Method Development and Validation for the Simultaneous Estimation of Clonazepam and Paroxetine in Combined Dosage form using Colorimetry

 

Sheeja V.K., Swapna A.S.*, Sosamma Cicy Eapen and Kumar P.

Department of Pharmaceutical Analysis, Grace College of Pharmacy, Palakkad, Kerala, India

*Corresponding Author E-mail: swapnasurendran88@gmail.com

 

An analytical methodology was developed for the simultaneous estimation of Clonazepam and Paroxetine in combined dosage form. The method involves the use of N-naphthyl ethylene diamine (NED) as reagent. In this method the absorbance was measured at 535nm for Clonazepam and 398nm for Paroxetine. Linearity was observed in the range of 1-9µg/ml for Clonazepam 25-225µg/ml. The regression equation for the calibration data was y=0.001x + 0.653 with correlation coefficient of 0.99 for Clonazepam and y=0.047x + 0.130 with correlation coefficient of 0.981for Paroxetine. The limits of detection were 0.2µg/ml for Clonazepam and 2µg/ml for Paroxetine and the limit of quantitation were 0.6µg/ml and 16µg/ml for Clonazepam and Paroxetine respectively. The precision of the method was good. The values of the relative standard deviation did not exceed 2%. The proposed method was successfully applied for the simultaneous estimation of Clonazepam and Paroxetine in its combined tablet dosage form with good accuracy and precision. All the analytical validation parameters were determined and found in the limit as per ICH guidelines, which indicates the validity of the method. The developed method was found to be precise, accurate, rugged and robust for the simultaneous estimation of Clonazepam and Paroxetine in its combined dosage forms.

 

 

INTRODUCTION:

Clonazepam is a benzodiazepine drug which acts on Central Nervous System to produce a calm effect in panic disorders. Clonazepam is 5-(2-chlorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepine-2-one. Clonazepam is also used for epilepsy. Paroxetine is (3S, 4R)-3-[[1,3-benzodioxol-5-vloxy]methyl]-4-4-flurophenyl piperidine is a new generation antidepressant drug. It exerts its antidepressant effect through a selective inhibition for the reuptake of the neurotransmitter serotonin by the pre synaptic receptors. A fixed dose combination of Clonazepam and Paroxetine is used as the treatment option for the management of Co-morbid depression and anxiety. ^[1-3]

 

Literature survey reveals only a UV Spectrophotometric method of analysis for the simultaneous estimation of Clonazepam and Paroxetine in combined dosage form have been reported. ^[4] Since there was no colorimetric method developed for these drugs a new method was developed using NED reagent.

 

EXPERIMENTAL:

Instruments:

Shimadzu model Pharmaspec-1800 UV-Visible double beam spectrophotometer with 1 cm matched quartz cell was used for recording spectra and absorbance measurements.

 

Chemicals and Reagents:

Clonazepam pure drug was provided as gift sample (Pharma Fabrikon, Chennai), Paroxetine hydrochloride hemihydrates (Alkem Drug Laboratories, Mumbai), Tablet dosage form (Intas Pharmaceuticals Pvt. Ltd, Ahmadabad, India/). NED reagent was procured from SDF chemicals. Ammonium Sulphamate, Sodium nitrite, Hydrochloric Acid and the solvent Methanol were procured from Nice Chemicals. All chemicals and the solvent used were analytical grade.

 

 

Methods:

Preparation of Standard Stock Solutions:

Weighed accurately 5mg of Clonazepam and 125mg of Paroxetine in to a 50ml standard flask. 10ml of Concentrated Hydrochloric Acid and 20ml of Methanol were added in flask. Mixed well and made up with Methanol and kept in ice bath for 10minutes.

 

Plotting Calibration Curve:

For plotting Calibration curve first λmax of the colored complex of Clonazepam and Paroxetine were identified. At this wavelengths calibration curve were plotted (Figure 1 and 2).

 

Aliquot of the standard drug solution is pipette into separate 10ml standard flasks containing mixture of 1ml of 2N HCl and 0.3% Sodium nitrite for preparing five calibration standard solutions containing 2, 4, 6, 8 and 10mcg/ml of Clonazepam and Paroxetine. Mix well and 2.5ml of 0.1% Ammonium Sulphamate was added shake well for 1min. 1ml of 0.1% NED reagent was added and made up to volume with Methanol. Absorbance was measured against blank at 535nm and 398nm for Clonazepam and Paroxetine respectively. Absorbences are given in (Table 1). Overlain spectrum is shown in (Figure 3).

 

Figure 1 and 2: Calibration curve of Clonazepam and Paroxetine

 

Figure 3: Overlain Spectrum of Clonazepam and Paroxetine

 

Table 1: Absorbance Values for Calibration curve of Clonazepam and Paroxetine

Sl. No.	Clonazepam		Paroxetine
	Concentration (µg/ml)	Absorbance at 535nm	Concentration (µg/ml)	Absorbance at 398nm
1 2 3 4 5	1 3 5 7 9	0.195 0.259 0.353 0.477 0.564	25 75 125 175 225	0.661 0.750 0.784 0.846 0.932

 

Analysis of formulation:

20 tablets were weighed accurately and the average weight was calculated. The tablets were ground to a fine powder. An accurately weighed tablet powder equivalent to 5mg of Clonazepam and 5mg of Paroxetine transferred into 50ml standard flask. 10ml of Concentrated Hydrochloric Acid and 20ml of Methanol were added. Dissolved the contents and made up the volume with methanol. The solution was then filtered through Whatman filter paper. Appropriate aliquots within the Beer’s law limit were analyzed by the proposed method using the procedure described earlier. The concentration of Clonazepam and Paroxetine present in the formulation was calculated by simultaneous equation method.

 

Method validation;

Linearity and Range:

5 different concentrations of the standard Clonazepam and Paroxetine were prepared and the regression coefficients were found out. R²=0.99 and R²=0.981 for Clonazepam and Paroxetine respectively.

 

Accuracy:

The accuracy of the method was determined by recovery experiments. A known quantity of the pure drug was added to the pre-analyzed sample formulation at 80%, 100% and 120% levels. The recovery studies were carried out 3 times and the percentage recovery and percentage relative standard deviation of the percentage recovery was calculated (Table 2).

Precision:

In order to determine the precision of the proposed methods, pure drug solutions Clonazepam and Paroxetine) at a particular concentration level (within the working range) were prepared and analyzed in three replicates during the same day (intra-day precision) and on three consecutive days (inter-day precision). Percentage relative standard deviation (RSD %) of Clonazepam and Paroxetine indicates repeatability and   usefulness of the proposed methods in the routine analysis (Table 3).

 

Robustness of the method:

By introducing small changes in the time of analysis and percentage of NED reagent, the effects on the result were examined (Table 4).

 

Ruggedness of the method

Method ruggedness was evaluated by performing the analysis following the recommended procedures by two different analysts. From the %RSD values presented, one can conclude that the proposed method is rugged (Table 4).

 

Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ values were calculated to check the detection limit and quantification limit of the method by using following equations;

               

Where, s the standard deviation and S is the slope of the curve.

Table 2: Accuracy (Recovery studies) of Clonazepam and Paroxetine

Drug	Theoretical % Target level	Amount added (µg/ml)	Amount recovered (µg/ml)	% Recovery	% RSD
Clonazepam	80	4	5.00	100.00	0.55
			4.95	99.00
			5.00	100.00
	100	5	5.03	100.50	0.50
			5.01	100.20
			5.05	100.01
	120	6	5.10	102.00	0.50
			5.07	101.40
			5.12	102.42
Paroxetine	80	100	124	99.20	0.79
			127	99.00
			126	100.80
	100	125	125.87	101.60	0.49
			126.82	100.70
			126.25	100.80
	120	150	126.01	100.80	0.71
			124.81	99.84
			124.27	99.42

 

Table 3: Precision study

Drug	Amount (µg/ml)	Intraday		Interday
Clonazepam	5	% content	% RSD	% content	%RSD
		99.23	0.97	99.70	0.99
		101.008		98.16
		99.45		100.8
Paroxetine	25	100.208	0.46	100.00	0.59
		100.96		99.87
		100.11		100.96

 

 

Table 4: Robustness and Ruggedness

Drug	Amount taken (µg/ml)	Method robustness				Method ruggedness
Clonazepam	5	Parameter altered (Concentration of NED)	Amount Found (µg/ml)	% Content	%RSD	Parameter altered (analysts)	Amount Found (µg/ml)	% Content	%RSD
		0.08	4.924	98.48	1.3297	Analyst I	5.025	100.504	1.2115
			5.050	101.00
			4.952	99.04		Analyst II	4.961	99.23
		0.12	5.120	102.00	0.3295
			5.108	102.16		Analyst III	4.906	98.1
			5.102	102.05
Paroxetine	25	0.08	127.525	102.02	0.3994	Analyst I	124.93	99.94	0.3995
			126.690	101.35
			127.050	101.64		Analyst II	125.26	100.208
		0.12	126.005	100.84	0.4042
			127.001	101.61		Analyst III	125.91	100.728
			126.854	101.48

 

RESULTS AND DISCUSSION:

The goal of this present study was aimed at developing a sensitive, precise and accurate Colorimetric n method for the analysis of Clonazepam and Paroxetine in combined dosage form. Optical characteristics are shown in (Table 5).

 

The method is based on the diazotization of aromatic NH group and Coupling with NED reagent to form a violet colored complex (Scheme 1).^[5-7]

 

 

Table 5: Optical Characteristics:

Parameter	Clonazepam	Paroxetine
Detection wavelength	535 nm	398nm
Beer’s law Limit	1-9 µg/ml	25-225 µg/ml
Regression equation	y=0.047x + 0.130	y=0.001x +0.653
Correlation coefficient	0.99	0.981
Slope	0.047x	0.001x
Intercept	0.653	0.130
LOD	0.2µg/ml	2µg/ml
LOQ	0.6 µg/ml	16µg/ml

 

Scheme 1

 

 

CONCLUSION:

The present study described the successful evaluation of NED reagent in the development of Colorimetric method for simultaneous estimation of Clonazepam and Paroxetine in combined dosage form. All the validation parameters met the standard as per ICH guideline.

 

REFERENCE:

1.     R.B.Kakde and D.D,Satone. Spectrophotometric   Method for Simultaneous Estimation of Escitalopram Oxalate and Clonazepam in tablet dosage form. Indian Journal of Pharmaceutical Sciences. November- December 2009:702-705

2.     Moinuddin Syed, Seema Hashmi, Jitendra B Naik. UV spectrophotometric method development and validation for determination of Paroxetine hydrochloride in pharmaceutical dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. (2)2, 2010: 43-45.

3.     Tushar Jagawat. A comparative study to assess the efficacy and safety of combination capsules of Paroxetine and Clonazepam in comparison to Paroxetine in patients suffering from Co-morbid depression and anxiety. Delhi Psychiatry Journal 14(1), April 2011: 106-109.

4.     Janvika M  Boda, Hardik A  Bhalodiya, Parula B Patel. UV spectroscopic method for simultaneous estimation of Clonazepam and Paroxetine Hydrochloride Hemihydrate in combined Pharmaceutical Formulation. Available from file:///H:/3.htmhttp://www.inve nti.in/Article/ppaqa/333/12.aspx

5.     Padmarajaiah Nagaraja, Shailendra D. Naik, Ashwinee Kumar Shrestha and Anantharaman Shivakumar. A sensitive spectrophotometric method for the determination of sulfonamides in pharmaceutical preparations. Acta Pharm.  57 (2007): 333–342

6.     Nafisur Rahman, Masoom Raza Siddiqui, Syedd Najmul Hejaz Azmi. Quantitation of Nicorandil in Pharmaceutical Formulations by spectrophotometry using N-(1-naphthyl) Ethylenediamine Dihydrochloride. Yakugaku Zasshi, 2007, 127(2):367-374.

7.     P.D.Sethi. Quantitative analysis of pharmaceutical formulation methods with reaction mechanism, 4^th Edition, Volume 1,2,3 & 4

 

 

 

Received on 02.11.2013         Modified on 30.11.2013

Accepted on 08.12.2013         © AJRC All right reserved