Method Development and Validation for the Simultaneous Estimation of Clonazepam and Paroxetine in Combined Dosage Form using FT-IR

 

Sheeja V. K.1, Swapna. A. S. 2*

1Department of Pharmaceutical Analysis, Grace College of Pharmacy, Palakkad, Kerala, India

2Department of Pharmaceutical Chemistry, Sanjo College of Pharmaceutical Studies, Palakkad, Kerala, India

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

 

ABSTRACT:

A simple precise and rapid method has been developed for the simultaneous estimation of Clonazepam (CNZ) and Paroxetine (PRX) through Fourier Transform Infrared (FTIR) spectroscopy in tablet formulation. For the determination of the active pharmaceutical ingredients (API), KBr pellets containing known amount of standards and samples were used for acquisition of the FTIR spectra. The method involves the measurements of peaks of nitro group at 1340cm -1 (CNZ) and ring -NH group at 929 cm-1 (PRX). Linearity was observed in the range of 0.10- 0.26mg for Clonazepam 2.5-6.5mg for Paroxetine. The regression equation for the calibration data was y=3.283x + 0.019 with correlation coefficient of 0.995 for Clonazepam and y=0.258x+0.042 with correlation coefficient of 0.996 for Paroxetine. The limits of detection were 0.03mg for Clonazepam and 0.5mg for Paroxetine and the limit of quantitation were 0.5mg and 1.5mg 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. The developed method was found to be precise and accurate for the simultaneous estimation of Clonazepam and Paroxetine in its combined dosage forms. This technique does not use organic solvents, which is one great advantage over the most common analytical methods. This fact contributes to minimize the generation of organic solvent waste by the industry and thereby reduces the impact of its activities on the environment.

 

KEYWORDS: Clonazepam, Paroxetine Hydrochloride Hemihydrate, KBr, Fourier transform infrared spectroscopy, Co-Morbid depression.

 

 

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.[1,2,3]

 

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] Hence a simple, rapid and accurate FTIR method developed for simultaneous estimation of these drugs.

 

 

MATERIAL AND METHODS:

Chemicals and Reagents:

Clonazepam pure drug was provided as gift sample (Pharma Fabrikon, Chennai), Paroxetine hydrochloride hemihydrates (Alkem Drug Laboratories, Mumbai), Panazep 12.5mg Tablet (Sun Pharma Pvt. Ltd, Chennai, India). KBr used to formulate standard and sample pellets was of spectroscopic grade.

 

Instrumentation and analytical conditions:

Equipment:

The FT-IR spectrophotometer used was Shimadzu model IR Affinity-1, which allows spectral digitalization for obtaining electronic files of the analysis. The region comprised in the spectral analysis was from 4000 to 400cm-1. After obtaining the IR spectrum and with the assistance of the IR solution software, quantitative analysis is carried out in the spectral region 929 and 1342cm-1, related to a ring NH and NO2 bands of Clonazepam and Paroxetine respectively, and these bands had its height analyzed in terms of absorbance.

 

Obtaining of Analytical curve:

Equivalent amounts of 0.1, 0.14, 0.18, 0.22 and 0.26mg of Clonazepam and 2.5, 3.5, 4.5, 5.5 and 6.5mg of Paroxetine pure drug (previously diluted in potassium bromide) were taken and diluted with sufficient amount of potassium bromide to obtain 150mg pellets. The powder were mixed and ground until obtaining a homogeneous mixture. Thus, this mixture was compressed in a mechanical die press with 10 ton pressure for 2min to obtain translucent pellets, through which the beam of the spectrometer can pass.

 

Determination of Clonazepam and Paroxetine in combined dosage form:

Preparation of standard pellets:

Amount of powder equivalent to 0.26mg of Clonazepam and 6.5mg of Paroxetine were taken and diluted with sufficient amount of potassium bromide to obtain 150mg pellets.  The determinations were performed in triplicate.

 

Preparation of sample pellets:

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 25mg of Paroxetine mixed with 370mg of potassium bromide. Amount of powder equivalent to 0.26mg of Clonazepam and 6.5mg of Paroxetine were taken and make the pellet. The determinations were performed in triplicate.

 

Calculation of the amount of active pharmaceutical ingredient in the sample [5]

 

C sample = (A sample / A standard) x C standard

Amount of API = (C sample x equivalent weight)/ C standard

 

Percentage purity = (Amount of API/ Label claim) x 100

Method validation:

The method was validated by determining the following parameters: linearity, accuracy, detection limit and quantification limit.

 

Linearity:

With the intension of validate the method, five concentration of standard Clonazepam (0.10, 0.14, 0.18, 0.22 and 0.26mg) and Paroxetine (2.5, 3.5, 4.5, 5.5 and 6.5mg) were used. Linearity was evaluated by linear regression analysis.

 

Accuracy:

Accuracy was attained via the recovery assay, in which known quantity of pure drugs was added to known quantity of the sample. The recovery was performed in the three levels, 80%, 100% and 120%, and the pellets were prepared in three replicate.

 

Precision:

The precision of the method was evaluated in two requisites: repeatability and intermediate precision. Repeatability (intra-day) was studied by the performance of three determinations of the sample in a concentration 0.26mg Clonazepam and 6.5mg Paroxetine per pellet, all in the same day and identical working conditions. Intermediate precision (inter-assay) was assessed by performing the assay in three different days under the same experimental conditions. At the end of test, the percentage relative standard deviation (%RSD) values of the determinations were analysed.

 

Detection and quantification limit

 

The detection (LOD) and quantification (LOQ) limits were calculated based on the intercept standard deviation and the curve slope.

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

 

RESULT AND DISCUSSION:

The FT-IR spectrum of pure Clonazepam and Paroxetine mixture by KBr pellet method is given in Figure 1. The compound exhibited strong sharp signal 1340cm-1 (CNZ) and 929cm-1 (PRX). Which is due to the absorption of ring NH and NO2 group of CNZ and PRX respectively and this feature is taken for the quantitative analysis.

 

A calibration has been carried out for CNZ and PRX using known quantities of standard as mentioned in the experimental section. The results revealed that the drug followed Beer-   Lamberts law in the concentration range studied (0.1mg - 0.26mg for CNZ and 2.5mg- 6.5mg for PRX) (Table 1).

 

Table 1: Assay

 

Amount of drug in one tablet

% Purity

%RSD

 

CNZ

0.501

100.2

 

1.020

0.505

101.0

0.496

99.24

 

PRX

6.06

93.6

 

1.022

 

6.40

98.4

6.44

99.04

 

 

Figure 1: FT-IR spectrum of standard

 

 

Figure 2: FT-IR spectrum of formulation

 

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

Drug

Theoretical % Target level

Amount added

(mg)

Amount recovered

(mg)

% Recovery

% RSD

 

 

CNZ

 

80

0.208

0.503

100.6

0.11

0.502

100.4

0.503

100.6

100

0.26

0.500

100.0

0.39

0.504

100.8

0.502

100.4

120

0.312

0.5

100.0

0.30

0.501

100.2

0.503

100.6

 

 

PRX

 

80

5.2

13.070

104.56

0.98

12.823

102.58

12.904

103.23

100

6.5

12.498

99.98

1.22

12.300

98.4

12.201

97.61

120

7.8

12.498

99.98

0.29

12.481

99.84

12.427

99.42

 

 

 

The method was applied to formulation and the spectrum was recorded for Panazep 12.5mg tablet (Figure 2). From the spectra it was observed that the peak 1340cm-1 and 929cm-1 is free from interferences from other compounds present in the tablet (excipients).

 

Table 3: Precision study

Drug

Amount  (mg)

Intraday

Interday

 

 

CNZ

 

 

 

0.26

% content

% RSD

% content

% RSD

99.6

 

0.72

101.0

 

0.91

01.0

99.24

100.02

100.5

 

PRX

 

6.5

101.2

 

1.31

98.4

 

1.35

102.4

99.04

99.76

101.0

 

CONCLUSION:

The results showed that the Fourier Transform Infrared method for the Clonazepam and Paroxetine in combined dosage form quantitation presented good linearity, precision and accuracy in the range of concentrations from 0.1 to 0.26mg/pellet for Clonazepam and 2.5 to 6.5mg/pellet for Paroxetine. Therefore, it is a method interchangeable with other methods already described for the same purpose and can be used in routine tests for quality control of a pharmaceutical industry. Furthermore, it has some advantages over other methods described in the literature for, and the main one is that do not use organic solvents, which contributes to the non-generation of this type of residue, contributing to minimize the environmental impact of pharmaceutical industries. In addition, this study opens the possibility of applying the FT-IR spectroscopy to the quantification of other drugs.

 

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.inventi.in/Article/ppaqa/333/12.aspx

5.     Eliane Gandolpho Totoli, Herida Regina Nunes Salgado. Development and validation of the quantitative analysis of Ampicillin Sodium in powder for injection by Fourier-transform Infrared Spectroscopy (FT-IR). Physical Chemistry 2012, 2(6): 103-108

 

 

 

 

 

Received on 29.10.2019                    Modified on 13.12.2019

Accepted on 06.01.2020                   ŠAJRC All right reserved

Asian J. Research Chem. 2020; 13(1): 12-14.

DOI: 10.5958/0974-4150.2020.00003.6