INTRODUCTION:

Zotepine (ZOT) chemically, 2-[(8-chlorodibenzo(b,f)thiepin-10-yl)oxy]-N,N-dimethylethanamine)¹(Fig.1) is an atypical antipsychotic drug indicated for acute and chronic schizophrenia. According to literature survey, ZOTis not official in any of the Pharmacopoeia.

One of the UV spectrophotometric method²was observed in recent literature survey. Other methods which are found to be reported in literature surveyare HPLC²and LCMS/MS³. So, the objective of this work is to develop a new, simple, rapid, efficient and reproducible difference spectrophotometric method for the determination of ZOT in bulk and tablet dosage form.

Fig.1. Chemical structure of Zotepine

Difference spectroscopy:⁵

Selectivity and accuracy of spectroscopic analysis of sample containing absorbing interference may be markedly improved by the technique like difference spectrophotometry. The essential feature of difference spectrophotometric assay is that, the measured value is the difference in absorbance (A) between two equimolar solutions of the analyte in the different chemical forms which exhibit different spectral characteristics. This is the simplest and most commonly employed technique for altering spectral properties of an analyte by the adjustment of pH by means of aqueous solution of acid, alkali or buffer.

MATERIALS AND METHODS:

Instrumentation and materials:

Spectroscopic analysis was carried out on a V-530 double beam UV-Visible spectrophotometer containing 10 mm path length. All the apparatus and instruments were calibrated and validated before starting the experimental work. Pure ZOT procured from Symed Laboratory, Ahmadabad was used as standard drug without further purification. Commercial tablet formulation SIRILEPT (Sun Pharma) containing ZOT 50mg were purchased from local market.

Preparation of standard stock solution:

50mg of ZOT was accurately transferred to 50ml volumetric flask and dissolved in methanol and made up the volume to mark. The solution was further diluted with 0.1MHCl and 0.1M NaOH separately to get the concentration of 200μg/ml.

Selection of wavelength:

The above prepared solutions were scanned over the range of 400-200nm against reagent blank. From the spectrum obtained, the λ max was recorded at 264 nm and 235 nm in acidic and basic solution respectively. Difference in absorbance between these two maxima was calculated to find out the amplitude.

Study of calibration curve

Different aliquots from above acidic and basic standard solution were diluted with 0.1MHCl and 0.1M NaOH respectively to prepare a series of concentration from 5-35μg/ml as working standard solution. Difference spectrum was recorded by placing ZOT (at 264nm) (Fig. 2) in reference cell and ZOT (at 235 nm) (Fig. 3)in sample cell. For each concentration, differences in absorbance were calculated to find out the amplitude. The calibration curve was prepared by plotting absorbences vs. concentrations (Fig. 4).

Fig. 2. Overlay of 5-35 µg/ml of Zotepine in 0.1 M HCl at 264 nm

Fig. 3. Overlay of 5-35 µg/ml of Zotepine in 0.1 M NaOH at 235 nm

Assay of the marketed formulation

Twenty tablets were weighed accurately and triturate to fine powder. The powder equivalent to 50mg of ZOT was transferred in to 50ml volumetric flask, dissolved in 20 ml of methanol. The solution was filtered through Whatman filter paper and made up volume to the mark with methanol. From the resultant solutions further dilutions were prepared with 0.1N HCl and 0.1N NaOH separately to get final concentrations of ZOT. The absorbances were measured at 264nm and 235nm in acidic and basic solution and the concentration of each analyte was determined with the equation obtained from calibration curve. (Table 3)

Fig. 4. Calibration curve of Zotepine

Table 1: Analysis of the tablet formulation

Formulation (Tablet)	Concentration (μg/ml)	Percentage of drug estimated	Mean % estimated	% RSD
SIRILEPT	20	20.49	101.8	1.25
	20	19.90
	20	20.82

Table 2.Linearity range of ZOT

Sr. No.	Concentration	Absorbance
1	5	0.6525
2	10	0.827
3	15	1.0615
4	20	1.2412
5	25	1.5312
6	30	1.7807
7	35	2.0123

Method validation:

The method was validated for different parameters like linearity, accuracy, precision as per International Conference on Harmonization (ICH) guideline Q2 (R1).⁴

1. Linearity

The sample solutions for linearity and range study were prepared from stock solution to get linear concentration range 5–35 μg/ml. The absorbances were measured at 264nm and 235nm in acidic and basic solutions. For each concentration, differences in absorbance were calculated to find out the amplitude. ZOT showed good linear response in concentration range 5–35 μg/ml with correlation coefficient 0.995.

Table 3.Precision data of Zotepine

Para-meters	Fortified amount (μg/ml)	Amount found (μg/ml)	%RSD
Intra-day*	5	5.550933	1.2
	15	14.52061	0.99
	30	28.6269	1.02
Inter –day*	5	4.952278	1.54
	15	14.13666	0.97
	30	31.21258	1.7
Repeatability**	15	14.72061	1.2

*mean of three replicates ** mean of six replicates

Table 4:% Recovery

Formulation(Tablet)SIRILEPT
% level of drug added	Concentration (µg/ml)		Pure Drug Recovered	% of drug recovered	Mean	% RSD	mean RSD
	Tablet Powder	Pure Drug
50%	20	5	5.86	100.03	99.47	0.65	0.85
100%	20	15	15.11	100.5		0.60
150%	20	30	29.66	97.9		0.3

2. Precision

Precision study of the method was determined by performing intra-day variation, inter-day variation and repeatability studies and expressed in terms of % RSD. For intra-day and inter-day variation, three different absorbances of working standard solutions of ZOT (5, 10 and 15 μg/ml) were measured at three times a day and on three different days respectively. In repeatability study, six determinations of the fixed concentration (15 μg/ml) of both acidic and basic solutions of the drug were analyzed separately. The result of precision study is given in Table 2.

3. Accuracy

Accuracy of the proposed method was examined by recovery of the drug by standard addition technique. To the preanalysed formulation a known amount of the ZOT raw material was added in different concentration viz, 50%, 100%, 150% in both reference and sample solutions. The procedure was repeated as per the analysis of formulation. The amplitude was calculated and the amount of ZOT recovered was determined .This was prepared for six times (Table -4).

RESULT AND DISCUSSION:

A simple, precise and accurate difference spectrophotometric method has been developed for the estimation of ZOT in formulation. In this method the measured value is the difference in absorbance between two equimolar solutions of the analyte in different chemical forms which exhibit different spectral characteristics. The difference spectrum of ZOT in 0.1M NaOH was recorded by taking ZOT in 0.1M HCl as blank. The difference spectrum showed the maxima at 264nm in acidic solution and minima at 235nm in alkaline solution. Linear relationships between amplitude of maxima and minima of difference spectra verses the corresponding drug concentrations were observed.

The standard deviation of the slope and the intercept were low. The correlation coefficient exceeded 0.995.The mean percent label claims estimated for the marketed formulation close to 100 indicating the accuracy of the proposed method. The low value of the statistical parameter viz, standard deviation, percent co-efficient proves that the proposed method can be successfully employed for the routine analysis of ZOT in tablet formulation.

Table 5.Summary of validation parameters

Sr.No.	Parameters	Values obtained
1	Absorption maxima	264nm
2	Standard regression equation	y = 0.0461x + 0.3786
3	Regression Equation (r2)	0.9957
4	Accuracy (% Recovery)	99.47%.
5	System Precision (%RSD)	0.85
6	Linearity	5-35 µg/ml

CONCLUSION:

Based on the results obtained, it was found that, the proposed method was accurate, precise, reproducible and economical and can be employed for routine quality control of ZOT in tablet dosage forms.

ACKNOWLEDGEMENT:

The authors are thankful to Sinhgad Institute of Pharmaceutical Sciences, Lonavala to carry out the research work and Symed Lab, Ahmadabad, for providing the gift sample.

REFERENCES:

1. The Merck Index An Encyclopedia of chemicals, Drugs, and biological, 13^th edition, Merck Research Laboratories, Whitehouse station. New Jersey: 2001;10248.

2. A.S. Devi M. Validated UV spectrophotometric and HPLC methods for quantitative determination of ZOT.Research J. Pharm. and Tech,5(3);2012:342

3. Kazuyoshi N. Application of On-line electrochemistry/ electrospray/ Tandem mass spectrometry to a Quantification method for the Antipsychotic Drug ZOT in Human Serum. The Japan Society for Analytical Chemistry, Volume 25;2009:1197-1201

4. ICH, Q2B (1993). Validation of Analytical Procedure: Methodology, International Conference on Harmonization, Geneva, March1996.

5. Beckett, A. H.; Stenlake J.B., Practical Pharmaceutical Chemistry, 4^th ed.; Part 2, CBS Publishers and Distributors: New Delhi, 2002, 275-278, 281-300.

Received on 09.08.2013 Accepted on 05.09.2013

Asian J. Pharm. Ana. 3(3): July-Sept. 2013; Page 105-107