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 - IMPROVED METHODS FOR THE QUANTITATIVE
DETERMINATION OF ASENAPINE MALEATE AND ITS KNOWN IMPURITIES.
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 reversed-phase liquid chromatographic (RP-LC) method (Method-I) for the quantitative determination of Asenapine maleate and its known impurities (Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-VI, impurity-VIII, impurity-IX and impurity-X). The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
The present invention also relates to an improved reversed-phase liquid chromatographic (RP-LC) method (Method-II) for the quantitative determination of known impurities of asenapine maleate namely impurity-V and impurity-VII. The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
BACKGROUND OF THE INVENTION
Asenapine maleate is chemically known as Trans-5-chloro-2-methyl-2,3,3a,12b-
tetrahydro-lH-dibenz[2,3:6,7]oxepino[4,5-c]-pyrrole Maleate.
It is used in acute and maintenance treatment of schizophrenia in adults.
American Psychiatric Association (APA) considers most atypical antipsychotic agents
first-line drugs for management of the acute phase of schizophrenia.


The product mixture of a reaction rarely is a single compound pure enough to comply with pharmaceutical standards. Side products and byproducts of the reaction and adjunct reagents used in the reaction will, in most cases, be present. At certain stages during processing of the Asenapine maleate contained in the product mixture into an active pharmaceutical ingredient ("API"), the Asenapine maleate must be analyzed for purity, typically by UPLC, HPLC or GC analysis, to determine if it is suitable for continued processing or ultimately for use in a pharmaceutical product.
The U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER) has promulgated guidelines recommending that drug applicants identify organic impurities of 0.1% or greater in the active ingredient. "Guideline on Impurities in New Drug Substances," 61 Fed. Reg. 371 (1996); "Guidance for Industry AND As: Impurities in Drug Substances," 64 Fed. Reg. 67917 (1999). Unless an impurity has been tested for safety, is in a composition proven to be safe in clinical trials, or is a human metabolite, the CDER further recommends that the drug applicant reduce the amount of the impurity in the active ingredient to below 0.1 %. In order to obtain marketing approval for a new drug product, manufacturers must submit to the regulatory authority evidence that the product is acceptable for administration to humans. Such a submission must include, among other things, analytical data showing the impurity profile of the product to demonstrate that the impurities are either absent, or present in a negligible amount. Therefore, there is a need for analytical methods to detect impurities to identify and assay those impurities.
Generally, impurities (side products, 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" ("RRt") to identify impurities.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of asenapine maleate.
In another aspect, the present invention provides an HPLC method for asenapine maleate containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of total impurities.
In another aspect, the present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
In yet another aspect, the present invention provides a simple, accurate and well-defined stability indicating an High performance liquid chromatography (HPLC) method for the determination of asenapine maleate in the presence of degradation products.
In another aspect, the present invention provides a reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of impurity-V and impurity-VII.
In another aspect, the present invention provides an HPLC method for asenapine maleate containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of impurity-V and impurity-VII.
In another aspect, the present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.

In yet another aspect, the present invention provides a simple, accurate and well-defined stability indicating and High performance liquid chromatography (HPLC) method for the determination of impurity-V and impurity-VII in the presence of degradation products.
In one aspect, the HPLC method described in the present invention has the following advantages when compared with prior art methods for determining asenapine maleate and its related impurities:
i) Gradient profile to elute all related impurities and organic phase is 60% which
ensure the elution and detection of non polar impurities forming during the
process or stress study;
ii) The present method mobile phase pH is about 2.0 to 3.0 which is more stable
in all C18 HPLC columns;
iii) Consistency in specificity, precision & reproducibility with good peak shape;
and
iv) The degradation impurities from stress studies are well separated from the
known impurities.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 illustrates the HPLC chromatogram of spiked (Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VIII, impurity-IX and impurity-X spiked in asenapine maleate) sample.
Fig. 2 illustrates the spiked solution (Asenapine maleate + 0.15% of each Known impurity Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VIII, impurity-IX and impurity-X spiked solution) @ 220 nm.

Fig. 3 illustrates the HPLC chromatogram of spiked (Impurity-V and impurity-VII spiked in asenapine maleate) sample.
Fig. 4 illustrates the spiked solution (Asenapine maleate + 0.15% of each Known impurity Impurity-V and impurity-VII spiked solution) @ 220 nm.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, "limit of detection (LOD)" refers to the lowest concentration of analyte that can be clearly detected above the base line signal, is estimated is three times the signal to noise ratio.
As used herein, "limit of quantization (LOQ)" refers to the lowest concentration of analyte that can be quantified with suitable precision and accuracy, is estimated as ten times the signal to noise ratio.
As used herein, "gradient elution" refers to the change in the composition of the gradient eluent over a fixed period of time, stepwise or at a constant rate of change, as the percentage of the first eluent is decreased while the percentage of the second eluent is increased.
As used herein, "gradient eluent" refers to an eluent composed of varying concentrations of first and second eluents.
The eight main known impurities of asenapine maleate by method (I) are:
(i) Trans-2-methyl-2,3,3a, 12b-tetrahydro-l H-dibenz[2,3:6,7]oxepino[4,5-c] pyrrole (Impurity-I) which has the following structure:

The impurity-I is detected and resolved from aseanpine maleate by HPLC with an relative retention time (hereafter referred as RRT) of 0.68.
(ii) 3-(5-Chloro-2-(3-(l-methylpyrrolidin)phenoxy)phenyl)-4-(2-(4-chloro phenoxy) phenyl)-l-methylpyrrolidine (Impurity-II), which has the following structure:

Impurity-II is detected and resolved from asenapine maleate by HPLC with an RRT of 0.75.
(iii) Trans-5-bromo-2-methyl-2,3,3a,12b-tetrahydro-lH-dibenz [2,3:6,7] oxepino[4,5-cjpyrroie maIeate_(Impurity-III), which has the following structure:

The impurity-III is detected and resolved from asenapine maleate by HPLC with an RRT of 1.11.

(iv) Trans-5-chloro-2-methyl-2,3,3a,I2b-tetrahydro-lH- dibenz[2,3:6,7] oxepino[4,5-c]pyrrole-N-oxide (Impurity-IV), which has the following structure:

The impurity-IV is detected and resolved from asenapine maleate by HPLC with an RRT of 1.15.
(v) Trans-5-chIoro-2,3,3a,12b-tetrahydro-lH-dibenz[2,3:6,7] oxepino[4,5-c]-pyrro!e oxalate (Impurity-VI), which has the following structure:

The impurity-VI is detected and resolved from asenapine maleate by HPLC with an RRT of 0.97.
(vi) Trans-N- methyl-4-(2-bromophenyl) -3-(2-hydroxy-5- nitrophenyl)- pyrrolidine (Impurity-VIII) which has the following structure:

Impurity-VIII is detected and resolved from asenapine maleate by HPLC with an RRT of 0.89.
(vii) (R)-2-(3-(diisopropylamino)-l-phenylpropyl)-4-methylphenyl isobutyrate hydrogen fumarate (Impurity-IX) which has the following structure:


Impurity-IX is detected and resolved from asenapine maleateby HPLC with an RRTof 0.73.
(viii) Trans-2-methyl-5-amino-2,3,3a,12b-tetrahydro-lH-dibenz[2,3:6,7] oxepino[4,5-c]-pyrrole fumarate (Impurity-X) which has the following structure:

Impurity-X is detected and resolved from asenapine maleate by HPLC with an RRT of 0.21.
The known impurities of asenapine maleate by method -II are:
(i) Cis-5-chloro-2-methyl-2,3,3a,12b-tetrahydro-lH- dibenz[2t3:6,7] oxepino[4,5-c] pyrrole (Impurity-V) which has the following structure:

The impurity-V is detected and resolved from aseanpine maleate by HPLC with an relative retention time (hereafter referred as RRT) of 0.77.
(ii) Trans-N-methyl-3-(2-hydroxy-5-chlorophenyl)-4-phenyl-pyrrolidine hydrobromide (Impurity-VII), which has the following structure:


Impurity-VII is detected and resolved from asenapine maleate by HPLC with an RRT of 1.18.
According to one aspect of the present invention, there is provided a reversed-phase liquid chromatographic (RP-LC) method-I for quantifying, by area percent, the amounts of asenapine maleate and all impurities, preferably, Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-VI, impurity-VIII, impurity-IX and impurity-X present in a sample of asenapine maleate.
Preferably, the method-I for determining the amount of impurities in a asenapine maleate sample comprises the steps of:
a) combining a Asenapine maleate sample with buffer and acetonitrile in the ratio of 70:30 (v/v) to obtain a solution;
b) injecting the sample solution into a 150mmx4.6 mm column with 3.0 um Unison UK-C18 column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 80:20 (v/v) initial.
d) Preparing Eluent A by dissolving 6.90 g of sodium dihydrogen phosphate monohydrate in 1000 mL of water, add 2.0 mL of triethylarm'ne and dissolve and the pH adjusted was about 3.0 with diluted orthophosphoric acid and filter through 0.45 micron filter paper.
e) Measuring of the amounts of asenapine and each impurity at 220 run wavelength with a UV detector (having an appropriate recording device).
Preferably, the initial ratio of eluent A and eluent B in step-(c) may be continued at the same ratio for 30 minutes then changed linearly to 65:35 (v/v) followed by same ratio for

5 minutes. Again changed the ratio linearly to 40:60 (v/v) followed by same ratio for 3 minutes. After 2 minutes the initial gradient of 80:20 is for 5 minutes to be conditioned for every analysis. The column temperature may be maintained at about 30°C.
The LOD /LOQ values of asenapine maleate and its related impurities, Impurity-I, impurity-II, impurity-Ill, impurity-IV, impurity-VI, impurity-VIII, impurity-IX and impurity-X are summarized in Table 1.
Table 1

S.No Components LOD (%) LOQ (%)
1 Impurity-I 0.0050 0.0151
2 Impurity-II 0.0046 0.0140
3 Impurity-III 0.0049 0.0147
4 Impurity-IV 0.0097 0.0294
5 Impurity-VI 0.0046 0.0138
6 Impurity-VI II 0.0048 0.0145
7 Impurity-IX 0.0041 0.0125
8 Impurity-X 0.0093 0.0283
9 Asenapine maleate 0.0039 0.0118
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and degradation products. The specificity of the LC method for asenapine maleate. Intentional degradation was attempted to stress conditions of acid hydrolysis (using 1.0M HC1), base hydrolysis (using 1.0M NaOH), and oxidative degradation (using 3.0% H2O2), thermal and photo degradation to evaluate the ability of the proposed method to separate asenapine maleate from its degradation products. To

check and ensure the homogeneity and purity of asenapine peak in the stressed sample solutions, PDA-UV detector was employed.
Preferably, the limit of detection (LOD) and limit of quantification (LOQ) were estimated by signal to noise ratio method, by injecting a diluted solution with known concentration. The accuracy of the related substances method with the spiked impurities was evaluated at 0.15 % and 0.225 % of concentration levels.
According to another aspect of the present invention, there is provided a chromatographic method to get the separation of all impurities and stress studies degradants from analyte peak. Satisfactory chromatographic separation was achieved using the mobile phase consists of buffer (6.9 g of sodium dihydrogen phosphate monohydrate in 1000 mL of water, add 2.0 mL of triethylamine and dissolve it. Adjust the pH to 3.0 +_ 0.05 with diluted orthophosphoric acid) In the optimized conditions the asenapine maleate, Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-VI, impurity-VIII, impurity-IX and impurity-X were well separated and the typical retention times (RT) of asenapine, Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-VI, impurity-VIII, impurity-IX and impurity-X were about 20.15, 13.74, 15.19, 22.44, 23.20, 19.63, 18.02, 14.79 and 4.26 minutes respectively, and typically shown in Figure 1. The system suitability results and the developed LC method was found to be specific for asenapine and its eight impurities, namely Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-VI, impurity-VIII, impurity-IX and impurity-X.
The system suitability values and mass numbers of asenapine maleate and its impurities were summarized in Table 2.

Table 2

Compound (n=l) Rt Rs N T (m/z)[M+H]
Impurity-X 4.26 11179 1.19 267.1
Impurity-I 13.74 44.52 45787 1.11 252.1
Impurity-IX 14.79 4.00 53019 1.04 297.0
Impurity-II 15.19 1.39 39093 1.65 571.0
Impurity-VI II 18.02 9.62 74033 1.16 378.9
Impurity-V 18.57 1.97 64544 1.19 286.0
Impurity-VI 19.63 3.71 87494 1.16 272.0
Asenapine 20.15 1.14 15573 3.13 286.0
Impurity-III 22.44 4.67 78278 1A3 331.9
Impurity-IV 23.20 1.91 40353 1.44 302.0
*n=l: determination, Rt: retention time, Rs: USP resolution, N: number of theoretical plates (USP tangent method), T: USP tailing factor, m/z: mass number.
Degradation in test solution was observed using 3% hydrogen peroxide in extreme oxidative degradation condition. Degradation was not observed using 1 M sodium hydroxide at 60°C for 6 hours and 1M HC1 at 60°C for 6 hours. Impurities observed in stress condition using PDA detector,. Major degradants was impurity-IV .Other unknown were also specific in this method .The peak test results obtained from PDA & LC-MS/MS confirm that the aseanpine peak is homogeneous and pure in all analyzed stress samples.
The percentage recovery of asenapine of its impurities in bulk drug samples was done at 0.15 %. The percentage recovery of Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-VI, impurity-VIII, impurity-IX and impurity-X in bulk drugs samples was ranged from 90.00 to 110.00.

According to another aspect of the present invention, there is provided a reversed-phase liquid chromatographic (RP-LC) method-II for quantifying, by area percent, the amounts of impurity-V and impurity-VII present in a sample of asenapine maleate.
Preferably, the method for determining the amount of impurities in a asenapine maleate sample comprises the steps of:
a) combining a Asenapine maleate sample with buffer and acetonitrile in the ratio of 70:30 (v/v) to obtain a solution;
b) injecting the sample solution into a 150mm x1.6 mm column with 3.0 μm Unison UK-C18 column;
c) Isocratic eluting the sample with a mixture of Buffer preparation-1: Methanol: Tetrahydrofuran 700:230:70 (V/V/V).
d) Preparing Buffer preparation-1 by dissolving 6.90 g ( 0.05 M) of Sodium dihydrogen phosphate monohydrate and dissolve in 1000 mL of water, add 2.0 mL of triethylamine and mix well. Adjust the pH = 2.0 ± 0.05 with orthophosphoric acid_solution and filter through 0.45 micron filter paper.
e) Measuring of the amounts of impurity-V and impurity-VII at 220 nm wavelength with a UV detector (having an appropriate recording device).
The column temperature may be maintained at about 40°C.
The LOD /LOQ values of impurity-V and impurity-VII are summarized in Table I. Table 1

S.No Components LOD (%) LOQ (%)
1 Impurity-V 0.0065 0.0197
2 Impurity-VII 0.0086 0.0259
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and degradation products. The specificity of the LC method for

asenapine maleate. Intentional degradation was attempted to stress conditions of acid hydrolysis (using 1.0M HC1), base hydrolysis (using 1.0M NaOH), and oxidative degradation (using 3.0% H2O2), thermal and photo degradation to evaluate the ability of the proposed method to separate impurity-V and impurity-VII from the degradation products. To check and ensure the homogeneity and purity of asenapine peak in the stressed sample solutions. PDA-UV detector was employed.
Preferably, the limit of detection (LOD) and limit of quantification (LOQ) were estimated by signal to noise ratio method, by injecting a diluted solution with known concentration. The accuracy of the related substances method with the spiked impurities was evaluated at 0.15 % and 0.225 % of concentration levels.
According to another aspect of the present invention, there is provided a chromatographic method to get the separation of impurity-V and impurity-VII and stress studies degradants from analyte peak. Satisfactory chromatographic separation was achieved using the mobile phase consists of buffer (6.90 g ( 0.05 M) of sodium dihydrogen phosphate monohydrate and dissolve in 1000 mL of water, add 2.0 mL of triethylamine and mix well. Adjust the pH = 2.0 ± 0.05 with orthophosphoric acid solution.) In the optimized conditions the asenapine maleate, Impurity-V and impurity-VII were well separated and the typical retention times (RT) of asenapine, Impurity-V and impurity-VII were about 14.28, 11.00 and 16.79 minutes respectively, and typically shown in Figure 3. The system suitability results and the developed LC method were found to be specific for impurity-V and impurity-VII.
The system suitability values and mass numbers of impurity-V and impurity-VII were summarized in Table 2.
Table 2

Compound (n=l) Rt Rs N T (m/z)
Impurity-V 11.00 17682 1.10 286.0

Asenapine 14.28 6.31 6833 2.77 286.0
Impurity-VII 16.79 4.11 17202 1.05 288.0
*n=l: determination, Rt: retention time, Rs: USP resolution, N: number of theoretical plates (USP tangent method), T: USP tailing factor, m/z: mass number.
Degradation was not observed using 1 M sodium hydroxide at 60°C for 6 hours, 3% hydrogen peroxide at 60°C for 6 hours and 1M HC1 at 60°C for 6 hours. Other unknown were also specific in this method. The peak test results obtained from PDA & LC-MS/MS confirm that the asenapine peak is homogeneous and pure in all analyzed stress samples.
The percentage recovery of asenapine of its impurities in bulk drug samples was done at 0.15 %. The percentage recovery of Impurity-V and impurity-VII in bulk drugs samples was ranged from 90.00 to 110.00.
According to another aspect of the present invention, there is provided a stability indicating analytical method using the samples generated from forced degradation studies.
According to another aspect of the present invention, there is provided an accurate and well-defined stability indicating HPLC method for the determination of asenapine maleate in the presence of degradation products.
In deliberate varied chromatographic conditions (pH and column), the robustness of the method is confirmed.

Experimental -1 (method-I)
The LC system, used for method development and forced degradation studies and method validation was Waters-Alliance (manufactured by Waters India Ltd) LC system with a photo diode detector. The out put signal was monitored and processed using Empower software system (designed by Waters India) on IBM computer (Digital Equipment Co).
The chromatographic column used was a Unison UK-C18, 150mm x 4.6mm,_column with 3.0 μm particles. The mobile phase consists buffer (6.90 gm of sodium dihydrogen phosphate monohydrate in 1000 mL of water add 2.0 mL of triethylamine and dissolve it followed by pH-3.0 with ortho phosphoric acid), and solvent is acetonitrile. The flow rate of the mobile phase was kept at 1.0 ml/min. beginning with the gradient ratio of mobile phase buffer and solvent 80:20(v/v), system was continued at the same ratio for 30 minutes. The ratio was changed linearly 65:35(v/v) and again system was continued at the same ratio for 5 minutes. The ratio was changed linearly 40:60(v/v) and again system was continued at the same ratio for 3 minutes. After 2 minutes the initial gradient of 80:20 is for 5 minutes to be conditioned for every analysis. The column temperature was maintained at 30°C and the wavelength was monitored at a wavelength of 220 nm. The injection volume was 10 μL for related substances determination. Buffer : Acetonitrile :: 70:30 (v/v) was used as diluent during the standard and test samples preparation.
Preparation of reference solution-1:
7.5 mg each of Impurity-I, Impurity-II, Impurity- III, Impurity-IV, Impurity-V, impurity-VI, impurity-VIII, impurity-IX, impurity-X and 5.0 mg of Asenapine Maleate standard were accurately weighed and transferred to the lOOmL volumetric flask(BOROSIL-Class-A), separately; 10 mL of diluent, 10 mL of acetonitrile was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent Pipette out 1 mL of this solution and transferred in to a lOmL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent.

Preparation of reference solution-2.
7.5 mg of Asenapine Maleate standard was accurately weighed and transferred to the lOmL volumetric flask(BOROSIL-Class-A) , separately; add 1.5 mL system suitability stock solution and about 5 mL diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.
Preparation of reference solution-3.
Dilute 1.5 mL of reference solution-1 to 10 mL with diluent.
Preparation of reference solution-4;
Weigh and transfer about 37.5 mg of Asenapine Maleate standard into a 50mL volumetric flask, add about 30 mL diluent and sonicate to dissolve it. Make up the volume with diluent and mix well. Dilute 1.0 mL of this solution to 100 mL with diluent. Further dilute 1 mL of this solution to 10 mL with diluent.
Experimental-2 (method-II)
The LC system, used for method development and forced degradation studies and method validation was Waters-Alliance (manufactured by Waters India Ltd) LC system with a photo diode detector. The out put. signal was monitored and processed using Empower software system (designed by Waters India) on IBM computer (Digital Equipment Co).
The chromatographic column used was a Unison UK-C18, 150mm x 4.6mm,_coIumn with 3.0 urn particles. The mobile phase consists of buffer (6.90 g ( 0.05 M) of Sodium dihydrogen phosphate monohydrate and dissolve in 1000 mL of water, add 2.0 mL of triethylamine and mix well. Adjust the pH = 2.0 ± 0.05 with orthophosphoric acid solution.), and solvents methanol and tetrabydrofuran. The flow rate of the mobile phase was kept at 1.2 ml/min. The column temperature was maintained at 40°C and the wavelength was monitored at a wavelength of 220 run. The injection volume was 10 uL

for related substances determination. Buffer Preparation-2: Acetonitrile:: 70:30 (v/v) was used as diluent during the standard and test samples preparation.
System suitability stock solution preparation (Imps. @7.5 ppm):
Weigh and transfer about 7.5 mg each of impurity-V and impurity-VII standards into al00 mL volumetric flask, add about 10 mL diluent and sonicate to dissolve. Make up the volume with diluent and mix well. Further dilute 1.0 mL of this solution to 10 mL with diluent.
System suitability solution preparation (Imps. @ 0.15%, API @ 750 ppm):
Weigh and transfer about 7.5 mg of Asenapine Maleate standard into a lOmL volumetric flask, add 1.5 mL system suitability stock solution and about 5 mL diluent and sonicate to dissolve it. Make up the volume with diluent and mix well.
Reference solution preparation (API @ 7.5 ppm ):
Weigh and transfer about 37.5 mg of Asenapine Maleate standard into a 50mL volumetric flask, add about 20 mL diluent and sonicate to dissolve. Make up the volume with diluent and mix well. Dilute 1.0 mL of this solution to 100 mL with diluent. Further dilute 1.0 mL of this solution to 10 mL with diluent.

We Claim,
1. A HPLC method for analyzing Asenapine Maleate, wherein the mobile phase comprises two or more liquids, including a first eluent A and a second eluent B, and the relative concentration of the liquids is varied to a predetermined gradient (Method-I).
2. A HPLC method according to claim 1, wherein the first eluent A is buffer.
3. A HPLC method according to claim 1, wherein the first eluent B is acetonitrile.
4. A HPLC method according to claim 1, wherein gradient of A eluent and B eluent in the ratio of 80:20 (v/v) initial continued at the same ratio for 30 minutes then and progressively increased to linearly to 65:35 (v/v) followed by same ratio for 5 minutes than after changed the ratio linearly to 40:60 (v/v) followed by same ratio for 3 minutes.
5. A HPLC method according to claim 2, wherein buffer is about 6.90 g of sodium dihydrogen phosphate monohydrate and in 1 liter of water 2.0 mL of triethylamine and pH about 3.0.
6. A HPLC method-I determining the amount of impurities in Asenapine maleate sample comprises the steps of:

a) combining a Asenapine maleate sample with buffer and acetonitrile in the ratio of 70:30 (v/v) to obtain a solution;
b) injecting the sample solution into a 150mm x 4.6 mm column with 3.0 urn Unison UK-C18 column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 80:20 (v/v) initial.

d) Preparing Eluent A by dissolving 6.90 g of sodium dihydrogen phosphate monohydrate in 1000 mL of water, add 2,0 mL of triethylamine and dissolve and the pH adjusted was about 3.0 with diluted orthophosphoric acid and filter through 0.45 micron filter paper.
e) Measuring of the amounts of asenapine and each impurity at 220 nm wavelength with a UV detector (having an appropriate recording device).

7. A HPLC method for analyzing Asenapine Maleate, wherein the mobile phase comprises Buffer preparation-1: Methanol: Tetrahydrofuran 700:230:70 (V/V/V) (Method-I).
8. A HPLC method according to claim 7, wherein the Buffer preparation-1 is 6.90 g (0.05 M) of Sodium dihydrogen phosphate monohydrate and dissolve in 1000 mL of water, add 2.0 mL of triethylamine and pH = 2.0 ± 0.05
9. A HPLC method-II determining the amount of impurities in Asenapine maleate sample comprises the steps of:

a) combining a Asenapine maleate sample with buffer and acetonitrile in the ratio of 70:30 (v/v) to obtain a solution;
b) injecting the sample solution into a 150mm x1.6 mm column with 3.0 urn Unison UK-C18 column;
c) Isocratic eluting the sample with a mixture of Buffer preparation-1: Methanol: Tetrahydrofuran 700:230:70 (V/V/V).
d) Preparing Buffer preparation-1 by dissolving 6.90 g (0.05 M) of Sodium dihydrogen phosphate monohydrate and dissolve in 1000 mL of water, add 2.0 mL of triethylamine and mix well. Adjust the pH = 2.0 ± 0.05 with orthophosphoric acid_solution and filter through 0.45 micron filter paper.
e) Measuring of the amounts of impurity-V and impurity-VII at 220 nm wavelength with a UV detector (having an appropriate recording device).

10. A HPLC method for Asenapine maleate containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of total impurities.