INTRODUCTION:

Escitalopram oxalate (ESC) is an antidepressant drug belonging to the category of selective serotonin reuptake inhibitors (SSRI). It acts by inhibiting sodium dependent serotonin transporter thereby blocking the reuptake of serotonin and making it available in the brain. It also acts as an antagonist of the histamine H1 receptor. Chemically it is (S)1-3[3-(dimethylamino) propyl]-1-(4-fluorophenyl)-1, 3-dihydro-5-iso benzofuran 5 carbonitrile oxalate; official in Indian Pharmacopeia¹. It is used as the first-line drug in depression and also used in the treatment of other psychiatry disorders like anxiety, phobia, obsessive-compulsive disorder (OCD), and eating disorders like Bulimia nervosa. It improves the feeling of well-being, increases energy levels, and decreases the nervousness of patients. Flupentixol dihydrochloride (FLU) is an antipsychotic drug, belonging to the category of thioxanthenes. It acts as strong antagonists of D1 and D2 dopamine receptors and also antagonizes alpha-adrenergic receptors and serotonin receptors.

Chemically, it is (z)-4-[3-(2-(trifluoromethyl)-9H thioxanthene-9-ylidene] propel]-1-piperazine ethanol dihydrochloride. It is official in British pharmacopoeia^2. It is highly water and methanol soluble. It is primarily used in schizophrenia mainly in people with poor compliance to medications and with frequent relapses, since it is a long-acting drug it is given through an injection route once every two to three weeks, thus improving compliance and preventing relapses in Schizophrenia patients. A Combination of ESC and FLU is available in the tablet dosage form (Rexipra tablets) (Fig.1).

Literature survey revealed that several methods (HPLC, UV) are developed for ESC and FLU in combined dosage from^3-9, but there have not been any researches on the HPTLC method for the estimation of these drugs (ESC, FLU) in a combined dosage form; even though researches are available in this method (HPTLC) for these drugs individually^10-12; hence an attempt to develop accurate and precise method like HPTLC for their routine estimation was done.

Fig.1: Chemical structure of ESC oxalate and FLU dihydrochloride

MATERIALS AND METHODS:

Instrumentation:

The samples were applied in the form of a band of width 6mm with a Camag 50 µL sample syringe (CAMAG Muttenz, Switzerland) using Camag Linomat V (Switzerland) sample applicator on a pre-coated silica gel aluminum plate 60F 254(10cmx10cm with 0.2 mm thickness, E.Merck, Germany). Application positions are at least 15 mm from the side and 10 mm from the bottom of the plates. Mobile phase components were mixed before use and the development chamber was left for saturation with mobile phase vapor for 20 mins before each run. Development of the plates was carried out by the ascending technique to a migration distance of 7 cm. then the plates were dried on hot plates. The plate was analyzed photometrically. All the analysis was carried out in a laboratory with temperature control (20-4ºC). All the drugs and chemicals were weighed on Shimadzu electronic balance (AX 200, Shimadzu Corp, and Japan)¹³.

Chemicals and Reagents:

Analytically pure ESC and FLU were supplied by Shangri-La industries (p), Sikkim respectively. All the chemicals used such as methanol and chloroform were analytical grade (AR) and they were purchased from Himedia laboratory Limited, Mumbai, and Avantor performance materials India Limited, Maharashtra.

Methods:

Separation using chloroform: methanol:

Different ratios of the chloroform: methanol as tried and from a ratio of (4:6 v/v) was selected because it gave good separation from the application position and solvent front.

Preparation of standard solution:

Accurately weighed 10 mg of ESC and 1 mg of FLU were taken and transferred into a 10 ml volumetric flask contains 1000 µg/ml of ESC and 100 µg/ml of FLU. Aliquots of standard solution were applied in the concentration range of 3-7 µg/spot of ESC and 0.3-0.7 µg/spot of FLU by applying 3-7µl prepared from standard solution. The chromatogram was shown in (Fig. 2).

Preparation of sample solution:

For analysis of tablet dosage form, twenty tablets, each containing 5 mg of ESC and 0.5 mg of FLU, were weighed and their average weight was calculated. The tablets were finely powdered and powder equivalent to 10mg of ESC and 1mg of FLU were accurately weighed and dissolved in 10 ml methanol. The solution was sonicated for 20 mins, filtered through the whatman no.41 filter paper and the residue was washed with methanol, this solution was further diluted with methanol to get the same concentration as that of the final standard solution. The chromatogram was shown in (Fig.3).

Method validation:

The developed method was validated as per the international conference on harmonization (ICH) guideline for linearity and range, specificity, precision, accuracy, limit of detection, and limit of quantification¹⁴.

Linearity and range:

A stock solution was prepared for both ESC and FLU they were serially diluted to yield five standard solutions. A volume of 3-7µl of each solution was applied to the HPTLC plates to deliver 3, 4, 5, 6, 7µg/spot of ESC and 0.3,0.4,0.5,0.6,0.7µg/spot of FLU. The plate was developed in a developing chamber previously saturated with the mobile phase for 20 mins.

Accuracy:

Accuracy of the method was determined by replicates (n=3) analysis, carried out using three solutions prepared by standard solution addition of pure active pharmaceutical ingredient at three different concentration levels 80 %, 100 % 120 % accuracy was calculated by comparing the difference between spiked value (theoretical value) and that actual found value.

Precision:

Precision was evaluated in terms of intraday, interday, and repeatability. Intraday precision was determined by analyzing sample solution of ESC and FLU at three levels covering low, medium, high concentration of the same day (n=3). Interday precision was determined by analyzing sample solution of ESC and FLU at three levels covering low, medium, high concentration over a period of 3 days (n=3). The peak areas obtained were used to calculate mean and RSD values. Repeatability of measurement of peak area was determined by analyzing 5 µg/spot of ESC and 0.5µg/spot of FLU in six replicates without changing the position of the plates.

Limit of detection and limit of quantification

Limit of detection (LOD) and Limit of quantification (LOQ) was calculated using the equations of LOD=3Sa/b and LOQ=10Sa/b, where, Sa= the standard deviation of the peak area of analyze of the drug, b= slope of the corresponding calibration curve.

Specificity:

The specificity was estimated by spiking commonly used excipients into a pre-weighed quantity of the drug. The chromatogram was taken by proper dilutions. Developed peak area and R_Fvalue of ESC and FLU were determined and the effect of the interfering compound was investigated.

RESULTS AND DISCUSSION:

Fixed experimental condition:

Stationary phase	:	Pre-coated silica gel 60 F254 on aluminum sheets
Mobile phase	:	Chloroform: methanol (4:6)
Chamber saturation	:	20 min
Migration distance	:	7cm
Band width	:	8mm
Slit dimension	:	6×0.3mm
Wavelength of scanning	:	254nm

Rf values:

Escitalopram oxalate : 0.24±0.02

Flupentixol dihydrochloride : 0.44±0.02

Method validation:

Linearity:

The calibration plots revealed a good linear relationship between area and concentration over the range of 3-7 µg/spot for ESC and 0.3-0.7µg/spot for FLU, respectively. Data for both ESC and FLU is presented in table 1. The standard curve for ESC and FLU are shown in Fig 4 and Fig 5.

Table 1: Linearity data for ESC and FLU

Parameters	ESC	FLU
Linearity range(µg/spot)	3-7	0.3-0.7
Correlation (r)	0.998	0.999
Slope	343.32	8034
Intercept	1959	1672

Fig 4: Linearity graph of ESC

Fig 5: Linearity graph of FLU

Accuracy:

Accuracy was calculated by comparing the difference between the spiked value (theoretical value) and the actual found value. Results are represented in the temperature of % recovery of the active pharmaceutical ingredients and data for both ESC and FLU is presented in table 2.

Table 2: Accuracy data for ESC and FLU

Level	% Recovery		% RSD
Level	ESC	FLU	ESC	FLU
80%	98.7	97.8	0.371	0.271
100%	102.7	100.3	0.480	0.301
120%	99.2	101.6	0.412	0.481

Precision:

Precision and intermediate precision results are shown in table 3. The method is precise and repeatable within acceptable limits.

Table 3 (a): Intraday precision data for ESC and FLU

Level	Concentration (µg/ml)		Peak area		% RSD
Level	ESC	FLU	ESC	FLU	ESC	FLU
I	4	0.4	3358.03 3356.35 3389.65	1600.3 1613.3 1628.9	0.556	0.88
II	5	0.5	3664.43 3682.03 3698.04	2329.3 2299.2 2356.6	0.461	1.23
III	6	0.6	4045.56 4120.01 4123.06	3120.6 3110.9 3150.6	1.07	0.661

Table 3(b): Interday precision data for ESC and FLU

Day	Concentration		Peak area		% RSD
	ESC	FLU	ESC	FLU	ESC	FLU
1	4	0.4	3358.3	1600.3	1.70	0.86
2			3256.9	1616.3
3			3356.4	1628.3
1	5	0.5	3664.4	2329.3	1.74	1.2
2			3698.3	2356.4
3			3484.3	2389.4
1	6	0.6	4045.5	3120.6	1.1	0.78
2			4112.3	3156.1
3			4020.3	3168.2

Limit of detection and limit of quantification:

The limit of detection was found to be 500 ng/spot for ESC and 50 ng/spot for FLU respectively. The limits of quantifications were found to be 1000 ng/spot for ESC and 100 ng/spot for FLU respectively.

Specificity:

The chromatogram of the solution, which was not spiked with ESC and FLU, did not show any spot, while the chromatogram of the solution of the tablet matrix spiked with ESC and FLU showed clear, compact, and well- separated peaks of ESC and FLU. Moreover, no other peaks were eluted besides the two active compounds. The method was therefore considered to be specific.

Analysis of formulation:

The Calibration curve was plotted using the peak area of standard vs concentration of the standard solution. The Peak area of the sample chromatogram was compared and the amount of drug in the formulation was calculated and shown in table 4.

Table 4: Analysis of formulation

Drug	Amount of drug (mg/ Tablet)		% Label claim	% RSD
Drug	Labelled	Estimated	% Label claim	% RSD
Escitalopram	5	4.8	96	0.521
Flupentixol	0.5	0.45	90	0.223

CONCLUSION:

The proposed HPTLC analytical method was validated according to ICH guidelines. The test results of method validation comply with the acceptance criteria. The method was found to be sensitive and specific for the quantification of ESC and FLU in pharmaceutical formulation. The proposed method can be used in detail stability studies, bioanalytical studies of ESC and FLU. The main advantage of the proposed method was its suitability and routine determination of these drugs in pharmaceutical formulations.

ACKNOWLEDGEMENT:

The authors are thankful to Shangri-La (P), Sikkim for providing the gift samples of standards ESC and FLU respectively.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Indian Pharmacopoeia, The Indian Pharmacopoeia Commission, India 2014; Vol-II, 1797.

2. British Pharmacopeia. H.M. Stationary Office, London 2012; Vol-I, 944.

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Received on 23.05.2021 Modified on 04.12.2021

Research J. Pharm. and Tech 2022; 15(11):5255-5259.

DOI: 10.52711/0974-360X.2022.00885