Claims:WE CLAIM:

1. A HPLC method for analysing Sugammadex sodium, wherein the mobile phase comprises two or more liquids, including a liquid A and a liquid B, and the relative concentration of the liquids is varied to a predetermined gradient.
2. A HPLC method as claimed in claim 1, wherein the liquid A is a buffer solution of an inorganic acid selected from Orthophosphoric acid.
3. A HPLC method as claimed in claim 2, wherein the concentration of the buffer solution is in the range of 0.001 to 0.1 M.
4. A HPLC method as claimed in claim 2, wherein the pH of the buffer solution is in the range of 2 to 6.
5. A HPLC method as claimed in claim 1, wherein the liquid B comprises mixture of a liquid A (Buffer solution) and one or more organic solvents,
wherein the one or more organic solvents are selected from acetic acid, methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol, tetrahydrofuran, acetone, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine and acetonitrile, or a mixture thereof; and
wherein the buffer solution is 0.2% Orthophosphoric acid in water;
wherein the liquid B comprises 10 to 90% v/v of the liquid A (Buffer solution).
6. A HPLC method as claimed in any one of the preceding claims, wherein the flow rate of the mobile phase is in between 0.01 and 10 ml/min.
7. A HPLC method as claimed in one of the preceding claims, 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 having a run time in the range of 10 to 180 minutes.
8. A HPLC method as claimed in any one of the preceding claims, further comprises a stationary phase selected from octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel.
9. A HPLC method as claimed in any one of the preceding claims, which can be used for detecting and quantification of one or more impurities selected from Sulfoxide impurity, a-SGM impurity, SGM monohydroxy, Sugammadex acid methyl ester and ß-SGM impurity.
10. A HPLC method for analysing Sugammadex sodium having chromatographic conditions provided in the below table:
Liquid-A 0.2% Orthophosphoric acid in water
(buffer solution)
Liquid-B Mixture of Liquid-A and Acetonitrile in the ratio of 1:9 v/v
Stationary phase Kromasil C18 (250 x 4.6 mm), 3.5 µ.
pH of the range 2.0 - 2.5
Wave length 205nm
Flow rate 0.6mL/min
Sample concentration 2.5mg/mL
Column temperature 55°C
Injection volume 10µL
, Description:FIELD OF THE INVENTION
The present invention relates to new HPLC methods for the analysis of the drug substance Sugammadex sodium and related substances. In a first method the mobile phase comprises two or more liquids, and the relative concentration of the liquids is varied to a predetermined gradient. In a second method the mobile phase comprises a polar organic solvent, and the stationary phase comprises a gel. The present invention also relates to a method for analysing a substance, comprising the detection and optional quantification of one or more specific impurities.
BACKGROUND OF THE INVENTION
In order to secure marketing approval for a pharmaceutical product, a manufacturer must submit detailed evidence to the appropriate regulatory authorities to 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.
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, diastereomers or enantiomers), contaminants which are present in materials used in the synthesis of the API or in the preparation of the pharmaceutical composition, and unidentified adventitious substances. Other impurities which may appear on storage include degradants of the API, for instance formed by hydrolysis or oxidation.
The health authorities have very stringent standards and manufacturers must demonstrate that their product is relatively free from impurities or within acceptable limits and that these standards are reproducible for each batch of pharmaceutical product that is produced.
The tests that are required to demonstrate that the API or pharmaceutical compositions are safe and effective include a purity/ assay test, a related substances test, a content uniformity test and a dissolution test. The purity/ assay test determines the purity of the test product when compared to a standard of a known purity, while the related substances test is used to quantify all the impurities present in the product. The content uniformity test ensures that batches of product like a tablet contain a uniform amount of API, and the dissolution test ensures that each batch of product has a consistent dissolution and release of the API.
The technique of choice for the analysis of APIs or pharmaceutical compositions is usually High Performance liquid Chromatography (HPLC) coupled with a UV- Visible detector. The API and the impurities present, if any, are separated on the HPLC stationary phase and they can be detected and quantified using their response obtained from the UV- Visible detector.
HPLC is a chromatographic separation technique in which high-pressure pumps force the substance or mixture being analysed together with a mobile phase, also referred to as the eluent, through a separating column containing the stationary phase.
HPLC analysis may be performed in isocratic or gradient mode. In isocratic mode, the mobile phase composition is constant throughout. A gradient HPLC mode is carried out by a gradual change over a period of time in the percentage of the two or more solvents making up the mobile phase. The change in solvent is controlled by a mixer which mixes the solvents to produce the mobile phase prior to its passing through the column.
If a substance interacts strongly with the stationary phase, it remains in the column for a relatively long time, whereas a substance that does not interact with the stationary phase as strongly elutes out of the column sooner. Depending 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.
Sugammadex sodium is chemically known as 6A,6B,6C,6D,6E,6F,6G,6H-Octakis-S-(2-carboxyethyl)6A,6B,6C,6D,6E,6F,6G,6H-octathio-?-cyclodextrin sodium salt and alternate name is 6-Per-deoxy-6-per-(2-carboxyethyl)thio-?-cyclodextrin, sodium salt. Sugammadex sodium is the first selective relaxant binding agent (SRBA) for reversal of neuromuscular blockade by the agent rocuronium or vecuronium in general anesthesia. It was approved in 2008 by EMEA and also approved in 2015 by USFDA. It is marketed in the form of a sterile solution for intravenous injection under the brand name Bridion®.
Sugammadex was first disclosed in US RE44,733E (US 6670340).
The US ‘733 discloses a process for the preparation of Sugammadex sodium of Formula by reaction of ?-cyclodextrin with iodine in the presence of triphenylphosphine (PPh3) in DMF to produce Iodo-?-cyclodextrin. Further, Iodo-?-cyclodextrin is reacted with 3-mercapto propionic acid in the presence of sodium hydride and DMF to give Sugammadex sodium, which is purified using dialysis membrane.
US ‘733 does not disclose the HPLC method for analysing the Sugammadex sodium.

Julia et. al. (J.Med Chem 2002,45, 1806-1816) discloses a process for the preparation of Sugammadex sodium by reaction ?-cyclodextrin with bromine in the presence of the catalyst triphenylphosphine in N,N- dimethylformamide to obtained 6-deoxy--6-perbromination-?-cyclodextrin. The product with methyl 3-mercaptopropionate in the catalysis of anhydrous cesium carbonate to give methyl ester of sugammadex, and then hydrolyzed with sodium hydroxide to give the crude sugammadex sodium. Which is purified using dialysis membrane.
Julia does not disclose the HPLC method for analysing the Sugammadex sodium.
Wang Hai et. al. (Chem.Asian 2011,6,2390-2399) discloses a iodonation of ?-cyclodextrin in the presence of I2/ PPh3/DMF to give 6-deoxy-6-iodo-?- cyclodextrin crude, the crude product was reacted with acetic anhydride to an ester, by purified by silica gel column chromatography, and then hydrolyzing sodium methoxide, to obtain high purity full deoxy-6-iodo-6-[?]-cyclodextrin purified product, which is reacted with 3-mercaptopropionic acid in the final ether to give the desired product. The reaction intermediate high purity and low impurities, after treatment of the purified product is relatively simple. But with column chromatography process for preparing iodo-?-cyclodextrin, it increases the reaction step, time-consuming. When the method and the use of iodo-?-cyclodextrin prepared as starting material is more comfortable, sodium gluconate, and qualified products cannot be directly obtained, still face difficulties purified sodium gluconate product more comfortable.
Wang Hai does not disclose the HPLC method for analysing the Sugammadex sodium.
Marcel A.H. et al., (Journal of Chromatography B, 879 (2011) 1573–1586) discloses initial chromatography was obtained on a Hypersil BDS C18, polar end capped reversed phase column (Thermo Fisher Scientific Inc., Waltham, MA, USA). Due to the adsorption of sugammadex to metal surfaces and observed excessive peak tailing, chromatographic separation was subsequently achieved on a Varian Polaris C18, hydrophilic groups embedded, reversed phase column (Varian Inc. Lake Forest, CA, USA). Both column types are often applied to retain polar analytes and are stable over a wide pH range (1.5–10) and under hydrophilic conditions (from zero to100%aqueous). Sugammadex and its internal standard are both very polar compounds due to the polycarboxylic groups present. Various combinations of methanol or acetonitrile and 0.1% (v/v) formic acid in water were investigated and compared to identify the optimal mobile phase composition that produced the best sensitivity, efficiency, and peak shape. A linear mobile phase gradient was selected, starting at 30% methanol in 0.1% (v/v) formic acid (in water) and raising methanol content from 30 to 80% in 3.5 min.

The process disclosed in Journal of Chromatography B, 879 (2011) 1573–1586 suffers from the following disadvantages outlined below:
? Formic acid and methanol combination buffer used in above process is not suitable for related substances by HPLC, due to lower wavelengths of methanol and formic acid (UV cut off for Methanol is about 205nm and for Formic acid is about 210nm) producing inconsistence baseline and reproducibility issue during the gradient programme.
? Sugammadex is a non UV compound.
CN105348412A discloses HPLC method, A pump was 0.1% formic acid, B pump methanol, gradient elution; column was Agilent Proshell 120EC-C18 column (50mm * 3.0mm, 2 7µm); column temperature was 40oC; the flow rate of 0·8MI/ min; injection 5µ1, an ELSD detector, operating parameters: drift tube temperature was 105°C, carrier gas flow rate 2.6L/min. The pharmaceutically active ingredient is 8-substituted sodium succinate and 7-substituted sodium succinate. Therefore, the purity of the amount of 8-substituted sodium sulphate and 7-substituted sodium sulphate in the product is the percentage by mass of the product.
The process disclosed in CN105348412A suffers from the following disadvantages outlined below:
? On ELSD, the lower level of impurities will not detect. More stabilization is required to get the smooth baseline and is depends upon the purity of the gas. Generally ELSD technic is useful for content method.
Hence, 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 Sugammadex sodium, particularly with respect to the related substances formed during the synthetic process.

OBJECTIVE 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 Sugammadex sodium, 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 Sugammadex sodium synthesized by the process disclosed in below description.
SUMMARY OF THE INVENTION
The present invention provides a HPLC method for analysing Sugammadex sodium, wherein the mobile phase comprises two or more liquids including a liquid A and a liquid B, and the relative concentration of the liquids is varied to a predetermined gradient.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a HPLC method for analysing Sugammadex sodium, wherein the mobile phase comprises two or more liquids including a liquid A and a liquid B, and the relative concentration of the liquids is varied to a predetermined gradient.
Preferably the liquid A is aqueous based, such as water or buffer solution.
Preferably, the buffer solution is an inorganic acid selected from Orthophosphoric acid.
The buffer can be present at a concentration of 0.001 to 0.1 M, preferably at a concentration of 0.001 to 0.05 M, more preferably at a concentration of 0.005 to 0.05 M, most preferably at a concentration of approximately 0.02 M.
Buffer solution is 0.2% of Orth phosphoric acid in water (Liquid A)

Typically, the method of the first aspect of the current invention is carried out at a column temperature between approximately 15 to 150oC.
The liquid B comprises mixture of a liquid A and one or more organic solvents
The liquid B is preferably an organic solvent is selected from acetic acid, methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, sec-butanol or tert-butanol, tetrahydrofuran, acetone, dimethoxyethane, DMF, DMSO, 1,4-dioxane, pyridine and acetonitrile, or a mixture thereof. Most preferably acetonitrile.
In another embodiment the liquid B comprises 10 to 90% v/v, preferably 30 to 80% v/v, more preferably 50 to 70% v/v of the additional solvent. Most preferably the liquid B comprises approximately 60% v/v of the additional solvent.
Preferably a mobile phase flow rate of between 0.01 and 10 ml/min is used, more preferably a mobile phase flow rate of between 0.1 and 4 ml/min is used, more preferably a mobile phase flow rate of about 0.6 ml/min is used.
The method of the first aspect of the current invention may comprise 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 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 25 or 30 to 120 minutes,
As used herein in relation to any aspect of the present invention, unless stated otherwise all percentages given in relation to the concentration of liquids A and/or B refer to the percentage by volume.
Alternatively, the first aspect of the current invention may comprise a gradient programming so that the relative concentration of the liquids A and B is varied to a gradient from about 100% A : 0% B, or from about 95% A : 5% B, or from about 90% A : 10% B, or from about 85% A : 15% B, to about 100% A : 0% B, or to about 5% A : 95% B, or to about 10% A : 90% B, or to about 15% A : 85% B, or to about 50% A : 50% B. The variation in gradient may typically take place over 10 to 180 minutes, preferably over 30 to 120 minutes, more preferably over 30 to 70 minutes.
A particularly preferred method according to the first aspect of the current invention is when the liquid A is 0.2% of Orthophosphoric acid in water (Buffer solution) and the liquid B is mixture of Liquid A and Acetonitrile in the ratio of 1:9 v/v and the gradient is as follows:
Time (Min) Liquid –A (%) Liquid –B (%)
0 80 20
30 78 22
35 78 22
40 70 30
48 60 40
60 50 50
62 80 20
70 80 20

In one embodiment of the first aspect of the current invention the stationary phase is selected from octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel, cyanopropyl silica gel, aminopropyl silica gel or an alkyl-diol silica gel, A particularly preferred stationary phase comprises a Kromasil C18 (250 mm x 4.6 mm), 3.5µm column.
Preferably the stationary phase has a particle size of between 0.1 and 100µm, or between 0.5 and 25µm, or between 1 and l0µm. More preferably the stationary phase has a particle size of about 5µm.
Preferably the stationary phase has a pore size of between 10 and 1000A, or between 20 and 400A, or between 50 and 150A More preferably the stationary phase has a pore size of about 100A.
In one embodiment of the first aspect of the current invention, the chromatography is carried out in a column between 10mm and 5000mm in length, or in a column between 50mm and 1000mm in length, or between 100mm and 500mm in length. More preferably the chromatography is carried out in a column about 205 mm 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 l mm and 10mm in internal diameter. More preferably the chromatography is carried out in a column about 4.6mm in internal diameter.
The eluent may be analysed by a detector such as a UV or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.
The eluent may be analysed by a detector such as a UV or visible spectrophotometer, a fluorescence spectrophotometer, a differential refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a radioactivity detector.
In one embodiment of the first aspect of the current invention, the Sugammadex sodium analysed is for use in a pharmaceutical composition. Preferably the method is a method of analysing a pharmaceutical composition comprising Sugammadex sodium.
In one embodiment of the first aspect of the current invention, the HPLC method detects and optionally quantifies one or more impurities selected from:

Sulfoxide impurity
a-Sugammadex sodium
(a-SGM impurity)

ß-Sugammadex sodium
(ß-SGM impurity)

Sugammadex acid methyl ester

Mono hydroxy impurity (SGM monohydroxy)

In another embodiment of the third aspect of the current invention, the substance comprises less than 25 wt.% of the one or more impurities. Preferably, the substance comprises less than 10 wt.%, less than 5 wt.% or less than 2 wt.% of the one or more impurities. More preferably the substance comprises less than 0.5 wt.%, or less than 0.3 wt.% of the one or more impurities.
None of the current HPLC methods are suitable for the detection and quantification of all synthetic intermediates and other related substances that are present in a Sugammadex sodium sample, particularly a sample synthesized by the reacting the de-hydrated ?-cyclodextran with in-situly generated bromine-DMF-TPP complex (Vilsmeier Haack reagent) in N,N-dimethylformamide to obtain bromo ?-cyclodextrin, which is further reacted with 3-mercaptopropionic acid in the presence of sodium hydroxide powder in dimethyl sulfoxide (DMSO) solvent to produce crude Sugammadex sodium followed by crystallised from dimethyl sulfoxide (DMSO) and aqueous methanol to obtain Sugammadex sodium.
In the embodiment of the present invention, HPLC method for analysing Sugammadex sodium having chromatographic conditions provided in the below table:
Liquid-A 0.2% Orthophosphoric acid in water
(buffer solution)
Liquid-B Mixture of Liquid-A and Acetonitrile in the ratio of 1:9 v/v
Stationary phase Kromasil C18 (250 x 4.6 mm), 3.5 µ.
pH of the range 2.0 - 2.5
Wave length 205nm
Flow rate 0.6mL/min
Sample concentration 2.5mg/mL
Column temperature 55°C
Injection volume 10µL

The following examples are provided to illustrate the invention and are merely for illustrative purpose only and should not be construed to limit the scope of the invention.

EXAMPLE:
Experimental conditions:
Column: Kromasil C18 (250 x 4.6 mm), 3.5 µ.
Flow rate: 0.6 ml/min;
Detection: 205 nm;
Sample concentration: 2.5mg/mL;
Diluent: Water.
Liquid A: 0.2% Orthophosphoric acid in water (Buffer solution).
Liquid B: Mixture of Liquid A and Acetonitrile in the ratio of 1:9 v/v
Mobile phase: liquid A - liquid B gradient.
The gradient program is described below:
Time (Min) Liquid –A (%) Liquid –B (%)
0 80 20
30 78 22
35 78 22
40 70 30
48 60 40
60 50 50
62 80 20
70 80 20