INTRODUCTION:

Escitalopram oxalate (ESI) is utilized in the treatment of depression and anxiety, functioning by restoring balance of serotonin, a substance naturally found in the brain. It is officially recognized in Indian and British pharmacopeia^1,2. Flupentixol HCI (FLU) are employed to the alleviate indication associated with schizophrenia and similar mental health conditions, and it is also officially recognized in IP and BP. Numerous. There are analytical technique for evaluating pharmaceutical product in the different formulations. A comprehensive literatures review has disclosed a range of analytical methods for estimating ESI, both single and combined with others drug. Similarly, various methods available for FLU determination both single and combined with other drugs.

Although there is a use spectroscopic method for escitalopram and flupentixol in combination dosage form^18-22 and UHPLC simultaneous analysis of ESI and FLU in combined dosage form and with other drugs reported in the literature^3-17, UHPLC for with other combinations^23-27none have encompassed complete validation according to ICH guidelines. In light of this, endeavours have been made to establish a novel UHPLC method for analysing escitalopram and flupentixol tablets.

EXPERIMENTAL:

Method Development:

Various mobile phases were experimented with, incorporating combinations of water, methanol, acetonitrile, and buffers in a different proportion. Ultimately, mobile phase comprising methanol and 0.1% trifluroacetic acid. (TFAA) Triethylamine (TEA) in a 65:35 v/v ratio was select and the pH adjust to 4.2. This selected mobile phase demonstrated excellent resolution and yielded peakes parameter deemed acceptable for both flupenthixol and escitalopram. System Suitability Studies were carried out, and Table 1 presents the calculated values for separation between the standard solutions in a terms of peak asymmetry, resolution and a number of theoretical plates. These results affirm the system's suitability for the simultaneous analysis of those drugs. A visual representation of a chromatogram from the standard solution is illustrated in figure.1.

The separation was successful in terms of apparatus and chromatographic conditions achieved using AGILET (1100) and 20µl sample injection loop and U.V. visible absorbance detector. The mobile phase mixture of methanol: 0.1 % TFAA (pH4.2 WITH TEA). The sample was deliverd at flow rate 0.7 ml/min with detection carried out 235 nanometer. To ensure purity, a mobile phase underwent filtration through a 0.2 µm membrane filtration and degassing. A 20 µl injection volume was used for every analysis, and the process was done at room temperature.

Fig. 1: Chromatogram of Flupenthixol (3.044 min), Esitalopram 4.118 min respectively.

Table 1: System Suitability Studies

Parameter	Method
Column	id 4.6 x100 mm length
Particle size packaging.	2.5mm
Stationary phase	C1₈(AGILENT)
Mobile Phase	Methano l 0.1 % Tri fluro acetic acid
Wavelength detection	235nm
Flow rate	0.7 ml/min
Temprature.	Ambient.
Size of sample.	20ml

Table 2: Recovery study of flupenthixol (FLU) and esitalopram (ESI):

Level of % Recovery	% Mean Recovery		% R.S.D.
Level of % Recovery	FLU	ESI	FLU	ESI
80	100.68	99.68	0.49	1.5
100	101.54	101.44	0.32	0.20
120	102.01	99.14	0.67	0.61

Preparation of Standard Solutions:

Weighed into a 100ml volumetric flask, 10mg of esitalopram and 0.5mg of flupenthixol were added, to with 100ml of mobile phase. Pipette out 0.2ml of resulting using mobile phase to dilute the solution to 10 ml in a 10ml volumetric flask.

Calibration Curve:

When a system's concentrations were gradually reduced to yield flupenthixol at 1 µg/ml and esitalopram at 100 µg/ml and 20 µg/ml, the system's linearity was evaluated. Construction of calibration curves was made possible by plotting peak area against concentration. Figs. 2 and 3 display the resulting calibration curves for flupenthixol (FLU) and esitalopram (ESI). Here are the formulas for the esitalopram regression lines.

y = 26.073x -73.821 R² = 0.9999, and y = 143.83x + 30.146 R² = 0.9996 for flupenthixol.

Figure-2: Calibration Curve of Esitalopram

Figure-3: Calibration Curve of Flupenthixol

Sample Solution Preparation:

The method was used to analyse a commercial sample, REXIPRA-5. Average weight of twenty tablet was found to be 18mg. To achieve homogeneity, the tablet was crushed and 18mg of the crushed tablets was dissolved in 100 mL of methanol and sonicated for a moment. of fifteen minutes. The drug was extracted in solution by cyclomixing for 5 min. A Millipore syringe filter (0.42mm) was used to filter the resulting solution. Using the developed method, the resulting clear solution was injected into UHPLC in duplicate. The method was found to be specific, with no interference from common tablet excipients such as lactose, starch, or sucrose.and so on. For each drug, via regression line equation the assay was calculated. Table 2 shows the percentage of individual drugs detected in the formulation. The result of the analysis show that an amount of a drug were good with formulations label claims.

Method Validation:

In accordance with the method validation and the ICH guidelines process involved the examination of validation parameters, they validate the method as per fixed guidelines [Linearity, Accuracy, Limit of detection (LOD), Limit of quantitation (LOQ), Specificity, Robustness]

Range and linearity:

The linearity of the formulation was tested at five concentration ranges: 1 to 5g/ml for flupenthixol and 20 to 100g/ml for esitalopram. Line of regression equations were determined as follows: for Esitalopram,

y = 26.073x - 73.821 (R² = 0.9999), and for Flupenthixol, y = 143.83x + 30.146 (R² = 0.9996).

These results establish a strong correlation between peak area and drug concentration across the specific concentration range.

Table No. 3: Precision Studies, Intraday and Interday(method precision)

Conc (µg/ml)	Esitalopram		Conc. (µg/ml)	Flupenthixol
	% RSD			% RSD
	Intraday	Interday		Intraday	Interday
20	1.47	0.48	1	0.99	0.95
40	0.03	0.07	2	0.68	0.75
60	0.09	0.39	3	0.30	0.28

Precision and accuracy:

The accuracy of the method was evaluated through recovery experiments. Conducted at 3 levels 80%, 100%, 120%. The resulting percentage recovery, as shown in Table No.2 fell within the acceptable range of 100 ± 2%, indicating the method's accuracy. Precision was demonstrated through both studies on intraday and interday variations. For In intraday studies, relative standard deviation (RSD) % values were calculated following three consecutive. In a single day, sample solution injections were performed. The standard and sample solutions were used in the interday variation studiesfrequently injected three days in a row. The resulting % RSD values were computed and shown in table no 3.

The obtained data, with % RSD couldn’t exceeding 1.5% signifies the precision of the new developed UHPLC method. Turning to the limit of detection (LOD), defined as the smallest analyte concentration yielding a measurable response, The formula employed to calculate the Limit of Detection (LOD) was The formula for calculating the Limit of Detection (LOD) was LOD = (3.3 x standard deviation)/Slope of the calibration curve. The LOD values for escitalopram and flupentixol were 0.018g/ml and 0.22g/ml, respectively. The Limit of Quantification (LOQ), that was the lowest analyte concentration that is a precisely quantifiable response, was determined using a specific formula.

The Limit of Quantification (LOQ) was calculated as LOQ = (10 x standard deviation)/Slope of the calibration curve. Flupenthixol and escitalopram LOQ values were calculated to be, respectively, 0.67 and 0.057g/ml.

Robustness:

Robustness was evaluated through deliberate, minor adjustments in the experimental procedures. Specifically, changes in wavelength, pH, and flow rate were intentionally introduced, and the subsequent effects were closely monitored. The method exhibited robustness, demonstrating its resilience to variations in wavelength, pH, and flow rate.

RESULT AND DISCUSSION:

Within the range of 20-100g/ml, the proposed method demonstrated simplicity and linearity. For the concentration for esitalopram and 1 to 5µg/ml for flupenthixol. Accuracy and precision were confirmed through recovery study, with %RSD not exceeding 1.5additionally, Flupenthixol LOD and LOQ were determined to be 0.22µg/ml and 0.67µg/ml, respectively, for flupentixol, and 0.018µg/ml and 0.057µg/ml, respectively, for escitalopram. These findings affirm the specificity and sensitivity for the method.

CONCLUSION:

In this research we established the simple, precise, accurate, specific and cost-effective development of Simultaneous Estimation of flupenthixol and esitalopram in combination dosage form by UHPLC. This method developed by using methanol and water as mobile phase. In this method development all parameters are carried according to ICH guidelines all the developed method produces a result in acceptable limit. So, this UHPLC method developed will be reliable for analysis of both raw material and formulations in pharmaceutical quality control.

ACKNOWLEDGEMENT:

The author would like to express gratitude to Concept Pharma Ltd., Aurangabad, India, for providing the working standards of flupenthixol and esitalopram. Special thanks go to Dr. S. B. Bhawar for offering guidance and support throughout the research.

ABBREVIATIONS:

RP-HPLC: Reverse Phase High performance liquid chromatography, UV: Ultraviolet spectroscopy, FLU: flupenthixol, ESI: esitalopram

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 16.12.2023 Modified on 04.03.2024

Research J. Pharm. and Tech 2024; 17(10):4991-4994.

DOI: 10.52711/0974-360X.2024.00767