Betahistine dihydrochloride quantitative determination in dosage forms by the reaction with sodium 1 , 2-napthoquinone-4-sulphonate

Results. Different variables affecting the reactions between sodium 1,2-napthoquinone-4-sulphonate and the drug were carefully studied and optimized. It was established that betahistine dihydrochloride reacts with sodium 1,2-napthoquinone-4-sulphonate in alkaline medium with the colored reaction product formation with absorption maximum at 485 nm. The proposed method was subjected to validation tests. The method was validated for the parameters like linearity, precision, accuracy, robustness and scope of application. Beer’s law was obeyed over the concentration range of 2.40–4.00 mg/100 ml. Parameters of linear dependence were calculated with the help of regression analysis by means of least squares. Coefficients of stoichiometric relationship were also determined by means of molar ratios and continuous changes. The proposed procedure meets the requirements of State Pharmacopoeia of Ukraine that are made for dosage forms.


Introduction
Betahistine dihydrochloride (N-methyl-2-(2-pyridyl) ethylamine dihydrochloride) is a structural analogue of histamine by its pharmacological properties.The drug has mild agonistic action at H 1 -receptors and significant antagonistic one -at histamine H 3 -receptors in central and autonomous nervous system.It promotes blood circulation increase in cerebrum and inner ear vessels as well as normalizes neurotransmission in cerebral and vestibular nuclei [1].Betahistine dihydrochloride drugs are widely used in clinical practice for treatment of Meniere's syndrome and other vestibular disorders that are accompanied with faintness, sonitus and earache, decrement in hearing acuity [2].
Betahistine dihydrochloride quantitative determination procedures with application of various physical and chemical methods such as potentiometric titration [3], HPLC [4], colorimetry [5] are described.But spectrophotometry is the most widely used for the assay of the drug [6,7].
Spectrophotometric method is one of the most convenient methods in pharmaceutical analysis, as it is sensitive, precise, cost effective and available to quality control laboratories.Search of highly sensitive and low cost colored reagents is one of the problems of this method.Sodium 1,2-napthoquinone-4-sulphonate (NQS) meets these requirements.
For this purpose the development of highly sensitive, accurate and simple procedure of betahistine dihydrochloride quantitative determination in substance and dosage forms based on reaction with sodium 1,2-napthoquinone-4-sulphonate and validation of the worked out procedure was the objective of the work.
Sodium 1,2-napthoquinone-4-sulphonate of "chemically pure" qualification, NaOH of "reagent grade" qualification and distilled water were used as chemical reagents and a solvent.Betahistine dihydrochloride working standard (WS) was used as a standard.
General procedure of betahistine dihydrochloride quantitative determination 1,00 ml of betahistine dihydrochloride standard solution 0.08 % was placed into a measuring flask for 25 ml.Add 1 ml of 0.5 % NQS and 1 ml of 0.05 M NaOH and mix.Reaction mixture was allowed for 15 min at room temperature and brought up with distilled water to the mark.Absorption was measured at 485 nm against compensation solution blank that didn't contain investigated substance.

Procedure of betahistine dihydrochloride quantitative determination in tablets
Accurate quantity of tablet weight (300 mg) was transferred to 25 ml measuring flask and brought up to the mark with the distilled water.Received solution was mixed and filtered.First portions of filtrate were discarded.The aliquots of the received solution were analyzed according to the proposed procedure.

Results and discussion
Highly sensitive, precise and simple betahistine dihydrochloride quantitative determination by visible spectrophotometry was developed and validated.The procedure is based on interaction of the dosage form with sodium 1,2-napthoquinone-4-sulphonate.In alkaline medium, the colored reaction product with absorption maximum at 485 nm was obtained (Fig. 1).
Optimum conditions to carry out the reaction between betahistine dihydrochloride and sodium 1,2-napthoquinone-4-sulphonate has been established while developing this procedure.We established the influence of reagent concentration at the reaction path.Necessary quantity of reagent was determined experimentally by reaction product maximum yield, i.e. by maximum value of absorbance.Absorption maximum was at 0.5 % concentration of 1,2-napthoquinone-4-sulphonate (Fig. 2).That's why further investigation was carried out at 0.5 % reagent solution.
It was experimentally proved that alkaline medium creation was obligatory for successful reaction path between betahistine dihydrochloride and sodium 1,2-napthoquinone-4-sulphonate.The dependence of absorption from the NaOH solution concentration is given on Fig. 2. 0.05 M solution of NaOH was chosen as the optimum one.
Thereafter, the influence of time at the reaction product formation was studied.At room temperature the intensity of the investigated solution coloration increased in time, and absorption maximum was observed in 15 min.
Coefficients of stoichiometric relationship were also determined by means of molar ratios and continuous changes.Stoichiometric relationships of reacting species "sodium 1,2-napthoquinone-4-sulphonate -betahistine dihydrochloride" were uniquely agree with each other and form 1:1 (Fig. 3).
The border of determination at optimum conditions was 0.24 μg/ml, that showed the high sensitivity of the reaction.
Thereafter, the validation of the developed procedure was carried out according to the requirements of State Pharmacopoeia of Ukraine by means of standard [8,9].Principal validation features, namely, linearity, precision, accuracy, robustness and scope of application were established.
Linearity Linearity was determined in the range of concentration, in which submission to the Beer's law was observed, namely 2.40-4.00mg/100 ml.The calibration graph of the absorption from betahistine dihydrochloride concentration was plotted according to the obtained data.It is given in Fig. 4.

Вопросы фармации
Parameters of linear dependence were calculated using the regression analysis by means of least squares.Received values are given in the Table 1.
Calculated numbers show that all requirements of State Pharmacopoeia of Ukraine concerning linear dependence parameters are met.Procedure scope of application is 70-130 %.

Precision
Precision was determined at level of coincidence.9 samples, the concentrations of which were uniformly distributed in the investigated procedure range, were analysed.The comparison solution absorption was also measured.According to the State Pharmacopoeia of Ukraine requirements to precision, the procedure is accurate at level of coincidence, if unilateral confidence interval (Δx) doesn't exceed the maximum allowable analysis uncertainty (ΔAs%).The data of the table 2 show the accuracy of the developed procedure.

Accuracy
To establish the accuracy of the developed procedure, the standard addition method was used.Deferent amounts of betahistine dihydrochloride standard solution were added to three equal dosage forms samples and analysis was made three times.The results of determination were accurate, as significant systematic imprecision was absent, i.e. the real value was in the established confidence interval (Table 3).

Robustness
Robustness estimation was carried out at the stage of procedure development.Investigation of stability of analytical solutions in time was carried out for estimation of procedure robustness.Sample solution and comparison solution were stable within at least 30 min.

Conclusion
On the basis of the carried out investigations of betahistine dihydrochloride quantitative determination, spectrophotometric procedure has been developed.This procedure is successfully applied for dosage forms analysis.Investigation results show that the procedure is highly sensitive, precise, simple and relevant to be applied at the quality control laboratories for dosage forms.

Fig. 2 .Fig. 3 .
Fig. 2. I: the dependence of the absorption of the reaction product from reagent concentration, II: from the final NaOH solution.

Table 1 .
The calibration graph of the absorption against the concentration of betahistine dihydrochloride.Linear dependence data

Table 2 .
Determination of the results of coincidence of the betahistine dihydrochloride quantitative determination in tablets (n = 9, P = 0.95)

Table 3 .
Results of determination of accuracy of betahistine dihydrochloride quantitative determination in tablets by means of standard addition method