Development and Validation of Spectrophotometric Method for Analysis of Ziprasidone HCl

 

Arun Shirwaikar¹, Sarala Devi², K Premalatha³, Muhammed Maqbool³ and Sreejith K.R.³*

¹College of Pharmacy, Gulf Medical University, Ajman, U A E

²Department of Medicine, Pariyaram Medical College, Kannur, India

³Academy of Pharmaceutical Science, Pariyaram Medical College, Kannur, India.

*Corresponding Author E-mail: krsreejith_5228@yahoo.co.in

 

ABSTRACT:

A simple, precise and sensitive extractive Spectrophotometric method has been described for the assay of Ziprasidone HCl in pure and pharmaceutical preparations. This method is based on the formation of ion-pair complex between the basic nitrogen of the drug with bromothymol blue (BTB) in acidic buffer solution. The formed complex was extracted with chloroform and measured at 415 nm. Beer’s law was obeyed in the range 4-24 μg/ ml with correlation coefficient (n = 6) ≥ 0.9999. The molar absorptivity, Sandell sensitivity, detection and quantification limits were also calculated. The composition of the ion pairs was found 1:1 by Job’s method. The proposed methods have been applied successfully for the analysis of Ziprasidone in pure and in its dosage forms. These developed methods were validated for accuracy and precision.

 

 

1. INTRODUCTION:

Ziprasidone HCl is an Antipsychotic agent used in the management of CNS disorders. Chemically Ziprasidone HCl is 5-[2-[4-(1, 2-benzothiazol-3-yl) piperazin-1-yl] ethyl]-6-chloro-1, 3-dihydroindol-2-one hydrochloride (Figure 1). Ziprasidone HCl is a white or pale pink powder with a molecular weight of 449.4. It is freely soluble in methanol, ethanol and chloroform, soluble in ether, sparingly soluble in acetonitrile and octanol, and practically insoluble in water. It should be kept in a well closed container, protected from light [1-6].  Literature survey revealed that only few methods available for the estimation of Ziprasidone HCl alone, in combination with other drugs, in its dosage form and in plasma [7-16].

 

Figure 1

 

The present investigation was undertaken to develop a simple extractive Spectrophotometric method for the estimation of Ziprasidone HCl in bulk and in formulations.[17-20]

 

2. EXPERIMENTAL:

2.1Instrument:-

Shimadzu UV-Visible double beam Spectrophotometer (model 1700) was used for spectral studies.

Shimadzu analytical balance is used for weighing.

PH measurements are made with Systronics MKVI Digital PH meter

 

2.2 MATERIALS AND REAGENTS:

Materials –

Only A R grade reagents and solvents were used. The pure drug Ziprasidone HCL was obtained as a gift sample from Dr. Reddy’s laboratories, Hyderabad.

 

Pharmaceutical Formulation.

 

Zipsydon-20

Standard solution:-

 

Stock Standard Solution:

Accurately weighed quantity of Ziprasidone HCl (100 mg) was dissolved in 25 ml methanol in a volumetric flask. (4000mcg/ml)

 

Figure 2 Beers Plot

 

Standard Solution-A:

10 ml of stock standard solution was diluted to 100 ml with methanol (Concentration 400 mcg/ml)

 

Preparation of reagents.

Bromothymol Blue (0.1%) was prepared by dissolving 100 mg of bromothymol blue in 100 ml of distilled Water.

Phthalate buffer was prepared by dissolving 1.28g of Potassium Hydrogen Phthalate in 50 ml of water and the PH was adjusted to 4 with 0.2 M HCl and the volume was made up to 250 ml with water.

 

 2.3. Construction of calibration curves:

1–6 ml of standard solution A (400 mcg/ml) were transferred into a series of 10 ml standard flasks and made up with methanol. One ml from each standard flask is used for the formation of ion pair and is taken into a series of separating funnel. To each separating funnel 2.0 ml of Buffer solution and 2 ml of dye solution were added. The total volume of aqueous phase in each separating funnel was adjusted to 15 ml with distilled water. The solutions were mixed well and to it 5ml of chloroform were added. The contents in the separating funnel were shaken for 5 minutes. Chloroform layer were separated and the extraction procedure was repeated with another 5ml of chloroform. The extracts were dried using anhydrous sodium sulphite and collected to get 10 ml and absorbances of the separated organic layer were Measured at 415 nm against a reagent blank prepared under identical conditions. Calibration curve is plotted by taking concentration on X axis and Absorbance on Y axis. Beers plot is given figure 2

 

 

2.4. Procedures for dosage form.

Twenty capsules were weighed and average weight of one capsule was determined. A quantity of powder equivalent to 0.02g of Ziprasidone is  taken in a volumetric flask (100 ml) and shaken with methanol (25.0 ml) and was kept for ultra sonication for 5 min and  the volume was made up to the mark. The solution was then filtered through whatman filter paper No. 41 and 5ml of the filtrate was diluted with methanol in 10ml standard Flask to get sample solution (100mcg/ml) .5ml of this solution is again made up to 10 ml with methanol.4 ml of this solution is used for the formation of ion pair and was taken in a separating funnel. To the separating funnel added 2.0 ml each of buffer solution and dye solution. The total volume of aqueous phase in each separating funnel was adjusted to 15 ml with distilled water. The solution was mixed well and to it 5ml of chloroform was added. The contents in the separating funnel were shaken for 5 minutes. Chloroform layer was separated and the extraction procedure was repeated with another 5ml of chloroform. The extracts were dried using anhydrous sodium sulphite and collected to get 10 ml and absorbances of the separated organic layer were Measured at 415 nm against a reagent blank prepared under identical conditions to get the test absorbance.

 

3. RESULTS AND DISCUSSION:

Absorption spectra

When Ziprasidone HCl is allowed to react with bromo thymol blue in presence of phthalate buffer an ion pair is formed which can be  extracted with chloroform .The absorption spectra of ion pair were measured in the range 300-600nm against the blank solution. The ion pair complex show maximum absorbance at 415nm.

 

 

Figure 3 Absorption Spectrum

 

Table 1 Statistical analysis of calibration graphs and analytical data in the determination of the studied drug using the proposed method.

 

Parameters	Results
Wavelengths λ max (nm)	415
Beer’s law limits (μg mL−1)	4-24mcg/ml
Molar absorptivity ε,(L/mol-1 cm-1) x 104	1.4
Sandell, s sensitivity (ng cm-2)	31.84
Intercept (a)	0.002
Slope (b)	0.03145
Correlation coefficient (r)	0.9999
Limit of detection LOD (μg mL−1)	0.078
Limit of quantification LOQ (μg mL−1)	0.2384

 

The optimum reaction conditions for determination of the ion-pair complexes were established. Then linearity, accuracy, precision, sensitivity, and stability of proposed methods were studied and these developed methods applied to pharmaceutical preparations and obtained results evaluated statistically. Figure 3

 

3.1 Optimum conditions for complex formation

The optimization of the methods was carefully studied to achieve complete reaction formation, highest sensitivity and maximum absorbance. Reaction conditions of the ion-pair complex were found by studying with preliminary experiments such as pH of buffer, type of organic solvent, volumes of the dye, and shaking time for the extraction of ion-pair complexes.

 

3.1.1. Selecting of the Extracting Solvents

The effect of several extracting organic solvents on the ion-pair complex was examined. Chloroform, carbon tetrachloride, ethyl acetate, diethyl ether, toluene, were tried for effective extraction of the coloured ion pair complex from aqueous phase. Chloroform was found to be the most suitable solvent for extraction of colored complex because of its higher efficiency on color intensity, selective extraction of the ion-pair complex from the aqueous phase.

 

Figure 4 Effect of pH of phthalate buffer on the ion pair complex of drug with bromo thymol blue.

 

3.1.2. Effect of Time and Temperature.

The optimum reaction time was investigated from 0.5 to 10.0 min by following the color development at room temperature. Complete color intensity was attained after 5.0 min of mixing for all complexes. The absorbance remained stable for at least 12 hrs. At higher temperature absorbance started to decay.

 

Figure 5 Effect of volume of the reagent on the reaction of drug with dye

Table 2 Assay of tablets

Sl.No	Brand Name	Avg. Wt of tablet in g	Wt of Std Drug in g	Wt of tablet Powder in g	STD Absorbance	Test Absorbance	Content of drug in tablet in Mg	Average content in Mg	% RSD
1 2 3 4 5 6	Zypsidon	0.2281	0.1089	0.2280 0.2263 0.2296 0.2276 0.2132 0.2304	0.684	0.627 0.622 0.631 0.623 0.590 0.635	19.97 19.96 19.96 19.88 20.09 20.02	19.99	0.4520

 

Table 3 Data for recovery studies

Level    %	Brand Name	Avg Wt	Sl No	Amt present in µg/ml	Amt added in µg/ml	Mean Test absorbance*	Amt estimated in µg/ml	Percentage recovery	Mean %recovery	% RSD
80       100       120	Zypsidon	0.2281	1 2 3   1 2 3   1 2 3	9.9950 10.1005 10.0085   9.9950 10.1005 10.0085   9.9950 10.1005 10.0085	7.98 7.98 7.98   10.03 10.03 10.03   12.01 12.01 12.01	0.563 0.569 0.564   0.628 0.630 0.629   0.690 0.694 0.691	17.9511 18.1425 17.9830   20.0237 20.0874 20.0556   22.0005 22.1281 22.0324	99.70 100.77 99.93   99.98 99.57 100.17   99.96 100.14 100.11	100.03	0.3395

Mean standard absorbance =0.626*; * Mean of three observations ; Concentration of standard =19.96µg/ml

 

Table4 Data for intraday and interday precision (n=6)

Sl No	Intra day	Mean	SD	%RSD	Inter day	Mean	SD	%RSD
1 2 3 4 5 6	19.97 19.96 19.96 19.88 20.09 20.02	19.99	0.0904	0.4520	20.04 19.99 19.99 20.04 20.02 20.08	20.02	0.0339	0.1693

 

 

3.1.3. Effects of pH on the Ion-Pair Formation.

The effect of pH was studied by extracting the colored complexes in the presence of various buffers such as KCl-HCl (pH 1.5 - 4.5), NaOAc-HCl (pH 2.0 - 5.0), and potassium hydrogen phthalate-HCl (pH 2.0 - 5.0). The maximum color intensity and highest absorbance value were observed in potassium hydrogen phthalate- HCl buffer of pH 4.0 which is determined by adding buffer solution of varying pH to a series of solution containing unchanged drug and dye concentration. Further, 2.0 ml of buffer was sufficient to give maximum absorbance and reproducible results. Figure 4

 

3.1.4. Effects of Reagents Concentration.

The effect of the reagents was studied by measuring the absorbance of solutions containing a fixed concentration of drug and varied amounts of the reagent with 2ml buffer. Maximum color intensity of the complex was achieved with 2.0 ml of reagent solution. Figure 5

 

Figure 6    Job's method of continuous variation graph for the reaction of drug with dye [drug]=[dye]= 5 x 10-4 M

Table 5 Ruggedness of the proposed method

Parameter	Content of drug*	Std Deviation	%RSD
Analyst 1	19.9989	0.00813	0.0406
Analyst 2	20.0104

* Mean of six observations

 

 

3.1.5. Stoichiometric Ratio.

Job’s Method of Continuous Variation of equimolar solutions was employed[21]. A 0.0005 M standard solution of drug and 0.0005M solution of Bromo thymol blue were used. A series of solutions was prepared in which the total volume of drug and reagent was kept at 10 mL. The absorbance was measured at the optimum wavelength. The molar ratio of the reagent (drug: dye) in the ion-pair complex was determined by the Method of Continuous Variation (Job’s Method) and the results indicated that 1: 1 (drug: dye) ion pairs are formed. Figure 6

 

3.2. Method Validation.

3.2.1. Linearity.

A calibration graph was constructed by measuring the absorbance at six concentration levels. It showed a linear response of absorbance in relation to the concentration of drug over the range of 4 - 24 μg/ml. The statistical parameters were given in the regression equation calculated from the calibration graphs. The proposed method was validated as per the ICH guidelines [22-23] .The linearity of calibration graphs was proved by the high values of the correlation coefficient (r) and the small values of the y-intercepts of the regression equations. The apparent molar absorptivity (ε) was calculated and found to be 1.4 x 10⁴. Statistical analysis of calibration graphs and analytical data in the determination of the studied drug using the proposed method given in Table 1.

 

3.2.2. Sensitivity

The detection limit (LOD) for the proposed methods was calculated using the

following equation [24]:

LOD = 3.3s/S

Where s is the residual standard deviation of the least square regression line and S is the slope of the calibration graph. In accordance with the formula, the detection limit was found to be 0.078mcg/ml.

 

The limit of quantization, LOQ, is calculated as [24];

LOQ = 10 s / S

According to this equation, the limit of quantization was found to be 0.2384 mcg/ml.

 

3.2.3. Recovery study.

A quantity of powder equivalent to 0.02g of Ziprasidone is  taken in a volumetric flask (100 ml) and shaken with methanol (25.0 ml) and was kept for ultra sonication for 5 min and  the volume was made up to the mark. The solution was then filtered through Whatman filter paper No. 41. 1ml of the filtrate was mixed with definite concentration of standard solution and was taken in a separating funnel. To the separating funnel added 2.0 ml each of buffer solution and dye solution. The solution was mixed well and to it 4ml of chloroform was added. The contents in the separating funnel were shaken for 5 minutes. Chloroform layer was separated and the extraction procedure was repeated with another 4ml of chloroform. The extracts were collected and dried using anhydrous sodium sulphate and made up to  10 ml. Absorbances of the separated organic layer were Measured at 415 nm against a reagent blank prepared under identical conditions to get the test absorbance.

 

3.2.4.Precision.

The precision study of the developed method was done based on the data obtained from Table2. Results of six determinations from same batch were validated statistically and Standard deviation and Co-efficient of variation in% is found to be 0.0904 and 0.4520 respectively. The data for intraday and inter day precision is given in the Table 4

 

3.2.5. Ruggedness

The degree of reproducibility of test results obtained by the methods was checked by analyzing the drug sample by different analysts. To validate and confirm the results, six solutions of drug were prepared and analysis was carried out. The datas given in Table 5

 

3.3. Effects of excipients.

Effects of common excipients and additives were tested for their possible interferences in the assay of Ziprasidone by proposed method. It was observed that the excipients such as talc, lactose, starch and magnesium stearate did not interfere with the assay, since the formation of an ion-pair complex with anionic dyes needs a basic moiety.

3.4. Analysis of Pharmaceutical Preparations.

The suggested methods were applied successfully to the determination of Ziprasidone in formulation. Six replicate determinations were made. The assay results were in a good agreement with the label claims. The Recovery study was carried out to evaluate the accuracy of the method. Datas given in Table 2 and Table 3

 

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Received on 30.06.2012       Modified on 10.07.2012

Accepted on 20.07.2012      © RJPT All right reserved