Simultaneous Spectrophotometric Estimation of Paroxetine Hydrochlorides and Clonazepam in Bulk and Tablet Dosage Form

 

Sonali S. Gadge¹*, Madhuri D. Game², Vikrant L. Salode¹

 ¹P R Patil Institute of Pharmacy, Talegaon (SP), Dist - Wardha - 442202. (MS), India.

²Vidyabharati College of Pharmacy, Amravati – 444602 (MS), India.

 

ABSTRACT:

Two novel simple, accurate and precise spectrophotometric methods have been developed for quantitative estimation of Paroxetine hydrochloride and Clonazepam in the tablet dosage form. The standard stock solution was prepared by using the solvent methanol and further dilutions were carried out by using 0.1 N HCl. The method I is Vierodt’s Simultaneous method in which 291.60nm and 272.27nm was selected for estimation of Paroxetine and Clonazepam respectively, while method II is Absorbance Correction method, where 325 nm and 291nm was selected for estimation of Paroxetine hydrochloride and Clonazepam respectively. The calibration curve was plotted of the concentration range of 0.5-30μg/ml for both the drugs. Both the drugs obeyed Beer’s law in the concentration range 0.5-30μg/ml, correlation coefficient (r2<1). The precision of both the methods was found satisfactorily. The values of relative standards were not more than 2%. Both methods were validated statistically and recovery studies were carried out to confirm the accuracy. Commercial tablet formulation was successfully analyzed using the developed methods. Both these methods can be used for routine analysis for the estimation of Paroxetine hydrochloride and Clonazepam in the combined formulation. Due to the high sensitivity and simple sample preparation, the methods described can be used for undergraduate studies. Hence simple spectrophotometric methods have more advantages over sophisticated instrumental analysis such as HPLC. Hence these two simple and economical, instrumental methods always have a role in pharmaceutical analysis.

 

 

 

INTRODUCTION:

Clonazepam (CLO; 5 –(o – chlorophenyl )- 1,3 dihydro – 7 – nitro – 2 H – 1,4 – benzodiazepine (Figure 1 ) with prominent anticonvulsant, anxiolytic properties. It has been most effective in the treatment of typical and atypical absence, myoclonic, akinetic seizures, and infantile spasms. Co-administration of barbiturate may exacerbate the drowsiness caused by clonazepam. Clonazepam (CLZ) belongs to the drug class benzodiazepines. It is used in the treatment of anxiety and seizure disorders.

 

Mechanism of action involves Allosteric interactions between central anxiolytic drug receptors and gamma-aminobutyric acid (GABA) receptors heighten the consequences of neurotransmitter. As GABA is an inhibitory neurotransmitter, this results in increased inhibition of the ascending reticular activating system.^1,2

 

Paroxetine hydrochloride (PH) [(-)- Trans – 4R – (4’- fluorophenyl)-3S-[3’,4’ – methylenedioxyphenoxy) methyl] Piperidine hydrochloride ] (Figure 2) is a selective serotonin ( 5 – hydroxytryptamine, 5HT) reuptake inhibitor (SSRI) and potentiates 5 – HT in the CNS. PH is indicated for the treatment of major depressive disorder, social anxiety disorder, obsessive -compulsive disorder, panic disorder, generalized anxiety disorder, and posttraumatic stress disorder.^3,4

 

 

Depression and anxiety disorders are distinct illnesses that often coexist. Patients with combined depression and anxiety are more debilitated than patients with either condition alone. Currently, a fixed-dose combination of an antidepressant, such as Paroxetine, and an anti-anxiety drug, such as Clonazepam, is an available option for the treatment of co-morbid depression and anxiety.⁵

 

Clonazepam and Paroxetine are official in IP, BP and USP when used individually, but according to literature, the combination of Clonazepam and Paroxetine is not official in any Pharmacopoeia. Various analytical methods, such as spectrofluorimetry spectrophotometry, HPLC⁶ and HPTLC⁷, have been reported to detect Clonazepam and Paroxetine alone and in combination with other drugs in Pharmaceutical dosage forms. Spectrophotometric methods and the stability-indicating HPLC method have been reported for the estimation of Clonazepam and Paroxetine in combined pharmaceutical formulations.^8,9 However, development and validation of Paroxetine and Clonazepam in Pharmaceutical formulations by the method has not been yet reported.

 

Hence, this manuscript is the first to describe the development and validation of HPLC method as per the ICH guidelines ICH Q2 (R1) for the simultaneous estimation of Clonazepam and Paroxetine.

 

Figure1: Structure of Clonazepam

 

Figure 2: Structure of Paroxetine

 

MATERIALS AND METHODS:

The present work aimed to develop simple, precise, selective and economical instrumental spectrophotometric methods for the simultaneous estimation of Paroxetine hydrochloride and Clonazepam. Pure drugs of PXT HCl and CLZ were procured as a gift sample from Torrent Pharma Pvt. Ltd., Ahmedabad, Gujarat, India. For preparing solutions, A.R. grade methanol was issued from the college. PARI CR Plus, IPCA Lab (commercially available) containing 12.5mg of PXT HCl and 0.5mg Clonazepam per tablet, were randomly selected for the study and were procured from the market. The 0.1 N HCl solution was prepared in double-distilled water as per IP 1996 procedure. A Shimadzu UV/Vis 1601 double beam spectrophotometer with a fixed slit width (2 mm) and 1 cm matched quartz cells were used for all the spectral measurements.

 

Preparation of standard stock solution:

By dissolving 10mg of PXT HCl and CLZ respectively in 100ml methanol standard stock solutions (100μg/ml) of PXT HCl and CLZ were prepared separately. To obtain solutions of PXT HCl (25μg/ml) and CLZ (1 μg/ml), the standard stock solutions were diluted with 0.1 N HCl The resulting solutions were scanned in the range 200-400nm in 1 cm cells against solvent as blank for method I. The UV absorption overlay spectrum of PXT HCl and CLZ is depicted in the figure. From the overlain spectra the wavelengths 291.60nm (λ_max of PXT) and 272.27nm (λ_max of CLZ) were selected for Simultaneous equation method and for Absorbance correction method two wavelengths selected were 325 nm and 291nm.

 

Standard stock solutions of PXT HCl and CLZ were diluted with 0.1 N HCl to obtain a concentration range of 0.5 -30μg/ml and absorbances were measured at selected wavelengths. The concentration of drug against absorbance was plotted to obtain calibration curves and curves were found to be linear in the concentration range under study. The absorptivity values of PXT HCl at 291.60nm and 272.27nm were 10.26 and 14.156 while respective values for CLZ were 239.932 and 239.75. Amount of each drug was estimated by substituting the absorbance and absorptivity values in the following equations: C_PXT = A_2ay1 – A₁ay₂/ax₂ay₁- ax₁ay₂ and C_CLZ = A₁ax₂ – A₂ax₁/ax₂ay₁ – ax₁ay₂, where A₁ and A₂ are the absorbances of mixtures at 291.60 nm and 272.27nm. ax₁ is absorptivity value of PXT HCl at 291.60nm, ax₂ is absorptivity value of PXT HCl at 272.27nm, ay₁ is absorptivity value of PXT HCl at 291.60nm and ay₂ is absorptivity value of CLZ at 272.27nm and Qm = A₂/A₁, Qy = ay₂/ay₁ and Qx = ax₂/ax_1.

 

Figure 3: Overlain spectra of Paroxetine (25 µg/ml) and Clonazepam (1 µg/ml)

 

The main criteria for Absorbance correction method are based upon the determination of identity, concentration and absorptivity of the absorbing interferents and finally, it’s contribution is calculated from the total absorbance of the mixture. Based on this principle solution of PXT (37.5µg/ml) and CLZ (1.5µg/ml) were prepared separately by appropriate dilutions of standard stock solutions and scanned over the range of 200 -400 nm.

 

From the overlay spectrum (Figure 1) the wavelengths selected for estimation of drugs were 325 nm as detecting wavelength for Paroxetine HCl and 291 nm as detecting wavelength for Clonazepam (CLZ). Both the drugs obeyed Beer’s law individually and in the mixture within the concentration range of 5 – 30µg/ml for both drugs. The absorptivity values are recorded in Table 1. The sample absorbance, absorptivity and corrected absorbance were

 

determined and finally the concentration of each drug following equation C = A/A (1 %, 1 cm).

 

Figure 4: Overlain spectra of Paroxetine HCl (37.5 ug/ml) and Clonazepam (0.5ug/ml)

 

Amount of these drugs were calculated by using following equations:

A= abc

C_X = A1/ab

Cx = A1/ax1 *b

A2= A _PXT + A _CLZ

A2 = (ay2*cy*b) + (ax2*cx*b)

A2= (ay2*cy) + (ax2*cx)

Cy=[A2-(ax2*cx)]/ay2

 

Where A1, A2 absorbance of mixture at 291 nm (λ1) and 325 nm (λ2), ax1 and ax2 are absorptivities of PXT HCl at λ1 and λ2 respectively, ay1 and ay2 are absorptivities of CLZ at λ1 and λ2 respectively. Cx and Cy are concentrations of PXT HCl and CLZ respectively. Calibration curves of different concentrations at two wavelengths are plotted as follows

 

Figure 5: Plot of Beer – Lambert study for mixed standard at 325 nm and 291 nm

 

Twenty tablets were accurately weighed and the average weight was calculated, for the analysis of both PXT HCl and CLZ. Tablets were finely powdered and mixed thoroughly. Tablet powder quantity equivalent to 12.5 mg of PXT HCl was weighed accurately, dissolved in 100ml methanol and sonicated for 15 min. the solution was filtered through Whatman filter paper and transferred to the volumetric flask. For getting final concentration of about 12.5µg/ml of PXT HCl and 0.5 µg/ml of CLZ, aliquot portions of the filtrate were further diluted with 0.1 N HCl. Tablet sample solutions were prepared and analysed by scanning at a respective set of wavelengths and absorbance difference values were noted and the concentration of each drug was calculated from the respective calibration curve.

 

For method I, absorbances of tablet sample solutions were recorded at 239.932nm and 239.75nm and the concentration of each drug was obtained by using mentioned formulae. For method II, tablet sample solutions were prepared and analyzed at a respective wavelengths and the concentration of each drug was calculated from respective formulae.

 

VALIDATION:

Both the methods were validated statistically as per ICH/USP 16 guidelines for all the parameters like accuracy, linearity, precision, ruggedness and specificity. The recovery studies were carried out by standard addition method at three different levels (80, 100, 120% of test concentration), to study the accuracy of the proposed methods. To the pre-analyzed tablet powder, a known amount of drug was added and the percentage recovery was calculated.

 

Table 1: Recovery study data

Standard addition level (%)	% Recovery ± SD*
	Method I		Method II
	PXT HCl	CLZ	PXT HCl	CLZ
80	99.95 ± 0.41	98.47 ± 0.53	98.80 ± 0.37	98.99 ± 0.95
100	99.23 ± 0.48	98.95 ± 0.63	100.5 ± 0. 99	98.95 ± 0.98
120	99.93 ± 0.61	99.88 ± 0.47	99.20 ± 1.10	99.88 ± 1.15

Method I is Vierodt’s Simultaneous method and method II is Absorbance correction method*, mean of three determinations, SD is the standard deviation

 

Table 2: Summary of validation parameters

Parameters	Method I		Method II
	PXT HCl	CLZ	PXT HCl	CLZ
Linearity Range	± 20 % test conc.	± 20 % test conc.	± 20 % test conc.	± 20 % test conc.
Beer’s Law limit (μg/ml)	0.5 to 30	0.5 to 30	0.5 to 30	0.5 to 30
Precision, % Drug found ± SD (n=3)	99.47 ± 0.86	99.16 ± 1.066	99.97±0.96	99.97 ± 89
Ruggedness, % Label Claim (n=3)
Intraday	99.71	99.20	100.53	99.87
Interday	99.81	99.55	99.49	99.28
Different analyst	99.67	99.98	99.83	99.64

Method I is Vierodt’s Simultaneous method and method II is Absorbance correction method, (n=3) results are mean of three determinations, SD is the standard deviation.

 

Table 3: Results of tablet formulation analysis

Method	Drug	% Label Claim*	± SD*
I	PXT HCl	99.67	± 0.2888
	CLZ	99.98	± 0.7751
II	PXT HCl	99.87	± 0.8252
	CLZ	98.99	± 0.7636

Method I is Vierodt’s Simultaneous method and method II is Absorbance correction method, * results are mean of sample solutions, SD is the standard deviation.

 

RESULT AND DISCUSSION:

The results of recovery studies were calculated and presented in the table. Precision was studied by analyzing three replicates of sample solutions and concentrations were, precision was studied. By carrying out experiment at different conditions like intra-day, inter-day, and by the different analyst, ruggedness was carried out. By analyzing the standard drug and sample, the specificity of the method was ascertained. No interference of the excipients was observed present in the formulation. By observing the validation parameter, the methods described were found to be specific, accurate, precise, and economical and can be successfully applied to analyze commercially available tablets containing PXT HCl and CLZ. The results obtained were in good agreement with the labelled content.

 

CONCLUSION:

The analytical results of the pharmaceutical formulation by using the proposed methods are highly reliable and reproducible and are in good agreement with the label claim of the drug. The additives usually present in pharmaceutical formulations of the assayed samples did not interfere with the determination of PXT HCl and CLZ. These methods can be routinely used for the analysis of PXT HCl and CLZ in combined dosage form. Because of the high sensitivity and simple sample preparation, the methods described can be used for undergraduate studies. Hence simple spectrophotometric methods have more advantages over sophisticated instrumental analysis such as HPLC. As a consequence simple and economical, instrumental methods always have a role in pharmaceutical analysis.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Torrent Pharma Pvt. Ltd., Ahmedabad, Gujarat, India for providing drugs as gift samples for research. Also, we are thankful to P R Patil Institute of Pharmacy, Talegaon (SP), Wardha for providing necessary facilities for the research work.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest.

 

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Received on 28.05.2020           Modified on 02.07.2020

Accepted on 26.07.2020         © RJPT All right reserved