Development and validation of UV spectroscopic Q-absorbance ratio method for zonisamide and aripirazole in synthetic mixture

A simple, specific, accurate and precise Q-Absorbance ration spectrophotometric method was developed and validated for estimation of Zonisamide and Aripirazole in Synthetic Mixture. Zonisamide and Aripirazole showed and iso-absorptive point at 230.50nm in Distilled water. The second wavelength used was 240.60nm which is λmax of Aripirazole in distilled water. The concentration of the drugs was determined by using ratio of absorbance at iso-absorptive point (λ1 =240.60 nm) and at the λmax of Aripirazole (λ2 =230.50 nm). This method is linear for both drugs; in range 10-30 μg/ml at λ1 R2 = 0.996 at λ2 (R2 = 0.997) for Aripirazole, and in the range of 10–30 μg/mL for Zonisamide found at λ1 (R2 = 0.990) and at λ2 (R2 = 0.996). The % Recovery was 101.12 % of Aripirazole and 101.89 % of Zonisamide by standard addition method. The LOD was found to be 0.084 μg/mL and 0.110 μg/mL for both drugs at λ1 and λ2 respectively. The LOQ was found to be 0.257 μg/mL and 0.330 μg/mL for both drugs at λ1 and λ2 respectively. The method was found to be precise as % RSD was less than 2.00 in Repeatability, Interday and Intraday precision for Zonisamide and Aripirazole. The % assay of analyte drugs in synthetic mixture was found to be 100.48 % of Zonisamide which showed good applicability of the developed method.


Introduction
One of the most common Neurodegenerative disorders amongst the elderly, leading to Dementia.The term 'Dementia' refers to several illnesses, which affect the functioning of the brain, leading to disruptions in memory, reasoning and emotional stability.In India, dementia is commonly associated with cerebrovascular disease.Earlier for the loss of memory drugs that improve blood flow like amphetamine, Pentoxyphylline were prescribed.Evidence of decrease in cholinergic mechanisms in Alzheimer's disease led to use of cholinergic drugs.
Zonisamide with Aripirazole on ECT-and Benzodiazepine-Resistant periodic catatonia, J Neuropsychiatry Clin Neurosci 24:3, 2012.Hence, there is a scope to develop analytical methods for Zonisamide and Aripirazole in combination.
Literature review reveals that, various analytical methods have been reported for the estimation of Zonisamide and Aripirazole in biological fluids, pharmaceutical formulation and bulk drug include UV spectrophotometric, High-performance liquid chromatography method (HPLC), Stability indicating RP-HPLC method, HPTLC method, TLC method, LC/MS/MS method and UPLC method in individual and/or in combination of other drug.
Literature review shows that, there is no reported method available for Q-absorbance estimation of both the drugs in combination.Therefore it is thought of interest to developed simple, accurate, precise and rapid methods for Qabsorbance estimation of Zonisamide and Aripirazole in combination.

Reagents and material
All the Reagents and Solvents used were of AR or HPLC grades.

Standard Stock Solution of Zonisamide (ZON)
Accurately weighed quantity of ZON 10 mg was transferred to 100ml volumetric flask, add 5 ml of Methanol and 20 ml of water, sonicate it for 15min and dilute it up to the mark with Water to make 100µg/ml solution of ZON

Standard Stock Solution of Aripirazole (APZ)
Accurately weighed quantity of APZ 10 mg was transferred to 100ml volumetric flask, add 5 ml of Methanol and 20 ml of water, sonicate it for 15min and dilute it up to the mark with Water to make 100µg/ml solution of APZ.

Preparation of Standard Mixture Solution (ZON + APZ):
Take 10mg ZON and 10 mg APZ in 100ml volumetric flask.Add 5 ml of Methanol and 20 ml of water, sonicate it for 15min and dilute it up to the mark with Water to make 100µg/ml solution of ZON and 100µg/ml solution of APZ.

Calibration Curve for Cilostazol
This series consisted of five concentrations of standard APZ solution ranging from 10-30μg/ml.The solutions were prepared by pipetting out Standard APZ stock solution (1ml, 1.5ml, 2ml, 2.5ml, 3ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Water.A zero-order derivative spectrum of the resulting solution was recorded and, measure the absorbance at 240.60 nm against a reagent blank solution (Water).Calibration curve was prepared by plotting absorbance versus respective concentration of APZ.

Linearity and range
The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of 10-30μg/ml and 10-30μg/ml for Zonisamide and Aripirazole respectively (n=5).

Intraday Precision
The precision of the developed method was assessed by analyzing samples of the same batch in nine determinations with three Standard solutions containing concentrations 26,28,30 μg/ml for ZON and 26,28,30 μg/ml for APZ and three replicate (n=3) each on same day.Q-Absorbance Ratio was measured at 231.50 nm for ZON and 240.60nm for APZ.The % RSD value of the results corresponding to the absorbance was expressed for intra-day precision

Interday Precision
The precision of the developed method was assessed by analyzing samples of the same batch in nine determinations with three Standard solutions containing concentrations 26,28,30 μg/ml for ZON and 26,28,30 μg/ml for APZ and three replicate (n=3) each on different day.Q-Absorbance Ratio was measured at 231.50 nm for ZON and 240.60nm for APZ.The % RSD value of the results corresponding to the absorbance was expressed for inter-day precision.

Accuracy
It was determined by calculating the recovery of ZON and APZ by standard addition method.Accuracy was done by adding both API standard solution and test solution.
Each solution was taken and diluted with Distilled Water up to 10ml volumetric flask and scanned between 200nm to 400nm against Distilled Water as a blank.The amount of ZON and APZ was calculated at each level and % recoveries were computed.

LOD and LOQ
The Limit of detection and Limit of Quantification of the developed method was assessed by analyzing ten replicates of standard solutions containing concentrations 10 μg/ml for ZON and 10 μg/ml for APZ.
The LOD and LOQ may be calculated as Where,  SD = ten replicates of absorbance  Slope = the mean slope of the 6 calibration curves

Robustness & ruggedness
 Robustness and Ruggedness of the method was determined by subjecting the method to slight change in the method condition, individually, the:  Change in Analyst-1 and Analyst-2. Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450),  % RSD was calculated.

Assay by UV spectrophotometric method
 Synthetic mixture was taken in water.Take 10mg ZON and 10 mg APZ in 100ml volumetric flask.conc of Zonisamide and Aripirazole was 100 μg/mL.From which 2.5 ml transferred in 10 ml volumetric flask and made up to the mark with the water.Final formulation contained 25μg/mL ZON and 25μg/mL APZ. Label claim = 25 mg Zonisamide  Excipients = q.s. Dissolve 25 mg ZON in 250 ml distilled water. Take 2.5 ml in 10 ml volumetric flask and dilute up to the mark with distilled water. So Final formulation contained 25μg/mL ZON.

Result and discussion
The methods were validated with respect ICH Q2R1 guidelines.
The standard solution of ZON and A P Z were scanned separately between 200-400nm, and zero-order spectra were showed overlapping peaks.(figure: 1) Figure 1 Overlain zero order spectra of ZON and APZ (1:1) ratios, respectively Thus, obtained spectra were then processed to obtain Q-Absorbance Ratio Spectrophotometric Method Iso absorptive point is 231.50 nm.λmax of Zonisamide 289.60 nm and λmax of Aripirazole is 240.60 nm.

Figure 2
Overlain first order spectra of ZON and APZ in 1:1 ratio, Respectively Showing Iso absorptive point
This method obeyed beer's law in the concentration range 10-30 µg/ml and 10-30µg/ml for ZON and APZ, respectively.

Accuracy
Accuracy of the method was determined by recovery study from synthetic mixture at three levels (80%, 100%, and 120%) of standard addition.The % recovery values are tabulated in Table 5 and 6.Percentage recovery for ZON and APZ by this method was found in the range of 99.95 to 100.12% and 99.57 to 100.25%, respectively.The value of %RSD within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.

Conclusion
All the parameters for two substances met the criteria of the ICH guidelines for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms.The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification.Ruggedness and Robustness of method was confirmed as no significant were observed on analysis by subjecting the method to slight change in the method condition.Assay results obtained by proposed method are in fair agreement.The method is validated as per ICH Q2R1Guidelines.

Figure 3 Figure 4 Figure 5 Figure 6
Figure 3 Calibration curve for ZONISAMIDE at 231.50nm

Table 1
Lists of Instrument and Apparatus

Table 2
Working Standard API

Table 3
Spectrophotometric conditions for Spectroscopic Method

Table 4
Solutions for Accuracy Study

Table 6
Intraday precision data for estimation of ZON and APZ *(n=3)

Table 7
Interday precision data for estimation of ZON and APZ *(n=3)

Table 11
Robustness and Ruggedness data of ZON and APZ *(n=3)

Table 12
Robustness and Ruggedness data of ZON and APZ *(n=3)

Application of the proposed method for analysis of ZON and APZ in synthetic mixture Table 13
Analysis data of ZON and APZ in Synthetic Mixture *(n=3)

Table 15
Summary of validation parameters