FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention - An improved method for the determination of purity of 1-
hexanol in active pharmaceutical ingredients.
2. Applicant(s)
(a) NAME: ALEMBIC PHARMACEUTICALS LIMITED
(b) NATIONALITY: An Indian Company.
(c) ADDRESS: Alembic Campus, Alembic Road,
Vadodara-390, 003, Gujarat, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which is to be performed:

Field of the invention
The present invention relates to an improved gas chromatographic (head-on) method for the determination of chromatographic purity and related impurities and isomeric impurities of 1-hexanol in active pharmaceutical ingredients such as Dabigatran Etexilate.
Background of the invention
1-hexanol is a raw material being used in the manufacturing process of one of the active pharmaceutical ingredients such as Dabigatran Etexilate.
Several methods for the preparation of Dabigatran have been described like any synthetic compound, Dabigatran, or a pharmaceutically-acceptable salt thereof can contain process impurities, unreacted starting materials, chemical derivatives of impurities contained in starting materials, synthetic by-products, and degradation products. It is also known in the art that impurities present in an active pharmaceutical ingredient ("API") may arise from degradation of the API, for example, during storage or during the manufacturing process, including the chemical synthesis.
It is well known in the art that, for human administration, safety considerations require the establishment, by national and international regulatory authorities, of very low limits for identified, but toxicologically uncharacterized impurities, before an active pharmaceutical ingredient (API) product is commercialized. Typically, these limits are less than about 0.15 percent by weight of each impurity. Limits for unidentified and/or uncharacterized impurities are obviously lower, typically less than 0.1 percent by weight. Specific standards can be applied to certain drugs where a pharmacopoeia monograph has been established for that drug. Typically, for impurities that are present in an amount of greater than 0.1 percent by weight, the impurity should be fully identified and characterized.

Therefore, in the manufacture of active pharmaceutical ingredients (APIs) knowledge of the purity of the API. such as Dabigatran, is required before commercialization, as is the purity of the API in the manufactured formulated pharmaceutical product.
Impurities introduced during commercial manufacturing processes must be limited to very small amounts and are preferably substantially absent. For example, the ICH Q7A guidance for API manufacturers requires that process impurities be maintained below set limits by specifying the quality of raw materials, controlling process parameters, such as temperature, pressure, time, and stoichiometric ratios, and including purification steps, such as crystallization, distillation, and liquid-liquid extraction, in the manufacturing process.
The direct product of a chemical reaction is rarely a single compound with sufficient purity to comply with pharmaceutical standards. Intermediates and by-products will, in most cases, be present with the API. At certain stages during processing of an API, such as Dabigatran, it must be analyzed for purity, typically, by GC or HPLC or TLC analysis, to determine the presence of any intermediates or by-products. The API need not be absolutely pure, as absolute purity is a theoretical ideal that is typically unattainable. Rather, purity standards are set with the intention of ensuring that an API is as free of impurities as possible, and, thus, is as safe as possible for human use. As discussed above, in the United States, the Food and Drug Administration guidelines recommend that the amounts of some impurities be limited to less than 0.1 % wt.
Generally, by-products and intermediates (collectively hereinafter defined as "impurities") are identified spectroscopically and/or with another physical method, and then associated with a peak position, such as that in a chromatogram, or a spot on a TLC plate. [Strobel p. 953, Strobel, H. A.; Heineman, W.R., Chemical Instrumentation: A Systematic Approach, 3rd ed. (Wiley & Sons: New York 1989)]. Thereafter, the impurity can be identified, e.g., by its position in the chromatogram, where the position in a chromatogram is conventionally measured in minutes between injection of the sample on the column and elusion of the particular component through the detector.

Impurities generally found in pharmaceutically active agents and formulations containing them include residual amounts of synthetic precursors and their by products to the active agent, by-products which arise during synthesis of the active agent, residual solvent, isomers of the active agent, contaminants which were present in materials used in the synthesis of the active agent or in the preparation of the pharmaceutical formulation, and unidentified adventitious substances. Other impurities which may appear on storage include substances resulting from degradation of the active agent, for instance by oxidation or hydrolysis, the ICH Q7A guidance for API manufacturers requires that process impurities be maintained below set limits by specifying the quality of raw materials, controlling process parameters, such as temperature, pressure, time, and stoichiometric ratios, and including purification steps, such as crystallization, distillation, and liquid-liquid extraction, in the manufacturing process.
At certain stages during processing of an API, such as Dabigatran Etexilate, or a pharmaceutically-acceptable salt thereof, it must be analyzed for purity, typically, by HPLC or GC analysis, to determine if it is suitable for continued processing and, ultimately, for use in a pharmaceutical product. The API need not be absolutely pure, as absolute purity is a theoretical ideal that is typically unattainable. Rather, purity standards are set with the intention of ensuring that an API is as free of impurities as possible, and, thus, are as safe as possible for clinical use. In the International Conference on Harmonisafion of Technical Requirements for Registration of Pharmaceuticals for Human Use ("ICH") guidelines recommend that the amounts of unknown impurities be limited to less than 0.1 percent.
As is known by those skilled in the art, the management of process impurities is greatly enhanced by understanding their chemical structures and synthetic pathways, and by identifying the parameters that influence the amount of impurities in the final product.
Impurities in Dabigatran Etexilate including, but not limited to, unreacted materials, by-products of the reaction, products of side reactions, or degradation products are

undesirable and, in extreme cases, might even be harmful to a patient being treated with a dosage form containing the API. Thus, there is a need in the art for a method for determining the level of impurities in Dabigatran samples and removing the impurities.
While developing a process for the preparation of Dabigatran Etexilate, present inventors serendipitously found an improved method has been developed for the determination of purity and related impurites and isomeric impurities of 1-hexanol namely 3-hexanol, 2-Methyl-1-pentanol, 2-ethyl-l-butanol, 4-methyl-1-pentanol and 3-methyl-1-pentanol. for the preparation of highly pure Dabigatran which minimizes process impurity like.
Structure of 1-Hexanol

The product mixture of a reaction rarely is a single compound. Side products, positional isomers, byproducts of the reaction and adjunct reagents used in the reaction will, in most cases, be present. At certain stages during processing of the 1-hexanol, 1-hexanol must be analyzed for purity, typically by GC analysis, to determine if it is suitable for continued processing or ultimately for use in our desired reaction.
Generally, impurities (side products, positional isomers, byproducts, and adjunct reagents) are identified spectroscopically and by other physical methods and then the impurities are associated with a peak position in a chromatogram (or a spot on a TLC plate). Thereafter, the impurity can be identified by its position in the chromatogram, which is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector, known as the "retention time" ("Rt"). This time period varies daily based upon the condition of the instrumentation and many other factors. To mitigate the effect that such variations have upon accurate identification of an impurity, practitioners use "relative retention time" ("RRf') to identify impurities.

Summary of the invention
In one aspect, the present invention provides a gas chromatographic method for the purity of 1-hexanol and determination of its related impurities and isomeric impurities.
In another aspect, the present invention provides a gas chromatographic method for 1-hexanol containing less than about 1.0 % area by GC, preferably less than about 0.5 % area by GC, more preferably less than 0.1 % area by GC, of specified impurities. In yet another aspect, the present invention provides a simple, accurate and well-defined gas chromatography (GC) method for the determination of chromatographic purity and related impurities and isomeric impurities of 1-hexanol.
In one aspect, the GC method described in the present invention has the following
advantages:
i) 1-hexanol and its impurities were well separated with a resolution not less than 1.0.
ii) the method ensures the elution and detection of impurities upto a level of 0.02%.
iii) Consistency in specificity, precision & reproducibility with good peak shape;
Brief description of drawings
Fig. 1 illustrates the GC chromatogram of mixture of isomeric impurities.
Fig. 2 illustrates the GC chromatogram for 1-hexanol.


Table-1 Structure of impurities:
The isomeric impurities are detected and resolved from 1-hexanol by GC with a relative retention time (hereafter referred as RRT) as described in table-2,
Table 2

Impurity RT (min) RRT
3-Hexanol 24.5 0.82
2-Methyl-1 -pentanol 28.2 0.94
2-Ethyl-l-butanol 28.4 0.95
4-Methy 1-1 -pentano 1 28.6 0.96
3-Methyl-l -pentanol 29.1 0.97
1 -Hexanol 29.9 1.0
According to one aspect of the present invention, there is a gas chromatographic (GC) method for determining the amount of unwanted related and isomeric impurities present in a sample of 1-hexanol by area normalization method (%area).

Preferably, the method for determining the related and isomeric impurities in 1-hexanol sample comprises the steps of:
a) Combining a 1-hexanol sample with dichloromethane to obtain a solution;
b) injecting the sample solution into a ZB-WAX (60m X 0.53mm X LOμm) or equivalent column;
c) method comprised of initial oven temperature of 35°C for 15.0 min which is increased at a rate of 5 °C/min to the final oven temperature of 220°C. The final time is 5 min.
d) Flow is 7.0 mL per minute and split ratio is 20:1.
e) Injector temperature of 220°C and detector temperature 270°C.
f)Carrier gas is nitrogen, mode is constant flow and injection volume is 1.0 μL.
According to another aspect of the present invention, there is provided a chromatographic method to get the separation of 1-hexanol and related and isomeric impurities peaks. Satisfactory chromatographic separation was achieved using ZB-WAX (60m X 0.53 mm X 1.0 urn). In the optimized conditions, 1-hexanol and isomeric impurities were well separated with a resolution not less than 1.0 and the typical retention time (RT) of 1-hexanol is about 29.9 min. and typically shown in Figure 2. The system suitability results and the developed GC method was found to be specific for 1-hexanol and its isomeric impurities. The resolution values of impurities were summarized in Table 3.
Table 3

Compound Rt Resolution
3-Hexanol 24.5 -
2-Methyl-1 -pentanol 28.2 22.85
2-Ethyl-1-butanol 28.4 1.77
4-Methyl-1 -pentano 1 28.6 1.57
3-Methyl-l-pentanol 29.1 2.85
1 -Hexanol 29.9 5.44

Experimental
The GC system, used for method development was 6890N (manufactured by Agilent)
with flame ionization detector. The out put signal was monitored and processed using
Chemstation version Rev.A. 10.02 (1757) software. The chromatographic column used
was ZB-WAX (60m X 0.53mm X l.Oμm). Method comprised of initial oven temperature
of 35°C for 15.0 min which is increased at a rate of 5 °C/min to the final oven
temperature of 220°C. The final time is 5 min. Flow is 7.0 mL per minute and split ratio
is 20:1. Injector temperature is 220°C and detector temperature is 270°C. Carrier gas is
nitrogen, mode is constant flow and injection volume is 1.0 uL
Diluent preparation:
Dichloromethane as diluent.
Standard solution:
Prepare a solution of 50 mg of 1-hexanol standard in 1 mL of diluent.
Sample solution:
Prepare a working solution of 50 mg of 1-hexanol sample in 1 mL of diluent.

We Claim,
1. A gas chromatographic method for the purity of 1 -hexanol and determination of its related impurities and isomeric impurities.
2. A GC method according to claim 1, wherein diluent is dichloromethane.
3. A GC method according to claim 1, wherein injector temperature of 220°C and detector temperature 270°C.
4. A GC method according to claim 1, wherein flow is 7.0 mL per minute and split ratio is 20:1
5. A GC method according to claim 1, wherein method comprised of initial oven temperature of 35°C for 15.0 min which is increased at a rate of 5 °C/min to the final oven temperature of 220°C and final time is 5 min.
6. A HPLC method according to claim 1, wherein UV detector is set to 225nm wavelength.
7. A GC method according to claim 1, wherein impurities are separated with a minimum resolution 1.0
8. A GC method according to claim 1, wherein column ZB-WAX (60m X 0.53mm X l.Oum) or equivalent column.
9. A gas chromatographic method for 1-hexanol containing less than about 1.0 % area by GC, preferably less than about 0.5 % area by GC, more preferably less than 0.1 % area by GC, of specified impurities..
10. A GC method for determining the related and isomeric impurities in 1-hexanol sample comprises the steps of:

ii) Combining a 1-hexanol sample with dichloromethane to obtain a solution; iii) injecting the sample solution into a ZB-WAX (60m X 0.53mm X l.0μm) or
equivalent column; iv) method comprised of initial oven temperature of 35°C for 15.0 rnin which is
increased at a rate of 5 °C/min to the final oven temperature of 220°C. The
final time is 5 min. v) Flow is 7.0 mL per minute and split ratio is 20:1. vi) Injector temperature of 220°C and detector temperature 270°C. vii) Carrier gas is nitrogen, mode is constant flow and injection volume is 1.0 μL.