WO2011064574A1 - Hplc method for detecting lenalidomide - Google Patents

Hplc method for detecting lenalidomide Download PDF

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Publication number
WO2011064574A1
WO2011064574A1 PCT/GB2010/051951 GB2010051951W WO2011064574A1 WO 2011064574 A1 WO2011064574 A1 WO 2011064574A1 GB 2010051951 W GB2010051951 W GB 2010051951W WO 2011064574 A1 WO2011064574 A1 WO 2011064574A1
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Prior art keywords
hplc method
liquid
salt
lenalidomide
current invention
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Application number
PCT/GB2010/051951
Other languages
French (fr)
Inventor
Erra Koteswara Satya Vijayakumar
Santosh Takale
Manish Singh
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Generics [Uk] Limited
Mylan India Private Limited
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Publication of WO2011064574A1 publication Critical patent/WO2011064574A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems

Definitions

  • the present invention relates to new HPLC methods for the analysis of the drug substance lenalidomide and related substances.
  • the mobile phase comprises two or more liquids and the relative concentration of the liquids is varied to a predetermined gradient.
  • the mobile phase comprises formic acid, a formate salt or a mixture thereof.
  • the current invention also relates to lenalidomide and associated pharmaceutical compositions that have been analysed by the methods of the current invention and/ or are substantially free of specific impurities.
  • the likely impurities in APIs and pharmaceutical compositions include residual quantities of synthetic precursors (intermediates), by-products which arise during the synthesis of the API, residual solvents, isomers of the API (e.g. geometrical isomers, diastereomets 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.
  • isomers of the API e.g. geometrical isomers, diastereomets 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 tests that are required to demonstrate that the API or pharmaceutical compositions are safe and effective include a purity/ assay test, a related substances test, a content uniformity test and a dissolution test.
  • the purity/ as say 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.
  • HPLC High Performance liquid Chromatography
  • 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.
  • 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.
  • 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.
  • Lenalidomide ( ⁇ ) chemically known as 3-(4-amino-l-oxo-l,3-dihydro-2H-isoindol-2- yl)piperidine-2,6-dione, is an analogue of thalidomide and a potent immunomodulatory agent with anti-angiogenic and anti-neoplastic properties. It is used for the treatment of patients with a sub-type of myelodysplastic syndrome. Lenalidomide is currently marketed as a racemic mixture.
  • HPLC methods reported in die prior art are not particularly convenient or suitable for analysing lenalidomide API, particularly with respect to related substances.
  • 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 lenalidomide synthesized by the process disclosed in co-pending Indian patent application 383/KOL/2009 and co-pending international patent application WO 2010/100476.
  • lenalidomide as used herein throughout the description and claims means lenalidomide and/ or any salt, tautomer, solvate, isomer or enantiomer thereof.
  • the current invention is particularly useful for the analysis of lenalidomide.
  • a first aspect of the current invention provides a HPLC method for analysing lenalidomide, 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.
  • the mobile phase comprises a first liquid A which is aqueous based, such as water or an aqueous solution of a buffer.
  • the buffer is an acid or an organic acid or an inorganic salt or a mixture thereof.
  • the buffer is a phosphate salt, an acetate salt, a trifluoroacetate salt, a formate salt, acetic acid, trifLuoroacetic acid, formic acid, a phosphoric acid or a mixture thereof. More typically the buffer is a phosphate salt, an acetate salt, a formate salt or trifluoroacetic acid.
  • the buffer is a salt
  • the counter cation is an ammonium cation
  • the buffer is formic acid, a formate salt or a rnixture thereof.
  • the buffer is a formate salt, such as ammonium formate.
  • the buffer can be present at a concentration of 0.001 to 0.02 M, preferably at a concentration of 0.001 to 0.01 M, more preferably at a concentration of 0.001 to 0.005 M, and most preferably at a concentration of about 0.002 M.
  • the buffer is ammonium formate present at a concentration of 0.001 to 0.005 M. Most preferably, the buffer is ammonium formate present at a concentration of approximately 0.002 M.
  • the pH of the buffer is approximately 2 to 7. More preferably the pH of the buffer is approximately 4 to 7. More preferably still the pH of the buffer is approximately 5 to 7. Most preferably the pH of the buffer is approximately 6.1.
  • the method of the first aspect of the current 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, such as methanol, acetonitrile, propanol or isopropanol or a mixture thereof.
  • the second liquid B comprises or is a dipolar aprotic solvent such as acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF.
  • the second liquid B comprises acetonitrile.
  • the second liquid B is acetonitrile.
  • the second liquid B is substantially water miscible.
  • 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 ⁇ ⁇ — 0.05.
  • the magnitude of ⁇ ⁇ 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.
  • 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.
  • the mobile phase contains less than 10%, less than 5% or less than 1% methanol by volume. In one embodiment, the mobile phase contains no methanol.
  • the mobile phase contains less than 10%, less than 5% or less than 1% of any alcohol by volume. In one embodiment, the mobile phase contains no alcohols.
  • the mobile phase contains less than 10%, less than 5% or less than 1% of any organic polar protic solvent by volume. In one embodiment, the mobile phase contains no organic polar protic solvents.
  • a preferred embodiment of the first aspect of the current invention is when the first liquid A is an aqueous solution of ammonium formate and the second Equid B is acetonitrile.
  • the method of the first aspect of the current invention 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.
  • the gradient is between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 120 minutes, more preferably between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 70 minutes.
  • the first aspect of the current invention may comprise a gradient programming so that the relative concentration of the liquids A and B by volume is maintained at a first ratio for a first period of time, and then varied to a gradient over a second period of time, finishing at a second ratio.
  • the first ratio may be 75-95 % A : 5-25 % B.
  • the first ratio is 80-90 % A : 10-20 % B.
  • 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 50-70 % A : 30-50 % B. Preferably the second ratio is 55-65 % A : 35-45 % B. Most preferably the second ratio is about 60 % A : 40 % B.
  • the second period of time may be from 5 to 90 minutes. Preferably the second period of time is from 15 to 45 minutes. Most preferably the second period of time is about 35 minutes.
  • a particukrly preferred embodiment of the first aspect of the current invention is when the first liquid A is 0.002 M ammonium formate and the second Equid B is acetonitrile.
  • 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, mote 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.
  • the stationary phase used is a gel, preferably a silica gel.
  • the stationary phase used is chiral and/ or the mobile phase further comprises a chital selector.
  • the stationary phase used in the first aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel.
  • Particularly suitable stationary phases include octadecylsilyl silica gel or octylsilyl silica gel.
  • a particularly preferred stationary phase comprises a Purospher Star RP 18e (250 mm x 4.6 mm), 5 ⁇ column.
  • the stationary phase has a particle size of between 0.1 and ⁇ , ⁇ , or between 0.5 and 25 ⁇ , or between 1 and ⁇ , or between 4.5 and ⁇ . More preferably the stationary phase has a particle size of about 5 ⁇ . ⁇ .
  • the stationary phase has a pore size of between 10 and ⁇ , or between 25 and 500A, or between 50 and 200A. More preferably the stationary phase has a pore size of between 80 and 150A, or between 110 and 13 ⁇ . Most preferably the stationary phase has a pore size of about 120A.
  • 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 current invention is when the first liquid A is 0.002 M ammonium formate and the second liquid B is acetonitrile and the gradient is as follows:
  • a more preferred method according to the first aspect of the current invention is when the first liquid A is 0.002 M ammonium formate and the second liquid B is acetonitrile 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.
  • 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.
  • the HPLC method detects and optionally quantifies in a single run the impurity 3-(l-oxo-4-nitro-l,3-dihyclro-isoindol- 2-yI)piperidine-2,6-dione.
  • the HPLC method according to the first aspect of the current invention efficiently detects and quantifies in a single run all impurities, including 3-(l-oxo- 4-nitro- 1 ,3 -clihydro-isoindol-2-yl) pipeiidine-2,6-dione.
  • 3-(l-oxo-4-nitro-l,3- dihydro-isomdol-2-yl)pipeddine-2,6-dione is used as internal or external reference marker, or as internal or external reference standard.
  • the HPLC method is used for the analysis of lenalidomide that is suitable for use in a pharmaceutical composition.
  • the HPLC method is used for the analysis of lenalidomide that has not entered the human or animal body.
  • the lenalidomide that is analysed is not in contact with a human or animal bodily fluid such as plasma.
  • the lenalidomide that is analysed is not in solution.
  • the HPLC method is used for the analysis of a pharmaceutical composition comprising lenalidomide.
  • the HPLC method is used for the analysis of a substance comprising at least 5% lenalidomide by weight.
  • the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% lenalidomide by weight.
  • the substance comprises at least 95% lenalidomide by weight.
  • the HPLC method is used for the analysis of a substance comprising lenalidomide as the only active pharmaceutical ingredient.
  • a second aspect of the current invention provides a chromatographic method for analysing lenalidomide, or a salt thereof, wherein the mobile phase comprises formic acid, a formate salt or a mixture thereof.
  • the mobile phase comprises a formate salt such as ammonium formate.
  • the mobile phase further comprises water. More preferably, the mobile phase comprises an aqueous solution of the formic acid, formate salt or mixture thereof. Most preferably, the mobile phase comprises an aqueous solution of a formate salt such as ammonium formate.
  • the formic acid, formate salt or mixture thereof can be present at a concentration of 0.001 to 0.02 M, preferably at a concentration of 0.001 to 0.01 M, more preferably at a concentration of 0.001 to 0.005 M, and most preferably at a concentration of about 0.002 M.
  • the formic acid, formate salt or mixture thereof is ammonium formate present at a concentration of 0.001 to 0.005 M. Most preferably, the formic acid, formate salt or mixture thereof is ammonium formate present at a concentration of approximately 0.002 M.
  • the pH of the aqueous solution is approximately 2 to 7. More preferably the pH of the aqueous solution is approximately 4 to 7. More preferably still the pH of the aqueous solution is approximately 5 to 7. Most preferably the pH of the aqueous solution is approximately 6.1.
  • 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 formic acid, formate salt or mixture thereof.
  • the first liquid A is an aqueous solution of the formic acid, formate salt or mixture thereof.
  • the second liquid B preferably comprises or is an organic solvent, such as methanol, acetonitrile, ptopanol or isopropanol or a mixture thereof.
  • the second liquid B comprises or is a dipolar aprotic solvent such as acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF.
  • the second liquid B comprises acetonitrile.
  • the second liquid B is acetonitrile.
  • the second Uquid B is substantially water miscible.
  • the mobile phase contains less than 10%, less than 5% or less than 1% methanol by volume. In one embodiment, the mobile phase contains no methanol.
  • the mobile phase contains less than 10%, less than 5% or less than 1% of any alcohol by volume. In one embodiment, the mobile phase contains no alcohols. In yet another embodiment of the second aspect of the current invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any organic polar protic solvent by volume. In one embodiment, the mobile phase contains no organic polar protic solvents.
  • a preferred embodiment of the second aspect of the current invention is when the first liquid A is an aqueous solution of ammonium formate and the second liquid B is acetonitrile.
  • a particularly preferred embodiment of the second aspect of the current invention is when the first Uquid A is 0.002 M ammonium formate and the second Uquid B is acetonitrile.
  • 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 Hquids 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 90%A : 10%B and 50%A : 50%B. More preferably still the relative concentration of the liquids A and B by volume is about 70%A : 30%B.
  • the relative concentration of the liquids in the mobile phase may be varied to a predetermined gradient.
  • 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.
  • the gradient is between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 120 minutes, more preferably between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 70 minutes.
  • the second aspect of the current invention may comprise a gradient programming so that the relative concentration of the liquids A and B by volume is maintained at a first ratio for a first period of time, and then varied to a gradient over a second period of time, fmishing at a second ratio.
  • the first ratio may be 75-95 % A : 5-25 % B.
  • the first ratio is 80-90 % A : 10-20 % B.
  • 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 50-70 % A : 30-50 % B. Preferably the second ratio is 55-65 % A : 35-45 % B. Most preferably the second ratio is about 60 % A : 40 % B.
  • the second period of time may be from 5 to 90 minutes. Preferably the second period of time is from 15 to 45 minutes. Most preferably the second period of time is about 35 minutes.
  • a particularly preferred method according to the second aspect of the current invention is when the first liquid A is 0.002 M ammonium formate and the second liquid B is acetonitrile and the gradient is as follows:
  • a mote preferred method according to the second aspect of the current invention is when the first liquid A is 0.002 M arrrmorrium formate and the second liquid B is acetonitrile and the gradient is as follows:
  • the method of the second aspect of the current invention is carried out at a column temperature between approximately 15 to 40°C.
  • 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 nJ/min is used, most preferably a mobile phase flow rate of about 1 ml/min is used.
  • the stationary phase used is a gel, preferably a silica gel.
  • the stationary phase used is chiral and/or the mobile phase farther comprises a chiral selector.
  • the stationary phase used in the second aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, 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 Purospher Star RP 18e (250 mm x 4.6 mm), 5 ⁇ . column.
  • the stationary phase has a particle size of between 0.1 and ⁇ , or between 0.5 and 25 ⁇ , or between 1 and ⁇ , or between 4.5 and 6 ⁇ . More preferably the stationary phase has a particle size of about 5 ⁇ m.
  • the stationary phase has a pore size of between 10 and lOOOA, or between 25 and 50GA, or between 50 and 20 ⁇ . More preferably the stationary phase has a pore size of between 80 and 150A, or between 110 and 130A. Most preferably the stationary phase has a pore size of about 12 ⁇ .
  • 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 refractometet, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.
  • a detector such as a UV and/ or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometet, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.
  • the chromatographic method detects and optionally quantifies in a single run the impurity 3-(l-oxo-4-nitro-l,3- dihydro4soindol-2-yl)piperidine ⁇ 2,6 ⁇ dione.
  • the chromatographic method according to the second aspect of the current invention efficiently detects and quantifies in a single run all impurities, including 3-(l-oxo ⁇ 4-nitt"o-l,3-dihydro-isoindol-2-yl)piperidinc-2,6-dione.
  • 3-(l-oxo-4-nitro-l,3- dihydro-isomdol-2-yl)piperidine-2,6-dione is used as internal or external reference marker, or as internal or external reference standard.
  • the chromatographic method is used for the analysis of lenahdomide that is suitable for use in a pharmaceutical composition.
  • the chromatographic method is used for the analysis of lenalidomide that has not entered the human or animal body.
  • the lenalidomide that is analysed is not in contact with a human or animal bodily fluid such as plasma.
  • the lenalidomide that is analysed is not in solution.
  • the chromatographic method is used for the analysis of a pharmaceutical composition comprising lenalidomide.
  • the chromatographic method is used for the analysis of a substance comprising at least 5% lenahdomide by weight.
  • the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% lenahdomide by weight.
  • the substance comprises at least 95% lenahdomide by weight.
  • the chromatographic method is used for the analysis of a substance comprising lenalidomide as the only active pharmaceutical ingredient.
  • a third aspect of the current invention provides lenahdomide which has been subjected to a method according to either of the first or second aspects of the current invention.
  • the lenaEdomide is substantially free of 3-(l-oxo-4-nitro-l,3-dlhydfo-isoindol-2- yl)piperidinc-2,6-dione.
  • a fourth aspect of the current invention provides lenalidomide which is substantially free of 3-(l-oxo-4-niteoT,3-dihydro4soindol-2-y ⁇
  • Lenalidomide 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 fifth aspect of the current invention provides a pharmaceutical composition comprising lenalidomide according to the third or fourth aspects of the current invention.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.
  • any embodiment of a given aspect of the current invention may occur in combination with any other embodiment of the same aspect of the current invention.
  • any preferred or optional embodiment of any aspect of the current invention should also be considered as a preferred or optional embodiment of any other aspect of the current invention.
  • the current invention can be used to analyse lenalidomide and/ or its salts as an API or lenalidomide and/ or its salts when prepared as a pharmaceutical composition.
  • compositions that can be analysed by the current invention include solid and liquid compositions and optionally comprise one or more pharmaceutically acceptable carriers or excipients.
  • Solid form compositions include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • Liquid compositions include solutions or suspensions which can be administered by oral, injectable or infusion routes.
  • 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.
  • HPLC methods reported in the prior art are not suitable for analysing lenalidomide, particularly with respect to the related substances formed in the synthesis of lenalidomide and/or its salts prepared by the process disclosed in co-pending Indian patent application 383/KOL/2009 and co-pending international patent application WO 2010/100476.
  • a particularly preferred embodiment of the current invention solves this problem and efficiently detects and quantifies, in a single run, all impurities and intermediates formed in the synthetic process.
  • the present invention is advantageous as the gradient method allows the elution of all polar to non-polar impurities.
  • the current invention is also advantageous as the method is selective, linear, precise and stability -indicating for the analysis of related substances in lenalidomide and/or its salts.
  • the current invention is highly sensitive and allows detection and quantification of related substances in lenahdomide and/ or its salts at levels much lower than acceptance limits specified by health authorities.
  • the method of the current invention can be used to easily detect and quantify all degradation impurities formed on storage of samples of lenaHdomide. 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 optionatty 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.
  • the buffer can be an organic salt such as the ammonium salt of acetate ot formate and mixtures thereof.
  • the buffer can be a mineral acid or a carboxylic acid, such as acetic acid or tri flu oroa eerie acid.
  • the first liquid A is ammonium formate, preferably at a concentration of 0.002 M.
  • the organic solvent(s) used as the second liquid B can be organic solvents like C r C 6 alcohols, such as methanol, efhanol, propanol, butanol or isoptopanol or mixtures thereof.
  • the organic solvent(s) may be tetrahydrofuran, ethyl acetate or acetonitrile or any suitable organic solvent(s).
  • the organic solvent is acetonitrile.
  • the stationary phase used in the method of the current invention is selected from octadecylsilyl silica gel (RP-18) or octylsilyl silica gel ( ⁇ . ⁇ -8).
  • An internal standard reference compound may be used in the method of the current invention if required.
  • the concentration of the components analysed may be determined by comparison with one or more external reference compounds.
  • the methods of the invention disclosed herein can also be used for the analysis of compounds with similar chemical structures and/or similar chemical or physical properties to lenalidomide, thalidomide and their salts and/ or isomers or enantiomers.
  • Diluent A methanol-acetoninale (50:50 v/v);
  • Diluent B 0.002 M ammonium formate-acetonittile (85:15 v/v);
  • the sample is initially dissolved in a small volume of diluent A, then diluted with diluent B to give a sample solution; the sample solution is then injected into the column which is run using the mobile phase outlined below;
  • First Liquid A 0.002 M aqueous ammonium formate
  • Second liquid B acetoniteile
  • RT Retention times
  • RRT Relative retention times
  • LOD Limit of Detection
  • LOQ Limit of Quantitation
  • % of LOD and LOQ are with respect to sample concentration of 200 ppm.

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Abstract

The present invention relates to new HPLC methods for the analysis of the drug substance lenaliclomide and related substances. In a first method the mobile phase comprises two or mote liquids and the relative concentration of the liquids is varied to a predetermined gradient. In a second method the mobile phase comprises formic acid, a formate salt or a mixture thereof. The current invention also relates to lenalidomide and associated pharmaceutical compositions that have been analysed by the methods of the current invention and/or are substantially free of specific impurities.

Description

HPLC METHOD FOR DETECTING LENALIDOMIDE
Field of the invention The present invention relates to new HPLC methods for the analysis of the drug substance lenalidomide 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 formic acid, a formate salt or a mixture thereof. The current invention also relates to lenalidomide and associated pharmaceutical compositions that have been analysed by the methods of the current invention and/ or 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 are absent from the active pharmaceutical ingredient (API) at the time of manufacture, or are present only in acceptable levels, and that the storage stability of the pharmaceutical composition is acceptable.
The likely impurities in APIs and pharmaceutical compositions include residual quantities of synthetic precursors (intermediates), by-products which arise during the synthesis of the API, residual solvents, isomers of the API (e.g. geometrical isomers, diastereomets 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 a purity/ assay test, a related substances test, a content uniformity test and a dissolution test. The purity/ as say 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. a 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 stationary7 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 on 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.
Lenalidomide (Ί), chemically known as 3-(4-amino-l-oxo-l,3-dihydro-2H-isoindol-2- yl)piperidine-2,6-dione, is an analogue of thalidomide and a potent immunomodulatory agent with anti-angiogenic and anti-neoplastic properties. It is used for the treatment of patients with a sub-type of myelodysplastic syndrome. Lenalidomide is currently marketed as a racemic mixture.
Figure imgf000004_0001
Several methods have been published in the prior art for the analysis of lenahdomide in bulk pharmaceutical formulations, but these methods have not been primarily developed for the detection and quantitation of impurities in lenalidomide (see, for example, B.S. Sastry et al, Int. J. PharmTech Res., 2009, vol. 1(3), pages 416-419). Additional HPLC methods have also been reported in the literature, which have been developed for the analysis of lenalidomide in biological fluids (see, for example, T.M. Tohnya et al, J. Chromatography B, 2004, vol. 811, pages 135-141; and L. Qing et al. Therapeutic Drug Monitoring, 2008, vol. 30(5), pages 620-627). A HPLC method suitable for the assay of lenahdomide API was published by G. Saravanan et al, Chromatographia, 2007, vol. 66(3-4), pages 287-290.
However, the current HPLC methods ate not suitable for the detection and estimation of total impurities, especially with respect to unknown impurities that are present in a lenahdomide sample, particularly a sample synthesized by the process disclosed in co- pending Indian patent application 383/KOL/2009 and co-pending international patent application WO 2010/100476.
Therefore the HPLC methods reported in die prior art are not particularly convenient or suitable for analysing lenalidomide API, particularly with respect to related substances.
Consequently, although several HPLC methods have been reported in the prior art for the analysis of lenalidomide and its impurities, there is still a need for an alternative method which avoids the problems associated with die 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 lenalidomide, 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 lenalidomide, 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 lenalidomide synthesized by the process disclosed in co-pending Indian patent application 383/KOL/2009 and co-pending international patent application WO 2010/100476.
Summary of the invention
The term "lenalidomide" as used herein throughout the description and claims means lenalidomide and/ or any salt, tautomer, solvate, isomer or enantiomer thereof. The current invention is particularly useful for the analysis of lenalidomide. A first aspect of the current invention provides a HPLC method for analysing lenalidomide, 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 an acid or an organic acid or an inorganic salt or a mixture thereof.
Typically, the buffer is a phosphate salt, an acetate salt, a trifluoroacetate salt, a formate salt, acetic acid, trifLuoroacetic acid, formic acid, a phosphoric acid or a mixture thereof. More typically the buffer is a phosphate salt, an acetate salt, a formate salt or trifluoroacetic acid.
Where the buffer is a salt, preferably the counter cation is an ammonium cation.
Preferably the buffer is formic acid, a formate salt or a rnixture thereof. Most preferably, the buffer is a formate salt, such as ammonium formate.
The buffer can be present at a concentration of 0.001 to 0.02 M, preferably at a concentration of 0.001 to 0.01 M, more preferably at a concentration of 0.001 to 0.005 M, and most preferably at a concentration of about 0.002 M.
Preferably the buffer is ammonium formate present at a concentration of 0.001 to 0.005 M. Most preferably, the buffer is ammonium formate present at a concentration of approximately 0.002 M.
Preferably the pH of the buffer is approximately 2 to 7. More preferably the pH of the buffer is approximately 4 to 7. More preferably still the pH of the buffer is approximately 5 to 7. Most preferably the pH of the buffer is approximately 6.1.
Typically, the method of the first aspect of the current 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, such as methanol, acetonitrile, propanol or isopropanol or a mixture thereof.
In one embodiment of the first aspect of the current invention, the second liquid B comprises or is a dipolar aprotic solvent such as acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF. Preferably the second liquid B comprises acetonitrile. Most preferably, the second liquid B is acetonitrile.
In another embodiment of the first aspect of the current 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, xY1 and xY2, wherein the magnitude of ΔχΥ (= xY2— xY1) is at least 0.05. For example, X and Y may form a single phase where the mole fraction of Y, xY, is from 0.40 to 0.45, or from 0.70 to 0.75; in both cases ΔχΥ— 0.05. Preferably the magnitude of ΔχΥ 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 current invention, the mobile phase contains less than 10%, less than 5% or less than 1% methanol by volume. In one embodiment, the mobile phase contains no methanol.
In another embodiment of the first aspect of the current invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any alcohol by volume. In one embodiment, the mobile phase contains no alcohols.
In yet another embodiment of the first aspect of the current invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any organic polar protic solvent by volume. In one embodiment, the mobile phase contains no organic polar protic solvents. A preferred embodiment of the first aspect of the current invention is when the first liquid A is an aqueous solution of ammonium formate and the second Equid B is acetonitrile.
The method of the first aspect of the current invention 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 between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 70 minutes.
Alternatively, the first aspect of the current invention may comprise a gradient programming so that the relative concentration of the liquids A and B by volume is maintained at a first ratio for a first period of time, and then varied to a gradient over a second period of time, finishing at a second 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 50-70 % A : 30-50 % B. Preferably the second ratio is 55-65 % A : 35-45 % B. Most preferably the second ratio is about 60 % A : 40 % B.
The second period of time may be from 5 to 90 minutes. Preferably the second period of time is from 15 to 45 minutes. Most preferably the second period of time is about 35 minutes.
A particukrly preferred embodiment of the first aspect of the current invention is when the first liquid A is 0.002 M ammonium formate and the second Equid B is acetonitrile. Preferably a mobile phase flow rate of between 0.01 and 10 ml/min is used, more preferably a mobile phase flow rate of between 0.1 and 4 ml/min is used, mote 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 current 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 chital selector.
Preferably the stationary phase used in the first aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel. Particularly suitable stationary phases include octadecylsilyl silica gel or octylsilyl silica gel. A particularly preferred stationary phase comprises a Purospher Star RP 18e (250 mm x 4.6 mm), 5μπι column.
Preferably the stationary phase has a particle size of between 0.1 and ΙΟΟμ,ιη, or between 0.5 and 25μηι, or between 1 and ΙΟμιη, or between 4.5 and όμΐπη. More preferably the stationary phase has a particle size of about 5μ.ηι.
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 80 and 150A, or between 110 and 13θΑ. Most preferably the stationary phase has a pore size of about 120A.
In one embodiment of the first aspect of the current invention, the chromatography is carried out in a column between 10mm and 5000mm in length, or in a column between 50mm and 1000mm in length, or between 100mm and 500mm in length. More preferably the chromatography is carried out in a column 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 current invention is when the first liquid A is 0.002 M ammonium formate and the second liquid B is acetonitrile and the gradient is as follows:
Figure imgf000010_0001
A more preferred method according to the first aspect of the current invention is when the first liquid A is 0.002 M ammonium formate and the second liquid B is acetonitrile and the gradient is as follows:
Figure imgf000010_0002
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 current invention the HPLC method detects and optionally quantifies in a single run the impurity 3-(l-oxo-4-nitro-l,3-dihyclro-isoindol- 2-yI)piperidine-2,6-dione.
In a preferred embodiment the HPLC method according to the first aspect of the current invention efficiently detects and quantifies in a single run all impurities, including 3-(l-oxo- 4-nitro- 1 ,3 -clihydro-isoindol-2-yl) pipeiidine-2,6-dione. In one embodiment of the first aspect of the current invention, 3-(l-oxo-4-nitro-l,3- dihydro-isomdol-2-yl)pipeddine-2,6-dione is used as internal or external reference marker, or as internal or external reference standard.
In another embodiment of the first aspect of the current invention, the HPLC method is used for the analysis of lenalidomide that is suitable for use in a pharmaceutical composition. Preferably the HPLC method is used for the analysis of lenalidomide that has not entered the human or animal body. Preferably the lenalidomide that is analysed is not in contact with a human or animal bodily fluid such as plasma. Preferably the lenalidomide that is analysed is not in solution. In another embodiment of the first aspect of the current invention, the HPLC method is used for the analysis of a pharmaceutical composition comprising lenalidomide.
In yet another embodiment of the first aspect of the current invention, the HPLC method is used for the analysis of a substance comprising at least 5% lenalidomide by weight. Preferably the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% lenalidomide by weight. Most preferably the substance comprises at least 95% lenalidomide by weight.
In yet another embodiment of the first aspect of the current invention, the HPLC method is used for the analysis of a substance comprising lenalidomide as the only active pharmaceutical ingredient.
A second aspect of the current invention provides a chromatographic method for analysing lenalidomide, or a salt thereof, wherein the mobile phase comprises formic acid, a formate salt or a mixture thereof.
In a preferred embodiment of the second aspect of the current invention, the mobile phase comprises a formate salt such as ammonium formate. Preferably the mobile phase further comprises water. More preferably, the mobile phase comprises an aqueous solution of the formic acid, formate salt or mixture thereof. Most preferably, the mobile phase comprises an aqueous solution of a formate salt such as ammonium formate.
The formic acid, formate salt or mixture thereof can be present at a concentration of 0.001 to 0.02 M, preferably at a concentration of 0.001 to 0.01 M, more preferably at a concentration of 0.001 to 0.005 M, and most preferably at a concentration of about 0.002 M.
Preferably the formic acid, formate salt or mixture thereof is ammonium formate present at a concentration of 0.001 to 0.005 M. Most preferably, the formic acid, formate salt or mixture thereof is ammonium formate present at a concentration of approximately 0.002 M.
Preferably the pH of the aqueous solution is approximately 2 to 7. More preferably the pH of the aqueous solution is approximately 4 to 7. More preferably still the pH of the aqueous solution is approximately 5 to 7. Most preferably the pH of the aqueous solution is approximately 6.1.
In one embodiment of the second aspect of the current 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 formic acid, formate salt or mixture thereof. Preferably the first liquid A is an aqueous solution of the formic acid, formate salt or mixture thereof.
The second liquid B preferably comprises or is an organic solvent, such as methanol, acetonitrile, ptopanol or isopropanol or a mixture thereof.
In one embodiment of the second aspect of the current invention, the second liquid B comprises or is a dipolar aprotic solvent such as acetone, acetonitrile, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine, or THF. Preferably the second liquid B comprises acetonitrile. Most preferably, the second liquid B is acetonitrile.
In another embodiment of the second aspect of the current invention, the second Uquid B is substantially water miscible.
In one embodiment of the second aspect of the current invention, the mobile phase contains less than 10%, less than 5% or less than 1% methanol by volume. In one embodiment, the mobile phase contains no methanol.
In another embodiment of the second aspect of the current invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any alcohol by volume. In one embodiment, the mobile phase contains no alcohols. In yet another embodiment of the second aspect of the current invention, the mobile phase contains less than 10%, less than 5% or less than 1% of any organic polar protic solvent by volume. In one embodiment, the mobile phase contains no organic polar protic solvents.
A preferred embodiment of the second aspect of the current invention is when the first liquid A is an aqueous solution of ammonium formate and the second liquid B is acetonitrile.
A particularly preferred embodiment of the second aspect of the current invention is when the first Uquid A is 0.002 M ammonium formate and the second Uquid B is acetonitrile.
In one embodiment of the second aspect of the current 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 Hquids 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 90%A : 10%B and 50%A : 50%B. More preferably still the relative concentration of the liquids A and B by volume is about 70%A : 30%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 between 100 % A : 0 % B to 0 % A : 100 % B over a period of 30 to 70 minutes.
Alternatively, the second aspect of the current invention may comprise a gradient programming so that the relative concentration of the liquids A and B by volume is maintained at a first ratio for a first period of time, and then varied to a gradient over a second period of time, fmishing at a second 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 50-70 % A : 30-50 % B. Preferably the second ratio is 55-65 % A : 35-45 % B. Most preferably the second ratio is about 60 % A : 40 % B. The second period of time may be from 5 to 90 minutes. Preferably the second period of time is from 15 to 45 minutes. Most preferably the second period of time is about 35 minutes.
A particularly preferred method according to the second aspect of the current invention is when the first liquid A is 0.002 M ammonium formate and the second liquid B is acetonitrile and the gradient is as follows:
Figure imgf000015_0002
A mote preferred method according to the second aspect of the current invention is when the first liquid A is 0.002 M arrrmorrium formate and the second liquid B is acetonitrile and the gradient is as follows:
Figure imgf000015_0001
Typically, the method of the second aspect of the current 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 nJ/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 current 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 farther comprises a chiral selector.
Preferably the stationary phase used in the second aspect of the current invention is reverse phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, 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 Purospher Star RP 18e (250 mm x 4.6 mm), 5μητ. column.
Preferably the stationary phase has a particle size of between 0.1 and ΙΟΟμηι, or between 0.5 and 25μΐη, or between 1 and ΙΟμιη, or between 4.5 and 6μηι. More preferably the stationary phase has a particle size of about 5μm.
Preferably the stationary phase has a pore size of between 10 and lOOOA, or between 25 and 50GA, or between 50 and 20θΑ. More preferably the stationary phase has a pore size of between 80 and 150A, or between 110 and 130A. Most preferably the stationary phase has a pore size of about 12θΑ.
In one embodiment of the second aspect of the current invention, the chromatography is carried out in a column between 10mm and 5000mm in length, or in a column between 50mm and 1000mm in length, or between 100mm and 500mm in length. More preferably the chromatography is carried out in a column 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 refractometet, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.
In one embodiment of the second aspect of the current invention the chromatographic method detects and optionally quantifies in a single run the impurity 3-(l-oxo-4-nitro-l,3- dihydro4soindol-2-yl)piperidine~2,6~dione. In a preferred embodiment the chromatographic method according to the second aspect of the current invention efficiently detects and quantifies in a single run all impurities, including 3-(l-oxo~4-nitt"o-l,3-dihydro-isoindol-2-yl)piperidinc-2,6-dione. In one embodiment of the second aspect of the current invention, 3-(l-oxo-4-nitro-l,3- dihydro-isomdol-2-yl)piperidine-2,6-dione is used as internal or external reference marker, or as internal or external reference standard.
In another embodiment of the second aspect of the current invention, the chromatographic method is used for the analysis of lenahdomide that is suitable for use in a pharmaceutical composition.
Preferably the chromatographic method is used for the analysis of lenalidomide that has not entered the human or animal body. Preferably the lenalidomide that is analysed is not in contact with a human or animal bodily fluid such as plasma. Preferably the lenalidomide that is analysed is not in solution.
In another embodiment of the second aspect of the current invention, the chromatographic method is used for the analysis of a pharmaceutical composition comprising lenalidomide.
In yet another embodiment of the second aspect of the current invention, the chromatographic method is used for the analysis of a substance comprising at least 5% lenahdomide by weight. Preferably the substance comprises at least 10%, at least 25%, at least 50%, at least 75% or at least 90% lenahdomide by weight. Most preferably the substance comprises at least 95% lenahdomide by weight.
In yet another embodiment of the second aspect of the current invention, the chromatographic method is used for the analysis of a substance comprising lenalidomide as the only active pharmaceutical ingredient.
A third aspect of the current invention provides lenahdomide which has been subjected to a method according to either of the first or second aspects of the current invention. Preferably the lenaEdomide is substantially free of 3-(l-oxo-4-nitro-l,3-dlhydfo-isoindol-2- yl)piperidinc-2,6-dione.
A fourth aspect of the current invention provides lenalidomide which is substantially free of 3-(l-oxo-4-niteoT,3-dihydro4soindol-2-y^
Lenalidomide 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 fifth aspect of the current invention provides a pharmaceutical composition comprising lenalidomide according to the third or fourth aspects of the current invention. Preferably the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.
For the avoidance of doubt, insofar as is practicable any embodiment of a given aspect of the current invention may occur in combination with any other embodiment of the same aspect of the current invention. In addition, insofar as is practicable it is to be understood that any preferred or optional embodiment of any aspect of the current invention should also be considered as a preferred or optional embodiment of any other aspect of the current invention.
Detailed description of the invention
The current invention can be used to analyse lenalidomide and/ or its salts as an API or lenalidomide and/ or its salts when prepared as a pharmaceutical composition.
The pharmaceutical compositions that can be analysed by the current invention include solid and liquid compositions and optionally comprise one or more pharmaceutically acceptable carriers or excipients. Solid form compositions include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid compositions include solutions or suspensions which can be administered by oral, injectable or infusion routes. The term "impurities" or "related substances" as used herein throughout the specification can mean either impurities formed in the manufacture of the API or the pharmaceutical composition and/or formed by degradation of the API or in the pharmaceutical composition on storage.
As discussed above, the HPLC methods reported in the prior art are not suitable for analysing lenalidomide, particularly with respect to the related substances formed in the synthesis of lenalidomide and/or its salts prepared by the process disclosed in co-pending Indian patent application 383/KOL/2009 and co-pending international patent application WO 2010/100476.
Howevei, a particularly preferred embodiment of the current invention solves this problem and efficiently detects and quantifies, in a single run, all impurities and intermediates formed in the synthetic process. The present invention is advantageous as the gradient method allows the elution of all polar to non-polar impurities.
The current invention is also advantageous as the method is selective, linear, precise and stability -indicating for the analysis of related substances in lenalidomide and/or its salts. In addition, the current invention is highly sensitive and allows detection and quantification of related substances in lenahdomide and/ or its salts at levels much lower than acceptance limits specified by health authorities.
In addition, the method of the current invention can be used to easily detect and quantify all degradation impurities formed on storage of samples of lenaHdomide. 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 optionatty 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 ot formate and mixtures thereof. Alternatively the buffer can be a mineral acid or a carboxylic acid, such as acetic acid or tri flu oroa eerie acid. Preferably the first liquid A is ammonium formate, preferably at a concentration of 0.002 M. The organic solvent(s) used as the second liquid B can be organic solvents like CrC6 alcohols, such as methanol, efhanol, propanol, butanol or isoptopanol or mixtures thereof. Alternatively, the organic solvent(s) may be tetrahydrofuran, ethyl acetate or acetonitrile or any suitable organic solvent(s). Preferably the organic solvent is acetonitrile. Preferably the stationary phase used in the method of the current invention is selected from octadecylsilyl silica gel (RP-18) or octylsilyl silica gel (Έ.Ρ-8).
An internal standard reference compound may be used in the method of the current invention if required. Alternatively the concentration of the components analysed may be determined by comparison with one or more external reference compounds.
The inventors have tested the methods of the current invention extensively to show that they ate reproducible, precise and linear with respect to concentration. While the current 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 current invention.
The methods of the invention disclosed herein can also be used for the analysis of compounds with similar chemical structures and/or similar chemical or physical properties to lenalidomide, thalidomide and their salts and/ or isomers or enantiomers.
The current invention is illustrated but in no way limited by the following example. Example
Experimental conditions:
Column: Purospher Star RP 18e (250 mm x 4.6 mm), 5μ; Flow rate: 1 ml/min;
Detection: 215 nin;
Sample concentration: 200 ppm;
Diluent A: methanol-acetoninale (50:50 v/v);
Diluent B : 0.002 M ammonium formate-acetonittile (85:15 v/v);
The sample is initially dissolved in a small volume of diluent A, then diluted with diluent B to give a sample solution; the sample solution is then injected into the column which is run using the mobile phase outlined below;
First Liquid A: 0.002 M aqueous ammonium formate;
Second liquid B: acetoniteile;
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 prepare the column for the next run:
Figure imgf000021_0001
Retention times (RT), Relative retention times (RRT), Limit of Detection (LOD) and Limit of Quantitation (LOQ) obtained for all the intermediates and lenalidomide are given in Table 1.
Figure imgf000021_0002
% of LOD and LOQ are with respect to sample concentration of 200 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

Claims
1. A HPLC method for analysing lenalidomide, 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.
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 method according to claim 3, wherein the buffer is an acid or an organic salt or an inorganic salt 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 or trifluoroacetic acid.
6. A HPLC method according to claim 5, wherein the buffer is a formate salt.
7. A HPLC method according to claim 6, wherein the buffer is ammonium formate.
8. A HPLC method according to any one of claims 3 to 7, wherein the buffer is present at a concentration of:
(l) 0.001 to 0.02 M; and/of
(ii) 0.001 to 0.01 M; and/ or
(iii) 0.001 to 0.005 M; and/or
(iv) approximately 0.002 M.
9. A HPLC method according to any one of claims 3 to 8, wherein the pFI of the buffer is approximately 2 to 7.
10. A HPLC method according to any preceding claitn, wherein the mobile phase comprises a second liquid B which is an organic solvent.
11. A HPLC method according to claim 10, wherein the second liquid B is selected from methanol, acetonitrile, propanol or isopropanol or a mixture thereof.
12. A HPLC method according to claim 11, wherein the second liquid B is acetonitrile.
13. A HPLC method according to any one of claims 2 to 12, wherein the first liquid A is an aqueous solution of ammonium formate and the second liquid B is acetonitrile.
14. A HPLC method according to any one of claims 2 to 13, which comprises a gradient programming so that 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 run over:
(i) 10 to 180 minutes; and/ or
(ii) 30 to 120 minutes; and/ or
(iii) 30 to 70 minutes.
15. A HPLC method according to any one of claims 2 to 14, wherein the first liquid A is 0.002 M aqueous ammonium formate and the second liquid B is acetonitrile.
A HPLC method according to claim 15, wherein the gradient is as follows
Figure imgf000024_0001
17. A HPLC method according to any preceding claim, wherein the stationary phase used:
(i) is reverse phase; and/ or
(ii) is octadecylsilyl silica gel or octylsilyl silica gel; and/or
(iii) comprises a Purospher RP 18e (250 mm x 4.6 mm), 5μ column.
18. A HPLC method according to any preceding claim, wherein the chromatography is carried out at temperature between approximately 15 to 40°C.
19. A HPLC method according to any preceding claim, wherein the lenalidomide is in the form of any salt, enantiomer, solvate, hydrate or anhydrate.
20. A HPLC method according to claim 19, wherein die lenahdomide is anhydrous.
21. A HPLC method according to any preceding claim, which detects and quantifies in a single run the impurity 3-(l-oxo-4-nitto-l,3~dihydro-isomdol-2-yl)piperidine-2,6-dione.
22. A HPLC method according to any preceding claim, which detects and quantifies in a' single run all impurities including 3-(l-oxo-4-mtro-l,3-dmydro4somdol-2-yl)pipendine- 2,6-dione and any other unknown impurities.
23. A chromatographic method for analysing lenalidomide, or a salt thereof, wherein the mobile phase comprises formic acid, a formate salt or a mixture thereof.
24. Lenahdomide which has been subjected to a method according to any one of claims 1 to 23.
25. Lenalidomide which is substantially free of 3-(l-oxo-4-nitto-l,3-dihydro4soindol-2- yl)pipetidine-2,6-dione.
26. A pharmaceutical composition comprising lenahdomide according to claim 24 or claim 25.
PCT/GB2010/051951 2009-11-24 2010-11-23 Hplc method for detecting lenalidomide WO2011064574A1 (en)

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