INTRODUCTION:

Ropinirole hydrochloride (Fig. 1) is an indole derivative chemically known as 4-[2-(dipropylamino) ethyl]-1,3-dihydro-2H-indol-2-one monohydrochloride which acts as a non-ergot D_2,D₃and D₄ dopamine receptor agonist which have actions that are similar to bromocriptine^1-2. It has been used in the treatment of Parkinson’s disease³. Review of literature revealed a few UV spectroscopic methods^1-2, a spectrofluorimetric method⁴, two visible spectrophotometric techniques^5,6, few liquid chromatographic techniques^7-12 along with capillary liquid chromatography¹³ and capillary zone electrophoresis¹⁴ for the assay of Ropinirole. The availability of an economical HPLC method with high sensitivity will be very useful for the determination of Ropinirole hydrochloride in pure and pharmaceutical formulations.

The aim of the study was to develop a simple, economical, precise and accurate reverse-phase HPLC method for the estimation of Ropinirole hydrochloride in bulk drug samples and pharmaceutical dosage forms.

ig. 1: Structure of Ropinirole hydrochloride

EXPERIMENTAL:

MATERIALS AND METHODS:

Ropinirole hydrochloride (standard) was obtained as a gift sample from Chemeca Drugs Private Limited, Visakhapatnam. Marketed dosage form of the drug (Ropark 2.0mg/tablet, Sun pharma Laboratories) was purchased from local pharmacy. Chemicals (AR grade) like orthophosphoric acid, triethanolamine, hydrochloric acid, sodium hydroxide and hydrogen peroxide (30% w/v) were procured from Thermo Fisher scientific India Pvt Ltd. HPLC grade methanol and water obtained from Qualigens was used in the study.

Instrumentation:

The study was performed on a Shimadzu HPLC system equipped with LC 20 AD binary pump and SPD M20A prominence photo diode array detector. Rheodyne manual injector with a fixed loop of 20µL was used. Chromatographic separation was achieved on a C8 Phenomenex column (250 × 4.6mm, 5µm) maintained at ambient conditions. The HPLC system was equipped with LC solutions software. The samples were weighed on a single pan electronic balance (Aczel CG 203L).

HPLC Conditions:

The mobile phase comprised of 0.1% orthophosphoric acid (pH adjusted to 2.61 with triethanolamine) and methanol in the ratio of 50:50 v/v. This was filtered before use through a 0.45µm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 1.2mL/min. The run time was set at 10.0min and the column temperature was ambient. The column was equilibrated for at least 30 min. with the mobile phase flowing through the system prior to the injection of the drug solution. The eluents were monitored at 248nm.

Preparation of Stock Solution and Working Standard Solutions:

A stock solution of the drug was prepared by dissolving 25.0mg of Ropinirole hydrochloride (standard) in methanol and the volume was made up to 25mL (1 mg/mL). 2mL of stock solution was taken in 20mL volumetric flask and thereafter made up to 20mL with diluent (0.1% orthophosphoric acid (pH 2.61): methanol, 50:50 v/v) to get a working standard of 100µg/mL. All the standard solutions were suitably diluted from this solution as and when required.

Preparation of Sample Solution:

Twenty tablets (Ropark 2.0mg) were weighed, powdered and powder equivalent to 25mg of the active ingredient was transferred to a volumetric flask and mixed with 15mL of methanol. The mixture was allowed to sonicate for 1 hour to ensure complete solubility of the drug, and then filtered through a 0.45 µm membrane filter and the solution was made up to 25 mL with methanol. 2.5mL of the solution was transferred to 25mL volumetric flask from stock solution and the volume was made up with diluent (100µg/mL). Further dilutions were made from this solution.

Method Optimization:

A 10µg/mL solution of Ropinirole hydrochloride was prepared from the working standard and injected into the HPLC where in a variety of mobile phases and compositions at varied flow rates were employed. After a series of trial and errors that were carried out, the best chromatographic parameters were achieved using 0.1% orthophosphoric acid (pH 2.61): methanol (50:50 v/v) at a flow rate of 1.2mL/min. The complete optimized conditions are given in table 1.

Validation:

The optimized method was validated as per ICH guidelines¹⁵ to check for precision, accuracy, LOD, LOQ, robustness and specificity.

System Suitability:

The system suitability test was performed at the beginning of each validation parameter by injecting a standard solution of Ropinirole hydrochloride in replicates (n=6) into the HPLC system. The system suitability parameters were evaluated from the standard chromatograms obtained by calculating the % RSD of the retention time, tailing factor, theoretical plates and peak areas from the replicate injections and are found to be within the acceptable range.

Linearity:

Aliquots of working standard Ropinirole hydrochloride solution were taken in different 10mL volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Ropinirole hydrochloride are in the range of 1-100µg/mL. Each of these drug solutions (20µL) was injected three times into the column, the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 248nm and a calibration graph was obtained by plotting peak area versus concentration of Ropinirole hydrochloride. Slope obtained was 40236 and Y-intercept was -10950. Correlation coefficient was found to be 0.9991. Linearity plot is shown in figure 2 and results are given in table 2.

Accuracy:

This parameter is performed to determine the closeness of test results obtained to that of the true value and expressed as percent recovery. The accuracy of the method was performed by calculating the recovery experiments at three levels (50%, 100% and 150%) following standard addition method. The test solution was injected three times for each spike level and the percentage recovery of Ropinirole was calculated. The results are summarized in table 3.

Precision:

Precision of an analytical method is the degree of agreement among individual test results. It was studied by analysis of multiple sampling of a homogeneous sample. This method was established by analyzing three different sample concentrations (10, 20 and 30µg/mL) of Ropinirole hydrochloride in replicates. Each prepared solution was analyzed thrice in order to record any intra-day and inter-day variation in the result. The results obtained for intra-day and inter-day variation are shown in table 3.

Robustness:

The robustness of an analytical procedure is a measure of its ability to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness study was performed by injecting replicates of standard and sample solutions into the HPLC system under altered chromatographic conditions such as flow rate (± 0.1mL/min), wavelength (±2nm), organic content in the mobile phase (±5%) and change in diluent ratio (± 5). The % RSD for the above conditions are calculated and shown in table 4.

Limit of Detection (LOD):

LOD is the lowest level of concentration of analyte in the sample that can be detected, though not necessarily quantitated. It was calculated using the regression equations obtained in the calibration curves and found to be 0.0007µg/mL when calculated using the formula, LOD = 3.3σ/S (σ = standard deviation of the response and S = slope of the calibration curve).

Limit of Quantification (LOQ):

Limit of Quantification is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied. It was found to be 0.0021µg/mL. LOQ was calculated using the formula, LOQ = 10σ/S (σ = standard deviation of the response and S = slope of the calibration curve).

Assay:

Within the linearity range sample solutions as discussed above were prepared from tablets and each time a 20µL of the solution was injected into the injector of the liquid chromatograph in replicates. The amount of drug obtained and assay (%w/w) were calculated.

Specificity:

The specificity of the method in the presence of excipients was established by comparing the chromatograms of blank, standard and sample solutions of the drug. There were no interferences at the retention time of the drug indicating that the method was highly specific to Ropinirole hydrochloride. A standard chromatogram is given in figure 3. The specificity can also be established in the presence of degradants created under a variety of forced degradations.

Forced Degradation Studies:

The degradation studies of the drug were carried out as per as the ICH guidelines, Q1A (R2)¹⁶. In order to determine the stability of Ropinirole hydrochloride, the drug was exposed to acid, alkali, hydrogen peroxide, heat and light. The results obtained are given in table 5.

a. Acid Degradation studies:

To 1mL of working standard solution of Ropinirole hydrochloride, 1mL of 0.5 N hydrochloric acid was added, refluxed for 3 hours at 60 – 65^ºC, the solution was cooled and neutralized with 0.5 N sodium hydroxide and diluted to obtain a 10µg/mL solution. This solution was injected into the system in replicates (n=3) and the chromatograms were recorded (Fig. 3a).

b. Alkali Degradation studies:

To 1mL of working standard solution of Ropinirole hydrochloride, 1mL of 0.5N sodium hydroxide was added and refluxed for 3 hours at 60-65^ºC, the solution was cooled and neutralized with 0.5 N hydrochloric acid and diluted to obtain a 10µg/mL solution. This solution was injected into the system in replicates (n=3) and the chromatograms (Fig. 3b) were recorded to assess the stability of the sample. Small peaks were observed without interfering with the drug peak.

c. Oxidation:

To 1 mL of working standard solution of Ropinirole hydrochloride, 1 mL of 30% v/v hydrogen peroxide was added and the solution was heated at 65 ^ºC for 3 hours in a hot air oven taking necessary precautions. For HPLC study, the resultant solution was cooled and diluted with mobile phase to obtain a 10 µg/mL solution and 20 µL was injected into the system in replicates. Small extra peak observed at retention time 2.74 minutes without affecting the shape of the drug peak. The chromatogram is shown in Fig. 3c.

d. Thermal Degradation Studies:

1 mL of working standard drug solution was placed in an oven at 60-65^ºC for three hours to study dry heat degradation. The resultant solution was diluted to 10 µg/mL solution and 20µL was injected (n=3) into the system and the chromatograms were recorded. No extra peaks were observed in the chromatograms (Fig. 3d) obtained indicating the stability of the drug at the mentioned temperature.

e. Photo Stability Studies:

The photochemical stability¹⁷ of the drug was also studied by exposing 1 mL of working standard solution of the drug to UV light at shorter wavelength (254nm) for 4 hours in a UV chamber. This solution was diluted with the diluent, each time a 20µL was injected into the system in replicates and the chromatograms (Fig. 3e) were recorded. No extra peaks were observed.

RESULTS AND DISCUSSION:

The current study was designed to develop a simple, precise, and rapid analytical RP-HPLC method, which can be used for the analysis of estimation of Ropinirole hydrochloride in bulk and pharmaceutical dosage forms. Initially the method optimization was performed on a trial and error method and the chromatographic conditions were selected in order to provide good performance of the method. To optimize mobile phase, various combinations of solvents such as water: methanol, 0.1% formic acid: methanol, boric buffer pH 9.1: methanol, boric buffer pH 9.1: acetonitrile, ammonium acetate buffer: methanol and 0.1% orthophosphoric acid: methanol were tried for Ropinirole hydrochloride taking some criteria from the literature and also the pKa values of the drug. Detection was carried out at several wavelengths in order to obtain enough sensitivity and at last the 248 nm, at which the drug showed good absorbance was selected as a detection wavelength. The flow rate was optimized at 1.2 mL/min, which is critical as it affects the peak symmetry parameters. The retention time for Ropinirole hydrochloride was found to be 2.91 ± 0.26 min. In the preparation of sample solution, the sonication time was also optimized after trying a series of sample preparations at varied sonication times and finally a time of 60 minutes was found effective in completely extracting the drug into the solvents. The method obeyed Beer’s law was in the range of 2-100µg/mL with the limit of detection 0.0007µg/mL and limit of quantification 0.0021µg/mL respectively. The proposed method was validated in accordance with the ICH guidelines and all the results were within the limits. The % RSD obtained in intraday and interday precision along with the recovery obtained in accuracy study indicates the validity of the method. Inspite of small deliberate variations created in the optimized method conditions, there was repeatability of the data without affecting the system suitability parameters indicating the robustness of the method. The degradation conditions were also started with the mild conditions (i.e 0.1 N hydrochloric acid and 0.1 N sodium hydroxide with just 30 min. heating) and since no degradations were recorded these conditions were more aggravated to 0.5 N and the heating time extended to 3 hours. However, the drug was found to be mostly stable (degradation less than 1.0%) in all the exposed conditions except in alkaline conditions which reported a minor degradation of 1.38%. There was no interference between the peaks obtained for the chromatogram of forced degradation preparations and the degradation peaks were well separated. The method was also applied to determine the purity of Ropinirole hydrochloride in marketed dosage forms. The assay was obtained as 99.32±0.83 %w/w without any interference from the excipients. All the prepared drug solutions were found to be stable over a period of 24 hours as observed from the %RSD of the assay obtained at various time intervals. Though some HPLC methods were reported in the literature (Table 6) the present method has several advantages like being more economical in terms of solvent usage and yet more sensitive as observed from the LOD and LOQ values while satisfying the precision, accuracy and robustness parameters.

Table 1: Optimized Chromatographic Conditions

Parameter	Optimized chromatographic conditions
Mobile phase	0.1% Orthophosphoric acid (pH 2.61): methanol (50:50 v/v)
Stationary phase	C8 Phenomenex column (250 × 4.6 mm × 5 µm)
Flow rate	1.2 mL/min.
Detection wavelength	248 nm
Column temperature	25 ± 2 ºC
Injection volume	20 µL
Detector	Photo diode array detector
Elution	Isocratic mode
Total run time	10 minutes
Retention time	2.91 ± 0.26 min.

Fig. 2: Calibration Curve

Table 2: Linearity Data

Conc. (µg/mL)	^*Peak area ± SD, % RSD
1	46854 ± 0.0321, 0.03
2	84109 ± 0.0641, 0.06
5	207382 ± 0.1538, 0.15
10	406868 ± 0.4569,0.45
20	703289 ± 0.6004, 0.60
30	1191238 ± 0.9243, 0.92
40	1545962 ± 0.7332, 0.74
50	2050891 ± 0.9653, 0.96
80	3254939 ± 1.5327, 1.53
100	3987620 ± 1.6992, 1.70

^*Mean of three replicates

Table 4: Robustness Data

Parameter	Condition	*Peak area	Tailing Factor	Theoretical Plates	^*Assay (% w/w) ± SD, % RSD
Flow rate (± 0.1 ml/min)	1.1	450354	1.908	3388.594	99.74 ± 0.4886, 0.48
	1.2	385204	1.962	3563.556
	1.3	390209	1.877	3168.890
Mobile phase composition (± 5% v/v)	55:45	389242	1.858	3289.654	99.87 ± 0.4754, 0.47
	50:50	381016	1.896	3692.855
	45:55	413970	1.548	2151.753
Diluent Ratio (± 5% v/v)	45:55	363143	1.893	5301.275	99.66 ± 0.7659, 0.76
	50:50	382456	1.949	3695.231
	55:45	425764	1.770	2894.145
Detection wavelength (± 2 nm)	246	385544	1.832	3481.632	99.39 ± 0.6300, 0.63
	248	391147	1.899	3233.951
	250	394652	1.796	3562.281

^*Mean of three replicates

Table 5: Data for Forced Degradation Study

Mode of Degradation	*Standard Average Area	*Sample Average Area	Assay (% w/w)*	Degradation (%)	Tailing Factor	Theoretical Plates
Control	423699	424187	99.90	-	1.739	3152.98
Acid Stress		422904	99.72	0.18	1.647	3980.986
Base Stress		417592	98.52	1.38	1.748	3378.952
Oxidation Stress		421762	99.49	0.41	1.686	3694.522
Thermal Stress		421478	99.4	0.5	1.662	3533.572
Photolytic Stress		422905	99.76	0.14	1.679	3286.747

* Mean of three replicates

Table 6: Comparative Table for the Present Method with the Existing LC Methods

Stationary Phase	Mobile Phase, Flow Rate	Linearity range (µg/mL)	LOD (µg/mL)	LOQ (µg/mL)	Rt (min.)	Reference
C18 ODS (250 × 4.6 mm, 5µ)	Buffer pH 6.0: Acetonitrile (50:50 v/v), 0.5 mL/min.	5-50	0.045	0.151	4.867	Sreekanth et al, 2009
Thermo BDS C18 (250 × 4.6 mm, 5µ)	0.02M KH₂PO₄: Acetonitrile (72:28 v/v), 1.1mL/min.	0.5-50	0.0011	0.0036	2.66	Lata et al, 2011
C18 (BDS) column ((250 × 4.6 mm, 5µ	0.05 M glacial acetic acid (pH 3): acetonitrile (50:50v/v),1mL/min.	4-12	0.045	0.139	4.03	Alivelu et al, 2014
C18 Hypersil BDS (250 × 4.6 mm, 5µ)	0.05 M glacial acetic acid: Acetonitrile (50:50 v/v); 1mL/min.	2-20	-	-	3.065	Abhishek et al, 2015
Gemini NX C18 (15 cm × 4.6 mm, 5 µ)	Methanol: Acetonitrile (70: 30 V/V); 0.7 mL/min.	2.5-160	0.045	0.15	2.718	Sadashivaiah et al, 2019
C8 Phenomenex (250 × 4.6 mm 5 µm)	0.1% Orthophosphoric acid (pH 2.61): methanol (50:50 v/v); 1.2 mL/min.	1-100	0.0007	0.0021	2.91	Present work

CONCLUSION:

A simple, sensitive and economic stability indicating RP-HPLC method was developed and validated as per the ICH guidelines for the analysis of Ropinirole hydrochloride in bulk and pharmaceutical dosage forms. The method was found to be linear, precise, accurate and robust. The degradation studies reveal the stability of the drug under the exposed conditions. Assay results from the study shows that this method can be used for the estimation of Ropinirole hydrochloride in tablets without any interference. Hence the proposed method can be conveniently used for the estimation of Ropinirole hydrochloride in bulk and pharmaceutical dosage forms.

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM (Deemed to be University), Visakhapatnam for providing the research facilities and Chemeca Drugs Private Limited, Visakhapatnam for supplying gift samples of Ropinirole hydrochloride.

CONFLICT OF INTEREST:

There is no conflict of interest.

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Received on 29.06.2020 Modified on 15.04.2021

Research J. Pharm.and Tech 2022; 15(4):1761-1767.

DOI: 10.52711/0974-360X.2022.00295

Conc. (µg/mL)	Intraday Precision		Interday Precision
Conc. (µg/mL)	^*Assay (%w/w) ± SD, % RSD
10	98.49 ± 0.3002, 0.30		98.91 ± 0.4806, 0.48
20	99.81 ± 0.1006, 0.10		99.59 ± 0.2810, 0.28
30	98.96 ± 0.5973, 0.60		99.27 ± 0.2270,0.60
Accuracy
Level (%)	Tablet (µg/mL)	Pure (µg/mL)	^*Recovery (%) ± SD, %RSD
50	10	5	101.37 ± 0.1662, 0.16
100	10	10	101.7 ± 0.5408, 0.53
150	10	15	101.64 ± 0.1058, 0.10