1. INTRODUCTION:

Flunarizine (FLU) is 1-[bis(4-fluorophenyl) methyl]-4-[(2E)-3- phenylprop-2-en-1-yl] piperazine. Structure¹shown in figure 1. Flunarizine is used in treatment of migraine.

Figure 1: Chemical structure of Domperidone

Domperidone (DOM) is 5-chloro-1-{1-[3-(2-oxo-2,3-dihydro-1H-1,3- benzodiazol-1-yl)propyl]piperidin-4-yl}-2,3- dihydro-1H-1,3-benzodiazol-2-one. Structure² shown in figure 2. Domperidone is used in treatment of antiemetic.

The goal of stability and related substance studies is to provide evidence regarding how a drug substance's or product's quality changes over time while being influenced by a number of environmental circumstances^3,4. Presence of impurities critically effect the stability and pharmacological action of pharmaceutical API and drug product.^5-8.An essential study for vaccine Formulation Development.UV Spectrophotometric methods (simultaneous equation methods/vierodts) and LC MS-MS are widely acceptable for simultaneous estimation of pharmaceutical combinations.Analytical quality by design [AQbD] and CCD help in regulatory compliance For RP-HPLC method development, stress testing or stability indicating methods.

Flunarizine (FLU) and Domperidone (DOM) are both available in market that can be used to treat migraine. Literature survey revealed that various analytical methods have been reported for estimation of individual drugs or in combination with other drugs including UV^9-11 and RP-HPLC^12-26. But there is no stability indicating analytical method has been reported for simultaneous estimation of Flunarizine and Domperidone using RP-HPLC. Hence it is worthwhile to develop and validate stability indicating analytical method for simultaneous estimation of Flunarizine and Domperidone in synthetic mixture by RP-HPLC.

2. MATERIAL AND METHOD:

2.1 Materials and instrumentation:

A standard sample of Flunarizine and Domperidone API was kindly gifted by Stallion Laboratories Private Limited, Ahmedabad. The method was developed using Water HPLC.

2.2 Optimized chromatographic condition:

Chromatographic separation was performed using isocratic elution mode with ACE Excel C18 (250mm × 4.6mm, 5µ) column and mobile phase used the composition of 10mM Potassium Dihydrogen Phosphate buffer: Acetonitrile: Methanol (40:30:30%v/v). Injection volume was set to 10µl. The column oven temperature was set to 40ºC. The flow rate was set to be 1.0ml/min. The detection wavelength for the UV detector was selected to 220nm.

3. PREPARATION OF SOLUTIONS:

3.1 Selection of Solvents:

Based on the solubility study, HPLC Grade methanol was selected as the solvent for dissolving FLU and DOM.

3.2 Preparation of Buffer solution:

Dissolve 1.36g of Potassium dihydrogen phosphate in 1000mL of water and adjust pH to 4.5.

3.3 Preparation of Mobile Phase:

Mix Buffer having 4.5pH, Acetonitrile and Methanol in the ratio of 40:30:30% v/v and filter through 0.44µm membrane filter paper.

3.4 Preparation of Standard solution of Flunarizine and Domperidone (500:1000μg/mL):

Transfer 50mg of Flunarizine and 100mg of Domperidone to a 100ml volumetric flask, add 70% of the methanol, sonicate to dissolve, then add the remaining methanol up to the mark, and mix well. Flunarizine concentration will be 500µg/ml and Domperidone concentration will be 1000µg/ml in the resulting solution. Take 1 ml from above stock solution in 10ml of volumetric flask and dilute with mobile phase. Flunarizine concentration will be 50µg/ml and Domperidone concentration will be 100µg/ml in the resulting solution.

4. FORCED DEGRADATION STUDIES:

Acid hydrolysis was carried out using 1.0N HCl for 12 hours and it was then neutralized using 1.0N NaOH. Basic hydrolysis was carried out using 1.0N NaOH for 12 hours, which was then neutralized with 1.0N HCl. then mixture was made upto the mark with diluent to attain final concentration and filter it. Oxidative hydrolysis was carried out using of 3% H2O2 for 6 hours then it was made upto the mark with diluent to attain final concentration and filter it. Thermal degradation was carried out at 24ºC for 6hours. Photo degradation carried out under UV light for 72hours.

For HPLC analysis, all the degraded sample solutions were diluted with the mobile phase to obtain a final concentration of 50μg/ml of Flunarizine and 100μg/ml of Domperidone.

5. METHOD VALIDATION:

Analytical validation parameters for this proposed method were determined according to ICH Q2(R2) guideline.

5.1 Linearity:

By plotting calibration curve of peak area v/s concentration, Linearity has been determined over 5 different concentrations.

5.2 Specificity

The percentage interference was calculated after injecting a blank (mobile phase), placebo, standard FLU and DOM solution spiked with API, and test Solution.

5.3 Accuracy:

Accuracy performed by drug-to-drug spiking at three levels at 80%, 100% and 120%. For each level three sets were prepared and percentage recovery was calculated.

5.4 Precision:

Repeatability was performed under 6 replicates of the assay concentration and intermediate precision studies were carried out at Three replicates of three standard assay concentration of 80%, 100% and 120%. The result was recorded in terms of Relative Standard Deviation (RSD).

5.5 Limit of detection (LOD) and limit of quantitation (LOQ)

LOD and LOQ were found by the equation as per ICH guidelines.

LOD= 3.3 x σ / S and LOD= 10 x σ / S

5.6 Robustness:

Robustness was performed by deliberated change in flow rate (±0.1ml/min), change in Wavelength and column oven temperature (40±1˚C). Robustness was calculated in terms of RSD.

5.7 Assay of Tablet:

To determine the content of Flunarizine and Domperidone in a tablet dosage form that claims to contain 5 mg and 10mg per tablet respectively. A single tablet was precisely weighed and transferred to a 100ml volumetric flask. 70ml of methanol was added and the mixture was sonicated. The solution was diluted with methanol and mixed the filtrate was sonicated. The solution was filtered through a 0.45µm membrane filter. 1ml of the filtrate was further diluted with the mobile phase to 10ml and mixed well. The resulting sample was then analyzed for assay.

6. RESULTS AND DISCUSSION:

6.1 Preliminary method development:

Preliminary studies were performed using different columns, different temperatures, various pH, and various combinations of mobile phase. From preliminary studies, it was found that 10mM Potassium Dihydrogen Phosphate buffer: Acetonitrile: Methanol (40:30:30 %v/v) composition and flow rate of 1.0mL/min with 40℃ column oven temperature shows good separation with acceptable suitability parameter. In optimized chromatographic condition Retention time of Domperidone was 4.4 min. and for Flunarizine 11.7min as mentioned in Figure 3.

Figure 3: Optimized Chromatogram of Standard Flunarizine and Domperidone.

Table 1: System Suitability Parameter of Optimized Condition

PARAMETERS	DOMPERIDONE	FLUNARIZINE
Retention time	3.4	9.7
Resolution	28.01	0.00
Theoretical Plates	16592	7859
Tailing Factor	1.3	1.7

6.3 Forced degradation study:

Conditions for degradation included acidic, basic, oxidative, thermal, and photolytic. The range of the observed percentage of degradation was 5.14-14.83%. It is mentioned in Figure 4 and results are shown in Table 2.

Table 2: Forced degradation summary

Degradation Condition	R.T (min)		%Degradation		Peak Purity Angle		Peak Purity Threshold
Name	FLU	DOM	FLU	DOM	FLU	DOM	FLU	DOM
Acid hydrolysis	9.7	3.4	8.4	11.7	0.181	0.101	0.227	0.275
Base hydrolysis	11.9	4.1	0.94	3.87	0.157	0.102	0.228	0.279
Oxidative hydrolysis	9.7	3.4	13.34	13.52	0.070	0.089	0.226	0.278
Thermal degradation	9.7	3.4	13.53	19.82	0.055	0.093	0.226	0.274
Photolytic degradation	9.7	3.4	19.21	14.56	0.050	0.083	0.224	0.260

6.4 METHOD VALIDATION:

6.4.1 Linearity:

Linear responses were found for Flunarizine in the concentration range of 25-75µg/mL and Domperidone in the concentration range of 50-150µg/mL. Figures 5 and 6 show the calibration curves for Flunarizine and Domperidone respectively.

6.4.2 Specificity:

It is proved that there is no any interference of excipient with the peak of Flunarizine and Domperidone by comparing the chromatogram of blank, mobile phase, and standard solution.

6.4.3 Accuracy:

Percentage recovery for Flunarizine was in the range of 98.89-101.84%, whereas the percent recovery for Domperidone was in range of 98.46-101.55% are shown in Table 3.

6.4.4 Precision:

Repeatability and intraday, interday precision for RP-HPLC method was measured in terms of RSD and was found to be less than 2. Thus, the method is precise. Results are shown in Table 3.

6.4.5 LOD and LOQ:

LOD and LOQ of Flunarizine and Domperidone were determined using average of slope and standard deviation of intercepts. LOD was found to be 0.22 µg/mL and 0.42µg/mL for FLU and DOM respectively. LOQ was found to be 0.68µg/mL and 1.29µg/mL for FLU and DOM respectively.

6.4.6 Robustness:

The method was found to be robust when different factors such as flow rate, temperature and mobile phase composition were deliberately changed. The relative standard deviation of peak area was less than 2 percent when the parameters were deliberately changed.

Table 4: Summary of Robustness

Parameter	Change	RSD	RSD
Parameter	Change	FLU	DOM
Flow Rate(ml/min)	0.9	0.25	0.21
	1.0	0.22	0.28
	1.1	0.65	0.32
Temperature	39	0.53	0.21
	40	0.27	0.34
	41	0.68	0.32
Wavelength	219	0.34	0.32
	220	0.26	0.51
	221	0.27	0.47

6.4.7 Assay of Tablet:

Tablet of Flunarizine and Domperidone containing 5 mg Flunarizine and 10 mg Domperidone was analyzed using the developed method, the results revealed that the Flunarizine concentration was 101.06% and the Domperidone concentration was 99.92%.

Table 5: Assay of Synthetic Mixture

Drug	Dose Strength	Concentration found	% Assay
FLU	5	4.88	101.06
DOM	10	9.92	99.92

Figure 7: Chromatogram of assay of Synthetic mixture

7. CONCLUSION:

The present work describes the development and validation of a stability indicating RPHPLC method for the detection of Flunarizine and Domperidone in tablet, which was successfully completed. Validation was carried out for Flunarizine and Domperidone as per ICH Q2(R2) guidelines. By RPHPLC, the accuracy of Flunarizine and Domperidone the fact that was discovered in the range of 98.89-101.84% and 98.46-101.55% it was discovered that precision was less than 2% in terms of RSD, with all other parameters falling within the ICH guidelines. Conditions for degradation included acidic, basic, oxidative, thermal, and photolytic. The technique is sufficient to distinguish between the peaks of active pharmaceutical ingredients (API) and degradants (generated during stress condition). The suggested stability indicating approach can therefore be used in pharmaceutical analysis for quality assurance and monitoring drug stability.

8. ACKNOWLEDGEMENT:

The authors are thankful to Stallion Laboratories Private Limited, Ahmedabad. for providing standard sample of Flunarizine and Domperidone.

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Received on 28.04.2024 Revised on 16.08.2024

Accepted on 02.11.2024 Published on 28.02.2025

Available online from March 04, 2025

Asian Journal of Pharmaceutical Analysis. 2025;15(1):25-30.

DOI: 10.52711/2231-5675.2025.00005

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

LEVEL	ACCURACY		PRECISION
	% Drug recovery		RSD
	% Drug recovery		Repeatability		Intra-day		Inter-day
	FLU	DOM	FLU	DOM	FLU	DOM	FLU	DOM
80	100.67	99.53	0.11	0.09	0.19	0.11	0.86	0.10
100	100.13	100.28			0.86	0.55	0.74	0.41
120	99.61	99.72			0.09	0.23	1.73	0.04