt such as ammonium formate, and an aUiylamine such as n-propylamine.

The second liquid B preferably comprises or is an organic solvent, preferably selected from an alcohol, preferably 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 C₆ alcohol. More preferably the alcohol is an alkyl alcohol. More preferably still the alcohol is a C_rC₄ 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, the 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 buffer comprising the formate salt mixed with n- propylamine, 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 0.02 M ammonium formate containing 0.1 % v/v n-propylamine 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.

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 50 % A : 50 % 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 70 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.

The first ratio may be 65-85 % A : 15-35 % B. Preferably the first ratio is 70-80 % A 30 % B. Most preferably the first ratio is about 75 % A : 25 % B. The first period of time may be from 0 to 120 minutes. Preferably the first period of time is from 30 to 90 minutes. Most preferably the first period of time is about 50 minutes. The second ratio may be 10-30 % A : 70-90 % B. Preferably the second ratio is 15-25 % A : 75-85 % B. Most preferably the second ratio is about 20 % A : 80 % B.

A particularly preferred method according to the third aspect of the present invention is when the first liquid A is 0.02 M ammonium formate containing 0.1 % v/v n-propylamine and the second liquid B is methanol and the gradient is as follows:

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. In other embodiments however the stationary phase used is non-chiral.

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 a Waters XTerra RP18 (250 mm x 4.6 mm), 5μηι column. Preferably the stationary phase has a particle size of between 0.1 and ΟΟμηι, or between 0.5 and 25μπι, 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 200A. More preferably the stationary phase has a pore size of between 100 and 175A, or between 125 and 15θΑ. Most preferably the stationary phase has a pore size of about 137A.

In one embodiment of the third aspect of the present invention, the chromatography is carried out in a column between lOmm 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.

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

(+)-3-amino-6-carboxamido-l,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxamido-3-N-memylamino-l,2,3,4-tetrahydrocarbazole. Preferably the chromatographic method detects and optionally quantifies in a single run both (+)-3-amino-6-carboxamido-l,2,3,4-tetrahydrocarbazole and (+)-3-N-benzyl-6- carboxamido-3-N-memylamino-l,2,3,4-tetrahydrocarbazole. In a preferred embodiment the chromatographic method according to the third aspect of the present invention efficiently detects and quantifies in a single run all impurities including those selected from the following compounds:

(+)-3-amino-6-carboxamido- ,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxamido-3-N-memylamino-l,2,3,4-tetrahydrocarbazole.

In one embodiment of the third aspect of the present invention, (+)-3-amino-6- carboxamido-l,2,3,4-tetrahydrocarbazole and/ or (+)-3-N-benzyl-6-carboxamido-3-N- memylamino-l,2,3,4-tetrahydrocarbazole is used as internal or external reference marker, or as internal or external reference standard.

In another embodiment of the third aspect of the present invention, frovatriptan and/or R- 3-emylammo-6-carboxamido-l,2,3,4-tetrahydrocarbazole is used as internal or external reference marker, or as internal or external reference standard. Preferably the method of the third aspect of the present invention is for analysing frovatriptan monosuccinate. More preferably the method of the third aspect of the present invention is for analysing frovatriptan monosuccinate monohydrate.

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

Preferably the chromatographic method is used for the analysis of frovatriptan or a salt thereof that has not entered the human or animal body. Preferably the frovatriptan or the salt thereof that is analysed is not in contact with a human or animal bodily fluid such as whole blood or plasma. Preferably the frovatriptan or the salt thereof that is analysed is not in solution. In another embodiment of the third aspect of the present invention, the chromatographic method is used for the analysis of a pharmaceutical composition comprising frovatriptan or a salt thereof. In yet another embodiment of the third aspect of the present invention, the chromatographic method is used for the analysis of a substance comprising at least 5% frovatriptan 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% frovatriptan or a salt thereof by weight. Most preferably die substance comprises at least 95% frovatriptan or a salt thereof by weight.

In yet another embodiment of the third aspect of the present invention, the chromatographic method is used for the analysis of a substance comprising frovatriptan or a salt thereof as die only active pharmaceutical ingredient.

A fourth aspect of the present invention provides a chromatographic method for analysing frovatriptan or a salt thereof, wherein the stationary phase is reverse phase and the mobile phase comprises an al lamine. The alkylamine may be selected from a monoalkylamine such as emylamine, n-propylamine or isopropylamine, a dialkylamine such as diediylamine, N-emyl-N-methylamine or N- emyl-N-n-propylamine, a cyclic-alkylamine such as piperidine, pyrrolidine or morpholine, or a triaUiylamine such as triethylamine, diisopropylethylamine or dimemylethylamine. Preferably the alkylamine is a monoalkylamine such as ethylamine, n-propylamine or isopropylamine. More preferably the aUcylamine is n-propylamine.

Preferably the mobile phase comprises an aqueous solution of the aUcylamine. The allcylamine is typically present at a concentration of 0.001 to 0.2 % v/v, preferably at a concentration of 0.005 to 0.2 % v/v, more preferably at a concentration of 0.01 to 0.2 % v/v, and most preferably at a concentration of about 0.05 % v/v.

In one embodiment of the fourth aspect of the present invention, the aqueous solution further comprises a carboxylate salt. Preferably the carboxylate salt is an acetate salt, a formate salt or a trifluoroacetate salt. More preferably the carboxykte salt is a formate salt such as ammonium formate. Where the aqueous solution comprises a carboxykte salt, preferably the counter cation to the carboxykte salt is an ammonium cation. The carboxykte salt is typically present in the aqueous solution 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 0.01 to 0.1 M, and most preferably at a concentration of about 0.02 M.

Preferably the pH of the aqueous solution is approximately 7.5 to 10. More preferably the pH of the aqueous solution is approximately 8.5 to 9.5. Most preferably the pH of the aqueous solution is approximately 9.2.

In one embodiment of the fourth aspect of the present invention, die 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 alkylamine. Preferably the first liquid A is an aqueous solution of the aUiylamine. More preferably the first liquid A is an aqueous solution of a mixture of the alkylamine and a carboxykte salt. Most preferably the first liquid A is an aqueous solution of a mixture of a formate salt such as ammonium formate, and an allcylamine such as n-propykmine.

The second liquid B preferably comprises or is an organic solvent, preferably selected from an alcohol, preferably an alkyl alcohol, such as methanol, ethanol, propanol or iso- propanol, or acetonitrile, or a mixture thereof. In one embodiment of the fourth 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_rC₆ alcohol. More preferably the alcohol is an alkyl alcohol. More preferably still the alcohol is a C_rC₄ 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 fourth aspect of the present invention, the second liquid B is substantially water miscible.

In one embodiment of the fourth 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 fourth 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 fourth aspect of the present invention is when the first liquid A is an aqueous solution of a buffer comprising a formate salt mixed with n- propylamine, and the second liquid B is methanol.

A particularly preferred embodiment of the fourth aspect of the present invention is when the first liquid A is 0.02 M ammonium formate containing 0.1 % v/v n-propylamine and the second liquid B is methanol.

In one embodiment of the fourth 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 50 % A : 50 % 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 70 minutes. Alternatively, the chromatographic method of the fourth 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. The first ratio may be 65-85 % A : 15-35 % B. Preferably the first ratio is 70-80 % A : 20- 30 % B. Most preferably the first ratio is about 75 % A : 25 % B.

The first period of time may be from 0 to 120 niinutes. Preferably the first period of time is from 30 to 90 minutes. Most preferably the first period of time is about 50 minutes.

The second ratio may be 10-30 % A : 70-90 % B. Preferably the second ratio is 15-25 % A : 75-85 % B. Most preferably the second ratio is about 20 % A : 80 % B.

A particularly preferred method according to the fourth aspect of the present invention is when the first liquid A is 0.02 M ammonium formate containing 0.1 % v/v n-propylamine and the second liquid B is methanol and the gradient is as follows:

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

In one embodiment of the fourth aspect of the present invention, the reverse phase 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. In odier embodiments however the stationary phase used is non-chiral.

Preferably the reverse phase stationary phase used in the fourth aspect of the present invention is selected from an octadecylsilyl silica gel, an octylsilyl silica gel, a phenylalkyl silica gel, a cyanopropyl silica gel, an 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 Waters XTerra RP18 (250 mm x 4.6 mm), 5μηι column.

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 100 and 175A, or between 125 and 15θΑ. Most preferably the stationary phase has a pore size of about 137A.

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

(+)-3-amino-6-carboxamido-l,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxarrddo-3-N-memylamino-l,2,3,4-tetrahydrocarbazole.

Preferably the chromatographic method detects and optionally quantifies in a single run both (+)-3-amino-6-carboxaniido-l,2,3,4-tetrahydiOcarbazole and (+)-3-N-benzyl-6- carboxamido-3-N-memylamino- 1 ,2,3,4-tetrahydrocarbazole. In a preferred embodiment the chromatographic method according to the fourth aspect of the present invention efficiently detects and quantifies in a single run all impurities including those selected from the following compounds:

(+)-3-amino-6-carboxamido-l ,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxamido-3-N-methylamino-l,2,3,4-tetrahydrocarbazole.

In one embodiment of the fourth aspect of the present invention, (+)-3-amino-6- carboxamido-l,2,3,4-tetrahydrocarbazole and/ or (+)-3-N-benzyl-6-carboxamido-3-N- memylamino-l,2,3,4-tetrahydrocarbazole is used as internal or external reference marker, or as internal or external reference standard.

In another embodiment of the fourth aspect of the present invention, frovatriptan and/ or R-3-emylamino-6-carboxamido-l,2,3,4-tetrahydrocarbazole is used as internal or external reference marker, or as internal or external reference standard. Preferably the method of the fourth aspect of the present invention is for analysing frovatriptan monosuccinate. More preferably the method of the fourth aspect of the present invention is for analysing frovatriptan monosuccinate monohydrate.

In another embodiment of the fourth aspect of the present invention, the chromatographic method is used for the analysis of frovatriptan or a salt thereof that is suitable for use in a pharmaceutical composition. Preferably the chromatographic method is used for the analysis of frovatriptan or a salt thereof that has not entered the human or animal body. Preferably the frovatriptan or the salt thereof that is analysed is not in contact with a human or animal bodily fluid such as whole blood or plasma. Preferably the frovatriptan or the salt thereof that is analysed is not in solution.

In another embodiment of the fourth aspect of the present invention, the chromatographic method is used for the analysis of a pharmaceutical composition comprising frovatriptan or a salt thereof. In yet another embodiment of the fourth aspect of the present invention, the chromatographic method is used for the analysis of a substance comprising at least 5% frovatriptan 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% frovatriptan or a salt thereof by weight. Most preferably the substance comprises at least 95% frovatriptan or a salt thereof by weight.

In yet another embodiment of the fourth aspect of the present invention, the chromatographic method is used for the analysis of a substance comprising frovatriptan or a salt thereof as the only active pharmaceutical ingredient.

A fifth aspect of the present invention provides a method for analysing a substance, comprising the detection and optional quantification of (+)-3-amino-6-carboxamido- 1,2,3,4-tetrahydrocarbazole and/ or (+)-3-N-benzyl-6-carboxamido-3-N-memylamino- 1,2,3,4-tetrahydrocarbazole, wherein the substance comprises an active pharmaceutical ingredient.

Preferably the method of the fifth aspect of the present invention comprises the detection and optional quantification of both (+)-3-aniino-6-carboxarnido-l, 2,3,4- tetrahydrocarbazole and (+)-3-N-benzyl-6-carboxamido-3-N-memylamino-l ,2,3,4- tetrahydrocarbazole.

In one embodiment of the fifth aspect of the present invention, the method further comprises the detection and optional quantification of frovatriptan or a salt thereof.

In another embodiment of the fifth aspect of the present invention, the substance is an active pharmaceutical ingredient. Preferably the substance comprises or is frovatriptan, optionally in the form of a salt, solvate, hydrate or anhydrate. More preferably the frovatriptan is in the form of frovatriptan monosuccinate. Most preferably the frovatriptan is in the form of frovatriptan monosuccinate monohydrate. Preferably the substance 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 whole blood or plasma. Preferably the substance that is analysed is not in solution.

In another embodiment of the fifth aspect of the present invention, the method is a method of analysing a pharmaceutical composition comprising frovatriptan or a salt thereof.

In yet another embodiment of the fifth aspect of the present invention, the substance comprises at least 5% frovatriptan 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% frovatriptan or a salt thereof by weight. Most preferably the substance comprises at least 95% frovatriptan or a salt thereof by weight. In yet another embodiment of the fifth aspect of the present invention, the substance comprises frovatriptan or a salt thereof as the only active pharmaceutical ingredient.

In a preferred embodiment of the fifth 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 (such as a diaUcylamine or a

or a mixture thereof.

Typically, the buffer is a phosphate salt, an acetate salt, a formate salt, a trifluoroacetate salt, a phosphoric acid, acetic acid, formic acid, trifluoroacetic acid, an aU<ylamine such as diemylamine, n-propykmine or triemylarnine, or a mixture thereof. More typically, the buffer is a phosphate salt, an acetate salt, a formate salt, acetic acid, trifluoroacetic acid, c ethylamine, n-propylamine, triethylamine or a mixture thereof. In one embodiment of the fifth aspect of the present invention, the buffer comprises a carboxylate salt and/ or an organic base. Preferably the carboxylate salt is an acetate salt, a formate salt or a trifluoroacetate salt. More preferably the carboxylate salt is a formate salt such as ammonium formate. Preferably the organic base is an amine such as an alfylamine. The alfylamine may be selected from a monoalliylamine such as ethylamine, n-propylarnine or isopropylamine, a dialliylamine such as cUethylamine, N-e yl-N-memylamine or N- ethyl-N-n-propylamine, a cyclic-alkykinine such as piperidine, pyrrolidine or morpholine, or a ttialkylamine such as triethylamine, dHsopropylethylamine or dimemylemylamine. Preferably the aUtylarnine is a monoaUcylainine such as emylamine, n-propylamine or isopropylamine. More preferably the aUcylamine is n-propylamine.

Where the buffer is or comprises a salt, preferably the counter cation is an ammonium cation. Most preferably the buffer is a mixture of a formate salt, such as ammonium formate, and a base, more preferably an organic base, such as n-propylamine.

The salt in 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 0.01 to 0.1 M, and most preferably at a concentration of about 0.02 M; and/ or d e base in the buffer can be present at a concentration of 0.001 to 0.2 % v/v, preferably at a concentration of 0.005 to 0.2 % v/v, more preferably at a concentration of 0.01 to 0.2 % v/v, and most preferably at a concentration of about 0.05 % v/v.

Preferably the pH of the buffer solution is approximately 7.5 to 10. More preferably the pH of the buffer solution is approximately 8.5 to 9.5. Most preferably the pH of the buffer solution is approximately 9.2. The second liquid B preferably comprises or is an organic solvent, preferably selected from an alcohol, preferably an alkyl alcohol, such as methanol, ethanol, propanol or iso- propanol, or acetonitrile, or a mixture thereof.

In one embodiment of the fifth 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_rC₄ alkyl alcohol such as methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol. Most preferably die second liquid B is methanol.

In another embodiment of the fifth aspect of the present invention, the second liquid B is substantially water miscible. In one embodiment of the fifth 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 fifth 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 fifth aspect of the present invention is when the first liquid A is an aqueous solution of a buffer comprising a formate salt mixed with n-propylamine, and the second liquid B is methanol. A particularly preferred embodiment of the fifth aspect of the present invention is when the first liquid A is 0.02 M ammonium formate containing 0.1 % v/v n-propylamine and the second liquid B is methanol.

In one embodiment of the fifth 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 d e fifth aspect of the present invention, said method detects and optionally quantifies in a single run (+)-3-amino-6-carboxairiido- 1 ,2,3,4-tetrahydrocarbazole and/or (+)-3-N-benzyl-6-carboxamido-3-N-memylamino- 1,2,3,4-tetrahydrocarbazole. More preferably said method also detects and optionally quantifies in the same run frovatriptan or a salt thereof.

Most preferably said method detects and optionally quantifies in a single run all three of: frovatriptan or a salt thereof;

(+)-3-arnmo-6-carboxamido-l,2,3,4-tetrahydiOcarbazole; and

(+)-3-N-benzyl-6-carboxarmdo-3-N-memylamino-l,2,3,4-tetrahydrocarbazole. In one embodiment of the fifth aspect of the present invention, (+)-3-amino-6- carboxamido-l,2,3,4-tetrahydrocarbazole and/ or (+)-3-N-benzyl-6-carboxamido-3-N- memylamino-l,2,3,4-tetrahydrocarbazole is used as internal or external reference marker, or as internal or external reference standard.

In another embodiment of the fifth aspect of the present invention, frovatriptan and/or R- 3-emylaiTiino-6-carboxamido-l,2,3,4-tetrahydrocarbazole 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 50 % A : 50 % B.

Alternatively, the chromatographic method of the fifth 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.

The first period of time may be from 0 to 120 minutes. Preferably the first period of from 30 to 90 minutes. Most preferably the first period of time is about 50 minutes. The second ratio may be 10-30 % A : 70-90 % B. Preferably the second ratio is 15-25 % A : 75-85 % B. Most preferably the second ratio is about 20 % A : 80 % B.

A particularly preferred method according to the fifth aspect of the present invention is when the first liquid A is 0.02 M ammonium formate containing 0.1 % v/v n-propylamine and the second liquid B is methanol and the gradient is as follows:

Preferably the method of the fifth 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 fifth 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. In other embodiments however the stationary phase used is non-chiral.

Preferably the stationary phase used in the fifth 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 a Waters XTerra RP18 (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 25μηι, or between 1 and ΙΟμηι, or between 4.5 and 6μηι. More preferably the stationary phase has a particle size of about 5μηι.

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 100 and 75A, or between 125 and 15θΑ. Most preferably die stationary phase has a pore size ot about 137A.

In one embodiment of the fifth 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 O.lmm 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 sixth 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 fifth 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 sixth aspect of the present invention, the substance comprises or is an active pharmaceutical ingredient. Preferably the substance comprises or is frovatriptan, optionally in the form of a salt, solvate, hydrate or anhydrate. More preferably the frovatriptan is in the form of frovatriptan monosuccinate. Most preferably the frovatriptan is in the form of frovatriptan monosuccinate monohydrate. Preferably the substance is for use in a pharmaceutical composition.

In another embodiment of the sixth aspect of the present invention, the substance comprises or is a pharmaceutical composition. Preferably the pharmaceutical composition comprises frovatriptan, optionally in the form of a salt, solvate, hydrate or anhydrate. More preferably the frovatriptan is in the form of frovatriptan monosuccinate. Most preferably the frovatriptan is in the form of frovatriptan monosuccinate monohydrate. Preferably the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

Preferably the substance of the sixth 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 whole blood or plasma. Preferably the substance is not in solution. In another embodiment of the sixth aspect of the present invention, the substance comprises at least 5% frovatriptan 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% frovatriptan or a salt thereof by weight. Most preferably the substance comprises at least 95% frovatriptan or a salt thereof by weight.

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

A seventh aspect of the present invention provides a batch of frovatriptan or a salt thereof which has been prepared by a process according to the sixth aspect of the present invention. Preferably the frovatriptan is substantially free of (+)-3-arrrmo-6-carboxamido- 1, 2,3,4- tetrahydrocarbazole and/ or (+)-3-N-benzyl-6-carboxamido-3-N-methylamino- 1 ,2,3,4-tetrahydrocarbazole. Frovatriptan 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.

An eighth 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 frovatriptan or a salt thereof which has been prepared by a process according to the sixth aspect of the present invention.

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

A tenth aspect of the present invention provides a batch of one or more pharmaceutical compositions which have been prepared by a process according to the sixth aspect of the present invention, wherein the pharmaceutical composition(s) comprise frovatriptan 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 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 present invention

The present invention can be used to analyse frovatriptan and/or its salts, in particular frovatriptan monosuccinate monohydrate, 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 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 particularly convenient for analysing frovatriptan, particularly with respect to the related substances formed in the synthesis of frovatriptan and/ or its salts prepared by the process disclosed in co-pending Indian patent application IN 657/KOL/2009 and the corresponding international patent application WO 2010/122343. 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 frovatriptan and/ or its salts. In addition, the present invention is highly sensitive and allows detection and quantification of related substances in frovatriptan 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 frovatriptan. 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, acetate or formate and mixtures thereof. Alternatively the buffer can be an organic salt such as the 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 or trifiuoroacetic acid. Alternatively the buffer can be an organic base such as a mono-, di- or ttiaUcylamine, such as diemylamine, n-propylamine or trie ylamine. Preferably the first liquid A is a mixture of 0.02 M ammonium formate and 0.1 % v/v n-propylamine.

The second liquid B is an organic solvent such as an alcohol, preferably a C, to C₆ 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 (RP-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: Waters XTerra RP18 (250 mm x 4.6 mm), 5μ, 137A pore size;

Flow rate: 1 ml/min;

Detection: 245 ntn;

Sample concentration: 250 ppm;

Injection volume: 20 μΐ;

Diluent: water - methanol (50:50 v/v);

The sample of frovatiiptan monosuccinate monohydrate is initially dissolved in a small volume of the diluent; the sample solution is then injected into die column which is run using the mobile phase outlined below;

First Liquid A: a mixture of 0.02 M ammonium formate and 0.1 % v/v n-propylamine; Second Liquid B: methanol;

Mobile phase: First Liquid A— Second Liquid B gradient; the gradient program is described below, with the program between 50 and 60 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 frovatriptan are given in Table 1. Component Approximate Approximate LOD LOQ

RT (min) RRT (%r (%)^*

Frovatriptan 7.2 1.00 0.007 0.02

(+)-3-Amino-6-carboxamido-l,2,3,4- 6.2 0.86 0.007 0.02 tetrahydrocarbazole

(+)-3-N-Benzyl-6-carboxarnido-3-N- 33.4 4.65 0.01 0.03 methylamino- 1,2,3,4- tetrahydrocarbazole

of LOD and LOQ are with respect to sample concentration of 250 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.

Claims

1. A HPLC method for analysing frovatriptan 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.

2. A HPLC method according to claim 1, wherein the mobile phase comprises a first liquid A which is aqueous based.

3. A HPLC method according to claim 2, wherein the first liquid A comprises water or an aqueous solution of a buffer.

4. A HPLC med od according to claim 3, wherein the buffer is selected from an acid, an organic salt, an inorganic salt, an organic base (such as a cUalfylamine or a

or a mixture thereof.

5. A HPLC method according to claim 4, wherein the buffer is a phosphate salt, an acetate salt, a formate salt, a trifluoroacetate salt, a phosphoric acid, trifluoroacetic acid, acetic acid, formic acid, an aUcylamine such as diemylamine, n-propylamine or ttiemylamine, or a mixture thereof.

6. A HPLC method according to claim 4 or 5, wherein the buffer is a mixture of a formate salt and a base.

7. A HPLC method according to claim 6, wherein the buffer is a mixture of ammonium formate and n-propylamine.

8. A HPLC method according to claim 6 or 7, wherein:

(i) the formate salt is present at a concentration of 0.001 to 0.2 M and/ or the base at a concentration of 0.001 to 0.2 % v/v; and/or

(ii) the formate salt is present at a concentration of 0.005 to 0.1 M and/or the base at a concentration of 0.005 to 0.2 % v/v; and/ or (iii) the formate salt is present at a concentration of 0.01 to 0.1 M and/or the base at a concentration of 0.01 to 0.2 % v/v; and/ or

(iv) the formate salt is present at a concentration of about 0.02 M and/or the base at a concentration of about 0.1 % v/v.

9. A HPLC method according to claim 8, wherein:

(i) the buffer is ammonium formate present at a concentration of 0.01 to 0.1 M and/ or the base is n-propylamine at a concentration of 0.01 to 0.2 % v/v; and/ or

(ii) the buffer is ammonium formate present at a concentration of approximately 0.02 M and/ or the base is n-propylarnine at a concentration of approximately 0.1 % v/v.

10. A HPLC method according to any preceding claim, wherein the mobile phase comprises a second liquid B which is or comprises an organic solvent.

11. A HPLC method according to claim 10, wherein the second liquid B is an alcohol, preferably an alkyl alcohol, such as methanol, ethanol, propanol or iso-propanol, or acetonitrile, or a mixture thereof.

12. A HPLC method according to claim 11, wherein the second liquid B is methanol.

13. A HPLC method according to any one of claims 10 to 12, wherein the first liquid A is a mixture of ammonium formate and n-propylamine and the second liquid B is methanol.

14. A HPLC method according to any one of claims 10 to 13, which comprises a gradient programming so that the relative concentration of the liquids A and B is varied to a gradient between 100 % A : 0 % B to 0 % A : 100 % B run over:

(i) 10 to 180 minutes; and/or

(H) 30 to 120 minutes; and/or

(iii) 30 to 70 minutes.

15. A HPLC method according to any preceding claim, wherein the stationary phase used: ® is reverse phase; and/ or

(ϋ) is octadecylsilyl silica gel or octylsilyl silica gel; and/ or

(iii) comprises a Waters XTerra RP18 (250 mm x 4.6 mm), 5μ column.

16. A HPLC method according to any one of claims 10 to 15, wherein the first liquid A is a mixture of 0.02 M aqueous ammonium formate and 0.1 % v/v n-propylamine and the second liquid B is methanol.

17. A HPLC method according to claim 16, wherein the gradient is as follows:

18. A HPLC method according to any one of claims 3 to 17, wherein the pH of the buffer solution is approximately 7.5 to 10.

19. A HPLC method according to any preceding claim, wherein the chromatography is carried out at a temperature between approximately 15 to 40°C.

20. A HPLC method according to any preceding claim, wherein the frovatriptan is in die form of any salt, solvate, hydrate or anhydrous.

21. A HPLC method according to claim 20, wherein the frovatriptan is the monosuccinate monohydrate salt.

22. A HPLC method according to any preceding claim, which detects and optionally quantifies in a single run one or more impurities selected from:

(+)-3-aniino-6-carboxamido-l,2,3,4-tetrahydrocarbazole; and

(+)-3-N-benzyl-6-carboxamido-3-N-methylamino-l,2,3,4-tetrahydrocarbazole.

23. A HPLC method according to any preceding claim, which efficiendy detects and quantifies in a single run all impurities including those selected from the following compounds:

(+)-3-amino-6-carboxamido-l,2,3,4-tetrahydrocarbazole;

(+)-3-N-benzyl-6-carboxamido-3-N-methylamino-l,2,3,4-tetrahydrocarbazole;

and any other unknown impurities.

24. A HPLC method according to any preceding claim, which is used to analyse frovatriptan or a salt thereof as an API or when prepared as a pharmaceutical composition.

25. A chromatographic method for analysing frovatriptan or a salt thereof, wherein the stationary phase is reverse phase and the mobile phase comprises an alcohol.

26. A chromatographic method for analysing frovatriptan or a salt thereof, wherein the mobile phase comprises a formate salt.

27. A chromatographic method for analysing frovatriptan or a salt thereof, wherein the stationary phase is reverse phase and the mobile phase comprises an aUtylamine.

28. A method for analysing a substance, comprising the detection and optional quantification of (+)-3-amino-6-carboxamido-l,2,3,4-tetrahydrocarbazole and/or (+)-3-N- benzyl-6-carboxamido-3-N-memylamino-l,2,3,4-tetrahydrocarbazole, wherein the substance comprises an active pharmaceutical ingredient.

29. 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 preceding claim; and

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

30. A batch of frovatriptan or a salt thereof, or a batch of one or more pharmaceutical compositions comprising frovatriptan or a salt thereof, which has been prepared by a process according to claim 29.

31. 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 frovatriptan or a salt thereof which has been prepared by a process according to claim 29.

32. A pharmaceutical composition which has been prepared by a process according to claim 31.