Quantitative determination of Asenapine in both bulk and formulations using neutralization titrations

Asenapine is used in the treatment of schizophrenia disease.Asenapine mainly control the psychotic symptoms’ mainly antagonist to various receptors like, serotonin (5-HT1A, 5HT1B,5-HT2A,5-HT2B,),histamine, and dopamine(D1,D2,D3,D4) receptors. It is also lower affinity towards muscaranic and acetylcholine receptors. In the present study, simple titrimetric method was developed. Respective quantities of Asenapine were taken in aqueous methanol titrated against 0.1N sodium hydroxide acid and 0.1N potassium hydroxide acid using phenolphthalein as an indicators for neutralization titration. This method were found to be sensitive and inexpensive, do not require any sample processing steps and can be utilized for estimation of asenapine in bulk and formulations.


INTRODUCTION
Asenapine (Org 5222, ASP) is a novel dibenzoxepinopyrrole 3,3:6,7) oxepino -(4, 5 -c) pyrrole (Z) -2butenedioate (1:1)] (Figure 1) with unique receptor pharmacology and is available as a fastdissolving tablet for sublingual administration.It has potent dopaminergic (D 1 -D 4 ), serotonergic (5 -HT 2A , 5 -HT 2C , 5 -HT 6 and 5 -HT 7 ), adrenergic (α 1 and α 2 ) and histaminergic (H 1 ) activity, but it lacks significant anti muscarinic activity (Shahid et al., 2009).ASP is an atypical antipsychotic approved in the USA in adults for the treatment of schizophrenia and for the acute treatment, as monotherapy or adjunctive therapy to lithium or valproate; of manic or mixed episodes associated with bipolar I disorder.ASP is indicated in the European Union for the treatment of moderate to severe manic episodes associated with bipolar I disorder in adults (European Medicines Agency, 2010).In short -term trials, ASP has demonstrated superiority over placebo in the treatment of schizophrenia (Cohen, 2007, Alphs et a., 2010).and acute manic episodes associated with bipolar I disorder (Cohem et al., 2009(Cohem et al., & 2010)).The proposed metabolism of ASP and the excretion profiles were recently published (Jacobs et al., 2011).
The present work aims to develop a simple, rapid and sensitive, accurate and economic titrimetric method for the determination of asenapine in pure form and pharmaceutical preparations using 0.1N sodium hydroxide and 0.1N a potassium hydroxide solvents.These methods do not require any sample processing and extraction steps and can be used for the quality control of these drugs in industry.The developed methods were validated as per ICH guidelines and USP requirements, suitable statistical tests were performed on validation data (S Bolton,1997,J.C,Miller,1984).

Preparation of 0.1N Sodium hydroxide
It was prepared by adding accurately weighed 4gm of sodium hydroxide was dissolved in 1000 ml of distilled water using standard volumetric flask.

Preparation of 0.1N Potassium hydroxide
It was prepared by dissolving accurately weighed quantity of 5.6gm of potassium hydroxide in distilled water and volume was made up to 1000 ml of distilled water using standard volumetric flask.

Standardization of 0.1N sodium hydroxide
Accurately measured quantity of 20.4gm of standard potassium hydrogen phthalate and dissolved in 1000 ml of distilled water, 5ml was pipette out into a clean conical flask and phenolphthalein indicator was added.Then the contents in the conical flask were titrated against standard solution of 0.1N sodium hydroxide, solution.Titration was carried out until color changes from colorless to pale pink.Results were obtained in triplicate for standardization using the following formula N 1 V 1 =N 2 V 2 , (Where N 1 and V 1 are the normality and volume of standard potassium hydrogen phthalate N 2 and V 2 are the unknown normality and consumed volume of sodium hydroxide).

Standardization of 0.1N potassium hydroxide
Accurately measured quantity of 20.4gm of standard potassium hydrogen phthalate and dissolved in 1000ml of distilled water, 5ml was pipetted into a clean conical flask and .phenolphthalein indicator was added.Then the contents in the .conical flask were titrated against standard solution of 0.1N potassium hydroxide, solution.Titration was carried out until color changes from colorless to pale pink.Results were obtained in triplicate for standardization using the following formula N 1 V 1 =N 2 V 2 , (Where N 1 and V 1 are the normality and volume of standard potassium hydrogen phthalate N 2 and V 2 are the unknown normality and consumed volume of potassium hydroxide.

Equivalent factors
The exact amount of base consumed by the drug can be determined by stoichiometric equations were described in Figures 2 & 3.In both of these steps, one mole of drug was undergone reaction with sodium hydroxide and potassium hydroxide.Therefore, Each 1 ml of 0.1N sodium hydroxide or 0.1N potassium hydroxide is equivalent to 0.04018 gm of asenapine.

Assay procedure using 0.1N sodium hydroxide
Aliquots of asenapine were prepared by dissolving different amounts of drug (10-100mg) in 20 ml of methanol (methanol was previously neutralized with 0.1N sodium hydroxide) Aliquots were titrated using previously standardized 0.1N sodium hydroxide using phenolphthalein as an indicator to pale pink was observed for end point identification.Results were obtained in triplicates and asenapine was assayed.Assay reaction for titration is shown in figure 2.

Assay procedure using 0.1N potassium hydroxide
Aliquots of asenapine prepared by dissolving different amounts of drug(10-100mg) in 20 ml of methanol was previously neutralized with 0.1N potassium hydroxide ) aliquots were titrated using previously standardized 0.1N potassium hydroxide using phenolphthalein as an indicator pale pink was observed for end point identification.Results were obtained in triplicates and asenapine was assayed.Assay reaction for titration is shown in figure 3.

Linearity
To establish the linearity of proposed methods, five separate series of solutions of drug ranging from 10mg-100mg were dissolved in 20 ml of previously neutralized methanol and add 10ml of distilled water.Then the resulting solution was titrated against 0.1N sodium hydroxide and 0.1N potassium hydroxide by using phenolphthalein as an indicator, end point pale pink color.

Specificity
Specificity is the ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample (Chandran et al., 2007).
The specificity of these methods were determined by adding inert excipients such as starch, microcrystalline cellulose, magnesium stearate and talc individually with known concentration of the drug and titrated by using standard solutions.

Estimation from excipient blends
The in-house prepared tablet formulation blends were prepared, since no marketed formulations were available.These tablet blends were prepared by adding immediate release excipients such as starch, microcrystalline cellulose, magnesium stearate and talc.The crushed blend equivalent 200 mg and 400 mg were transferred to conical flask and respective solvents were added; solutions were filtered through Whatman filter paper number (#40) and the filtrate was titrated with standard solutions using indicator.Simultaneous blank determinations were conducted to confirm specificity and to nullify the effect of each ingredient.Assay results were shown in Table 3 and 4

RESULTS & DISCUSSIONS
The mean five normality values are calculated and approximate values obtained, which were equivalent to normality of 0.1.Methanol was used to dissolve asenapine .Add 10ml of distilled water, did not produce any precipitate.The proposed reactions were found to be simple neutralization of asenapine using basic solvents like sodium hydroxide and potassium hydroxide.During the process of titration, the amount of base consumed was calculated.

Specificity & Linearity
An accurately consumed 1.33±0.04ml (RSD 1.3) of 0.1N sodium hydroxide is equivalent to 506.66 mg of asenapine and 1.34±0.05ml (RSD 1.61) of 0.1N potassium hydroxide is equivalent to 510.47±1.2mg of asenapine respectively.The correlation coefficient was found to be 0.9995 for 0.1N sodium hydroxide and 0.9997 for 0.1Npotassium hydroxide..

Assay & Recovery studies
The assay procedure was performed and percent recovery values were determined for actual drug and blend equivalents (Table 3 and 4).The estimated drug content with extremely low value of RSD established the precision of the proposed methods.Recovery experiments using the developed assay procedures further indicated absence of interference from pharmaceutical excipients used in the selected formulation blends.

CONCLUSION
A new titrimetric method has been developed to be routinely applied to estimate in asenapine bulk and formulation.
These methods have proved to be specific, linear, well recovered.Hence the method is recommended for routine quality control analysis.

Table . 1
: Aqueous titration Standardization values of the 0.1N sodium hydroxide.
Table.2: Standardization values of the 0.1N potassium hydroxide.