INTRODUCTION:

Escitalopram oxalate is chemically (1S)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-2-benzofuran-5-carbonitrile¹ shown in Figure 1. Escitalopram oxalate is a selective serotonin reuptake inhibitor (SSRI). The antidepressant, anti obsessive-compulsive, and antibulimic actions of escitalopram are presumed to be linked to its inhibition of CNS neuronal uptake of serotonin. Escitalopram blocks the reuptake of serotonin at the serotonin reuptake pump of the neuronal membrane, enhancing the actions of serotonin on 5HT autoreceptors.

SSRIs bind with significantly less affinity to histamine, acetylcholine, and norepinephrine receptors than tricyclic antidepressant drugs². Clonazepam is chemically 5-(2-chlorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin 2-one CLNZ is a benzodiazepine derivative. It is a highly potent anticonvulsant, amnesic and anxiolytic. Today’s pharmaceutical industries are looking for new ways to cut cost and shorten time for development of drugs while at the same time improving the quality of their products and analytical laboratories are not exception in this trend. High performance liquid chromatography (HPLC) is a well-established reliable technique used in controlling the quality and consistency of active pharmaceutical ingredients (API’s) and dosage forms^3,4,5

Literature review reveals that there were spectrophotometric^6,7HPTLC⁸ very few RP-HPLC^9,10,11and stability indicating^12,13available. Every RP-HPLC method have their own limitations like one method shows longer retention time of 6.8 and 12.7min. for ESC & CLNZ. The regular use of tri ethyl amine as a part of mobile phase can dissolve the silica under line and may reduce the column life. After repeating the reported methods some are giving unstable validations parameters and there is an ambiguity in optimized chromatogram. So keeping the above points in view it is necessary to develop and validate a reliable assay method for the routine analysis of Escitalopram and Clonazepam in combined dosage form as per the ICH Q2b guidelines¹⁴.

Figure 1. Chemical Structure of Escitalopram (A), clonazepam (B)

MATERIALS AND METHODS:

Reagents and materials:

Pure Escitalopram and Clonazepam were received from Shasun Pharma Limited, Pondichery, India.Tablets were purchased from local pharmacy, Hyderabad, India. Acetonitrile (HPLC grade) was obtained from Sigma-Aldrich, Hyderabad, India. Water (HPLC grade) was obtained from Merck.

Chromatographic equipment and conditions:

The development and validation was performed on HPLC system CYBERLAB (Salo Terrace, Millburry, USA). The chromatographic columns used for separation were Waters symmetry C18 (150 x 4.6 id) 3.5μm. The mobile phase utilized for the separation of both the drugs was phosphate buffer: acetonitrile in the ratio of 55:45 at pH 5.8. Column temperature was maintained at 30⁰C. Validation study, force degradation and solubility study were carried out using same optimised condition with suitable preparation of standard and sample solutions. Column temperature was maintained at 30⁰C.

Escitalopram and Clonazepam standard solution preparations:

Standard solution of escitalopram and clonazepam and were prepared separately by dissolving 100mg of each drug in 10mL acetonitrile and volume was made up to 100ml with water to achieve the primary concentration 1000µg/mL Further dilution was made by withdrawing the 10 mL of aliquot and volume was made up to 100 mL to achieve the final concentration of 100µg/mL

Preparation of working standard solution:

Working standard solutions were prepared by withdrawing 1mL of the stock solution from the escitalopram primary standard solution and 0.8mL aliquot from the Clonazepam primary stock solution and both aliquot were transferred in a single 10mL volumetric flask. The volume was made up to 10mL. Therefore the working standard solution contains 10µg/mL of escitalopram and 8µg/mL of Clonazepam.

Analysis of tablet dosage form:

Fixed dose combination tablet (Nexito Forte) contains 10mg of ESC and 0.5mg of CLNZ were analysed using optimised method. The tablets were weighed and finely powdered, accurately weighed portion of the powder equivalent to 100mg of escitalopram and 5mg of Clonazepam was transferred to 100ml of volumetric flask, followed by the addition of 25ml of mobile phase and subjected to sonication for 15minutes. The resulted solution was filtered through a 0.45µm membrane filter and diluted to the volume with mobile phase. An aliquot of 0.5ml from the above solution was transferred to 10 ml of volumetric flask and the volume was made up to mark with mobile phase to obtained a solution of 50ug/ml of Escitalopram and 2.5ug/ml of Clonazepam respectively.

Validation:

Accuracy:

Accuracy of the present method was determined¹⁵ by recovery study. Here fixed dose formulation was kept constant and standard escitalopram and clonazepam was spiked at three different concentration (80, 100 and 120 µg/ml) level, to obtained the concentration of ESC-50µg and CLNZ-2.5µg (80%), ESC-60µg and CLNZ-3.0µg (100%), ESC-70µg and CLNZ-3.5µg (120%) respectively.

Precision:

The intra- and inter-day precision was determined¹⁶ by analysing for six times on same day (intra-day study) and repeated on the second day (inter-day study). The chromatograms were recorded. The peak area and retention time of both the drugs under study was determined and relative standard deviation (RSD) was calculated.

Linearity:

Working standard solutions of escitalopram and clonazepam were prepared as describe earlier, aliquot from these solution was diluted with mobile phase in different concentrations from 1-200µg/ml of escitalopram and 1.5-150µg/ml of clonazepam. Calibration curve plotted for both the drug under study considering concentration versus peak area, obtained data was subjected to regression analysis.

System Suitability:

To verify that the analytical system is working properly and can give accurate and precise results. It was carried out¹⁷ by injecting the standard drugs of escitalopram and clonazepam six times. The RSD of the parameters like theoretical plates, peak area, retention time and asymmetric factor were calculated.

Specificity:

The specificity study¹⁸ was done to check the interference of extraneous components for that a solution containing a mixture of tablet and standard was prepared using sample preparation procedure and injected into the system, to evaluate possible interfering peaks.

Robustness:

Three sample solution was prepared and analysed under established condition¹⁹ by varying the analytical conditions like flow rate and detection wavelength at three different levels. One factor was changed at one time to estimate the effect. The content of the escitalopram and clonazepam was determined along with other factors like retention time, tailing factor and peak area.

Detection and Quantitation limit:

Standard solutions of escitalopram and clonazepam was prepared by sequential dilution and injected into the chromatographic system in decreasing order of concentration in the range of 1-10µg/ml of Escitalopram and 0.1-5µg/ml of Clonazepam.

Force degradation study of escitalopram and clonazepam:

Degradation study²⁰ was carried out using different ICH prescribed stress condition such as acidic, basic, oxidative, thermal and photolytic stresses. All study was carried using stock solution having escitalopram of 50 µg/ml and 2.5µg/ml of clonazepam. Individual drugs was exposed to all stressed conditions to confirm the assignment peaks observed due to degradation of ESC and CLNZ in the chromatogram of mixture of both the drugs.

Acid degradation Study:

The acid degradation study was performed in environmental test chamber (Acamus Technologies, India) at 60⁰C and 75% relative humidity using 1M HCL. 1 ml of stock solution was taken in 10 ml of volumetric flask, 1ml of 1M HCL was added to the flask, kept in environmental test chamber for 26 hour. After the stress period solution was neutralized using 1M NaOH and make up the volume with mobile phase.

Base degradation study:

Base degradation study was performed at 60⁰C and 58% relative humidity using same environmental chamber. 1 ml of stock solution was taken in 10ml volumetric flask mixed with IM 1ml of 1M NaOH for 26 hr. After the suitable stress period the solution was neutralized with I M HCL and make up the volume with mobile phase.

Oxidative degradation study:

The above study was performed in versatile environmental chamber at 40⁰C, 75% relative humidity using 6% H₂O₂. For this purpose 1ml of stock solution was taken in 10ml volumetric flask and 1ml of 6% H₂O₂ was added in to flask and kept at 60⁰C for 26 hr, finally make up the volume up to mark with mobile phase.

Thermal degradation study:

This study also has been carried out in environmental chamber at 40⁰C, 75% relative humidity in oven at 105⁰C, 1ml of stock solution was taken in 10ml volumetric flask and kept in chamber for 144 hr. and for dry heat thermolysis 1mg of dry drug in solid form was placed in oven at 110⁰C for 2 days.

Photo degradation study:

This study was carried out in sunlight (60000- 70000 lux) during day time and in U.V light at 254 nm for the period of 48 hr. 1ml of stock solution was taken in 10ml volumetric flask and make up the volume up to the mark with mobile phase was used for the study.

RESULT AND DISCUSSION:

Method development and Optimisation:

Before selecting the condition for optimization number of preliminary trials were conducted with different combination of solvents, buffer pH, flow rate, temperature, and columns in order to check the retention time, resolution, peak shape and other chromatographic parameters. All mobile phase used in optimization procedure were prepared by mixing different buffer system with the organic solvents. Several solvents (methanol, acetonitrile), buffers (ammonium acetate pH 5, orthophosphoric acid pH 3.5, 5, potassium dihydrogen orthophosphate, dipotassium hydrogen ortho phosphate pH 5.8, 6.8 )used in different volume ratio.analytical column was initially tested : Waters symmetry C18 (150 x 4.6 id x 3.5 μm particle size) with U.V detection at 231nm was used. The temperature of the column was varied between 20⁰C and 40⁰C. The samples were initially analysed using a mobile phase consisting of methanol: water (50: 50 v/v) p^H-4 at a flow rate of 1 ml/ min and UV detection at 231 nm. Under this condition only one drug was eluted. Methanol: water (70:30) of p^H-3 showed peaks were not well separated. Water(p^H-7): methanol: acetonitrile (40:40:20) showed separation of one peak. Phosphate buffer (p^H-3.5): methanol: acetonitrile (40:40:20) showed asymmetry factor beyond the limits and longer retention time. Phosphate buffer(p^H-5): methanol: acetonitrile (40:50:10) showed lack of theoretical plates and longer retention time. Phosphate buffer: methanol: acetonitrile (40:30:30) showed lack of theoretical plates with poor resolution. Buffer (ammonium acetate p^H-5) and acetonitrile (60:40) showed longer retention time with high resolution. mobile phase (A) contains KH₂PO₄+K₂HPO₄p^H-6.8and acetonitrile(B) (A: B 55:45 v/v). This mobile phase showed less tailing factor, less theoretical plates and longer retention time for both drugs. Further optimization was accomplished by investigating various pH. Finally a selected mobile phase consisted of potassium dihydrogen orthophosphate and acetonitrile in the volume ratio of 55:45(v/v) with apparent pH 5.8, 10 µl injection volume and UV detection at 231nm was found optimal to obtain good elution sensitivity, excellent peak shape with proper resolution. Under this condition adopted ESC and CLNZ were fully separated within 2.010minute retention time for ESC and 4.857 minute for CLNZ with a symmetrical and good peak shape shown in figure 2. This condition was adopted for subsequent analysis. The developed method was found to be specific and validated as per ICH guidelines.

Figure 2. Optimised chromatogram of ESC and CLNZ

Method Validation:

The accuracy of the method was evaluated by the determination of recovery of spiked pure drugs at three levels, 80%, 100% and 120%. The recovery studies were carried out three times and the %. Escitalopram mean recovery was 98.44 and 101.01 for clonazepam indicates the accuracy of the method. Validation results for accuracy are summarized in Table-1.

In intra-day precision study the relative standard deviation of 6 determinations of Escitalopram and Clonazepam for intraday precision was found to be within the acceptance criteria of less than 2.0%. which proves that the developed method was precise. In the study of linearity the linear correlation was found between the concentration and peak area in a specific range. The regression analysis data was presented in table 2.

The regression coefficient R² was found 0.998 for both ESC and CLNZ, indicates the linearity of the optimised method. The result of system suitability study shows that % RSD of several parameters like theoretical plate, retention time, peak area, asymmetric factor are within limit i.e. less than 2, results are shown in Table-3, which indicates the suitability of the analytical system.

In the study of specificity no peak were detected in the retention time corresponding to both ESC and CLNZ peak which clearly indicates there is no interference of excipients in the formulation which supports the specificity of the method. Statistical analysis of robustness study showed that there was no significant difference between the results obtain by little variation of some chromatographic conditions. The method showed robust on changing in flow rate from 0.8, 1, 1.2 ml/min, detection wavelength of 229, 231 and 233. The details of the study results were shown in table 4.

Table-1 Recovery study

Concentration of spiked level	Amount added (µg/ml)		Amount found(µg/ml)		% Recovery		Mean recovery
	ESC	CLNZ	ESC	CLNZ	ESC	CLNZ	ESC	CLNZ
80	50	2.5	49.64	2.55	99.2	101.9	98.4	101.01
100	60	3	58.80	3.0	98	100.04
120	70	3.5	68.64	3.5	98.06	100.6

Table 2: Summary of Validation Parameters

Validation parameters	Escitalopram	Clonazepam
Linearity	0.998	0.998
Precision (%RSD) Intraday (%)	0.99	1.42
Recovery (%)	98.44	101.01
LOD (µg/ml)	7.63	0.099
LOQ (µg/ml)	23.130	0.301

% RSD = percent of relative standard division, LOD= limit of detection, LOQ=limit of quantitation.

Table 3. System Suitability Study

Parameters	Escitalopram ±% RSD	Clonazepam ± %RSD
Retention time	2.009±0.042	4.86±0.057
Theoretical plate	2145.66±0.53	5023.66±0.50
Tailing factor	1.74±0.36	1.32±0.38
Peak area	4874.311±0.325	222.549±0.25

% RSD = percent of relative standard division

Table 4. Robustness study

Parameters	Escitalopram		Clonazepam
Parameters	T_R*	% RSD	T_R*	% RSD
Flow Rate (± 0.2 ml)	2.03	0.24	4.9	0.94
Wavelength (± 1nm)	2.003	1.11	4.8	0.92

* T_R = average of three retention time

The detection of limit was found to be 1.63µg/ml and 0.099µg/ml. Limit of quantitation of both the drug was found to be 3.130µg/ml for ESC and 0.301µg/ml for CLNZ presented in table of summary of validation parameters, indicates a very low level of ESC and CLNZ that can be easily detectable and can quantify using present development method, it also indicates the sensitivity of the method. To study the applicability of the developed method fixed dose combination tablet NEXITO FORTE was taken, which contains 10mg of escitalopram and 0.5mg of clonazepam were analysed. The amount of both the drug was found very close to the labelled claim amount. Result was presented in table 5 and the chromatogram was shown in Figure 3.

Table 5: Analysis of marketed formulation

Formulation	Label claim(mg)	Amount found(mg)	% of assay
Nexito Forte	Escitalopram 10mg	9.98	99.7
Nexito Forte	Clonazepam 0.5mg	0.4965	99.6

The % of assay of 99.7 for ESC and 99.6 for CLNZ, which indicates the efficiency of the assay procedure for the determination ESC and CLNZ Forced degradation study shows that the both the drugs were degraded under acidic condition and ESC was degraded in basic condition with the rise of several degradation peaks. The drugs were stable under oxidative, thermal and photolytic conditions. Stressed sample reveals that major degradation occurs under acidic stressed condition which shown 14.7% degradation of ESC and 13.6% of CLNZ followed by alkaline stressed condition shown 10.7% of ESC respectively CLNZ remain intact. In the above two degradation conditions both the drugs are well separated and very specific from the degradation peaks were shown in chromatogram figure 5 and Table 6. Which shows the specificity of the method.

Figure 3 Assay of marketed formulation

CONCLUSION:

Based on the above observation data of the present research work related to new method development of Escitalopram and Clonazepam was found satisfactory, simple, precise, accurate with good resolution, shorter retention time and among the other degradation products both Escitalopram and Clonazepam were well separated with all accurate results. In the present developed method the acceptable validation parameters makes this method of analysis more acceptable for the routine analysis in quality control department in industries. approved testing laboratories, bio-pharmaceutical and bio-equivalence studies and in clinical pharmacokinetic studies.

ACKNOWLEDGEMENT:

Authors are like to thank Shasun Pharma Limited, Pondichery, India for fro providing the ESCITALOPRAM and CLONAZEPAM as a gift sample. Authors also extend their thanks to management, Vaageswari college of Pharmacy, Karimnagar, A.P, India for providing laboratory and instrumental facility.

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Received on 11.08.2019 Accepted on 16.09.2019

Asian J. Pharm. Ana. 2019; 9(4):193-198..

DOI: 10.5958/2231-5675.2019.00032.2

Stress condition	Escitalopram			Clonazepam
Acidic	Area	%Assay	% Degradation	Area	%Assay	% Degradation
Acidic	3945.107	85	14.7	2003.2	86	13.6
Alkaline	3803.03	89	10.7	2568.1	99.6	-----
Photolytic	4784.60	99.7	----	2571.4	99.65	-----
Thermal	4685.32	99.6	-----	2565	99.6	-----
Oxidative	4682.75	99.7	-----	2574	99.60	-----