INTRODUCTION:

Clonazepam (CLO; 5 – (o – chlorophenyl)- 1,3 dihydro – 7 – nitro – 2 H – 1,4 – benzodizepin (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 prescribed for the treatment of anxiety and seizure disorders.

Mechanism of action involves Allosteric interactions between central anxiolytic drug receptors and gamma-minobutyric 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’- flurophenyl)-3S [3’,4’–methylenedioxyphenoxy) methyl] Piperidine hydrochloride] (Figure 1) is a selective serotonin (5 – hydroxy - tryptamine, 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. Mixed anxiety depression is gaining recognition as a separate diagnosis has been included in the 10th edition of the International Classification of Diseases. 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].

According to literature, Clonazepam and Paroxetine are official in IP, USP and BP when used individually [6], but the combination of Clonazepam and Paroxetine is not official in any Pharmacopoeia. Various analytical methods, such as Spectrophotometry, spectrofluorimetry, HPLC [7] and HPTLC [8], 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 [9,10]. However development and validation of Paroxetine and Clonazepam in Pharmaceutical formulations by 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.

Fig. 1 Structure of Paroxetine

Fig. 2 Structure of Clonazepam

MATERIALS AND METHODS:

Analytically pure PXT and CLZ were received as gift samples from Torrent Pharmaceutical, India). Marketed tablet formulations of Pari – CR Plus, IPCA Laboratories, India (Label claim 0.5 mg of PXT and 12.5 mg of CLZ) was procured from the local market. All the solvents and chemicals used were of analytical grade and issued from the college.

Preparation of standard solutions:

75 mg of standard PXT and 3 mg of CLZ were accurately weighed, transferred to two separate 10 ml volumetric flasks, dissolved in methanol and brought to volume with methanol to obtain a solution containing 7.5 mg of PXT and 3 mg of CLZ. From the aliquots of the stock solutions 0.1 ml solution was taken and diluted up to the mark with methanol in the two separate 10 ml volumetric flasks to get the concentration of 75 µg/ml of PXT and 3 µg/ml of CLZ.

Preparation of sample solution:

Twenty tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 20 tablets was transferred to 100.0 ml volumetric flask. Then to each flask about 50.0 ml of methanol was added and sonicated for 30 min, finally volume was made to the mark with methanol.

The extracts were filtered through Whatman paper No.1 and required dilutions were made with mobile phase to get final concentrations containing 75 µg/ml for Paroxetine HCl and 15 µg/ml for Clonazepam. The mobile phase was allowed to equilibrate with stationary phase until steady base line was obtained each (20 µl) volume of standard and sample solution were injected and chromatogram and peak areas were recorded as shown in figure 3.

Selection of chromatographic parameters:

The following chromatographic conditions were maintained throughout the method development.

Column: C₁₈Intersil, 250 × 4.6 mm column length

Particle size of packing: 5 µm

Mobile Phase : Acetonitrile: 0.1 % OPA (65: 35)

Detection wavelength : 239.65 nm

Flow rate : 0.8 ml/min

Temperature : Ambient

Sample size : 20 µl

METHOD VALIDATIION:

a) Accuracy:

The accuracy of an analytical method is the closeness of the test results obtained by that method to the true value for the sample. Accuracy of the proposed method was ascertained on the basis of recovery study performed by the standard addition method. The results of recovery studies were recorded.

b) Precision:

Precision of Associate in Nursing analytical methodology is that the degree of agreement among individual results once the tactic is applied repeatedly to multiple readings of a unvaried sample.

It is expressed as S.D. or R.S.D. of series of measurements. Repeatability, or intraday precision, was determined by performing three analyses at three concentrations on the same day.

c) Ruggedness:

The studies of ruggedness were carried out under three different conditions:

· Days

· Time

· Analyst

Different Interday study: The interday study was performed by applying the proposed method on sample of tablet on different days. The percent label claim were calculated using same formula as in analysis of tablet.

ii) Intraday study:

The intraday study was performed by applying the proposed method on same sample of tablet on same day at two hours interval. The percent label claim were calculated using same formula as in analysis of tablet.

iii) Different analyst:

The sample and standard solutions were prepared by different analyst and analysis was done by proposed method. The percent label claim were performed by using same formula as in analysis of tablet.

d) Linearity and range:

According to USP 80% to 120 % of test concentration were taken and dilution was done appropriately.

e) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ were calculable from the set of five standardisation curves wont to confirm methodology dimensionality.

LOD = 3.3 *σ /S and LOQ = 10 * σ/S

Where, σ = the standard deviation of y – intercept of regression lines, S = the slope of the calibration curves

Fig. 3: Chromatogram of Paroxetine and Clonazepam

Table 1. Multivariate analysis knowledge and outline of validation parameters for the projected technique

Parameters	PXT	CLZ
Detecting wavelength (nm)	239.65	271.23
Beer’s law limit (µg/ml)	75-375	3-15
Regerssion equation (y = mx+c)	y= 17.91x + 25.57	y = 68.40 x – 3.102
Slope	17.91	68.40
Intercept	25.57	3.102
Correlation coefficient(r²)	0.999	0.999
Retention time	4.766	7.666
Theorotical Plates	3149.8	3250.3
Tailing factor	1.444	1.3077
System precision 1. Intraday precision 2. Interday precision	99.96 100.16	100.02 100.80
Accuracy (% recovery)	101.61 ± 1.13	100.03 ± 1.15
LOD	2.99	0.1464
LOQ	9.0791	0.4438

CONCLUSION:

A validated HPLC method for the Simultaneous estimation of Paroxetine hydrochloride and Clonazepam in tablet was developed under experimental conditions described.

An essential analysis of projected methodology was performed by applied mathematics analysis.

Results of marketed formulation analysis shows % RSD values less than 2 % indicating reproducibility of the results.

ACKNOWLEDGEMENT:

We are thankful to Torrent Pharma Pvt. Ltd., Ahmedabad, Gujrat for providing the gifts of standard Clonazepam and Paroxetine hydrochloride. We are highly thankful to Vidyabharati College of Pharmacy, Amravati for providing the all facilities to carry out the work.

CONFLICT OF INTEREST:

The authors have no conflict of interest.

SYMBOLS AND ABBREVIATIONS:

PXT HCl	Paroxetine Hydrochloride
CLZ	Clonazepam
HPLC	High Performance Liquid Chromatography
OPA	Orthopthaladehyde
ICH	International Council for Harmonisation
μg/ml	Microgram per ml
μl	Microlitre
ml/min	Mililitre per minute
Nm	Nanometer
LOQ	Limit of Quantitation
LOD	Limit of Detection
Fig	Figure
SD	Standard Deviation
RSD	Relative Standard Deviation
%	Percentage
g/mol	Gram per mole
Λ_max	Maximum absorbance

REFERENCES:

1. The Indian Pharmacopoeia, The Indian Pharmacopoeia Commission, Ghaziabad, 6th edition 2010. Vol.-II: 1111-1114.

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3. Argrawal N, Joseph ER, Dubey NP, Durgbanshi A, Bose Devashish. Determination of Paroxetine in pharmaceutical preparation using HPLC with electrochemical detection. The Open Analytical Chemistry Journal. 2013; 7:1-5.

4. Athanasiou NV, Politou JA, Koupparis M, Spyropoulos J. Development and validation of an HPLC method, with fluorescence detection, for simultaneous determination of paroxetine and its metabolites in plasma. Journal of Liquid Chromatography and Related Technologies. 2007;30(11):1641-1655.

5. Jagawat T. A comparative study to assess the efficacy and safety of combination of capsules of paroxetine and clonazepam in comparison to paroxetine in patients suffering from co – morbid depression and anxiety. Delhi Psychiatry Journal. 2011;14(1):106 – 109.

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Received on 16.12.2019 Modified on 10.01.2020

Asian J. Research Chem. 2020; 13(2):113-116.

DOI: 10.5958/0974-4150.2020.00023.1