Assay of Ropinirole hydrochloride in Pharmaceutical Preparations by Visible Spectrophotometry

K. Raghubabu¹, V. Jagannadharao², B. Kalyana Ramu³

¹Department of Engineering Chemistry, AU College of Engineering (A), Andhra University, Visakhapatnam -530003 Andhra Pradesh (India)

²Department of Chemistry, Anil Neerukonda Institute of Technology and Sciences, Sangivalasa (AP) India.

³Department of Chemistry, Maharajah’s College (Aided and Autonomous), Vizianagaram-535002 (AP) India.

*Corresponding Author E-mail: drraghualways@yahoo.co.in

ABSTRACT:

Two simple and sensitive direct visible spectrophotometric methods (M₁ and M₂) have been developed for the determination of Ropinirole hydrochloride in bulk and solid dosage forms. These methods are based on the reaction of drug with aromatic aldehydes such as Vanillin or Para dimethyl amino Benzaldehyde (PDAB) in the presence of sulphuric acid in non aqueous medium and formed colored condensation products with an absorption maximum of 560nm for method M₁ and 660nm for method M₂. The Beer’s law obeyed in the concentration range of 5-20μg/ml for method M₁ and 25-150μg/ml for method M₂. The proposed methods are validated with respect to accuracy, precision, linearity and limit of detection. The suggested procedures are successfully applied to the determination of the drug in pharmaceutical preparation, with high percentage of recovery, good accuracy and precision. The results of analysis have been validated statistically by repeatability and recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of ropinirole hydrochloride in tablet dosage form without the interference of excipients.

KEYWORDS: Analysis, Beer’s Law, Condensation reactions, non-ergoline dopamine antagonist, Tablets.

INTRODUCTION:

Ropinirole hydrochloride (RPR) is an orally administered specific D₂and D₃ receptor non-ergoline dopamine antagonist. Chemically it is hydrochloride salt of 4-[2-(dipropyl amino) ethyl]-1,3-dihydro-2H-indol-2-one (Fig.1). It is used in the treatment of early and advanced Parkinson’s disease caused by deficit of dopamine. It has high relative in vitro specificity and acts by binding with higher affinity to D₃ than to D₂ or D₄ receptor subtypes. The mechanism of ropinirole induced postural hypertension is presumed to be due to a D₂-mediated blunting of the noradrenergic response to standing and subsequent decrease in peripheral vascular resistance. The drug is listed in Merck Index [1], Martindale the Complete drug reference [2].

Fig.1: Chemical structure of ropinirole hydrochloride

Several analytical techniques like HPLC [3-6], UPLC [7], TLC [8], Chemo metric [9], LC-MS [10], UV [11-13], capillary LC [14] visible spectrophotometric [15] and spectrophotometric and spectro fluorometric method [16] have been reported for its determination in plasma and tablet dosage forms. For routine analysis, simple, rapid and cost effective visible spectrophotometric methods are required and preferred. So the authors have made some attempts in developing visible spectrophotometric methods and succeeded in developing two methods (A and B) using aromatic aldehydes [17-18] such as vanillin (method M₁) or PDAB (method M₂) in the presence of sulphuric acid in non aqueous medium and colored condensation products are formed and stable for 30 minutes. These methods can be extended for the routine assay of RPR formulations.

MATERIALS AND METHODS:

Apparatus and chemicals

A Shimadzu UV-Visible spectrophotometer 1601 with1cm matched quartz cells was used for all spectral measurements. All the chemicals used were of analytical grade. Tablets were purchased from local market. Sulphuric acid (14M), Vanillin (BDH, 0.4%, w/v 2.63x 10^-2M); PDAB (E. Merck, 0.1% w/v 6.31x 10^-3M) in methanol was prepared.

Preparation of standard drug stock solution

About 100mg of RPR was dissolved in 100ml of methanol to get 1mg/ml stock solution. It was further diluted with the same solvent to get working standard solution (50μg/ml) for method M₁ and (100 μ g/ml) for method M₂. The prepared stock solution was stored at 4⁰c protected from light. From this stock solution, a series of standards were freshly prepared during the analysis day.

Preparation of Sample solution

About 20 tablets were pulverized and the powder equivalent to 100mg of RPR was weighed, dispersed in 25ml of IPA, sonicated for 30 minutes and filtered through Whatman filter paper No.41.The filtrate was evaporated to dryness and the residue was dissolved in 100 ml of methanol (1mg/ml). It was used as stock sample solution and was further diluted with the same solvent to get working standard solutions.

Table 1: Optical characteristics, precision and accuracy of proposed methods

Parameters	Method A	Method B
λ max (nm)	560	660
Beer’s law limit (µg/ml)	5- 20	25-150
Sandell’s sensitivity (µg/cm^2/0.001 abs. unit)	0.0054	0.0035
Molar absorptivity (Litre/mole/cm)	4.841x10^4	7.41x10^4
Regression equation (Y) ^*= a +b c
Intercept (a)	0.008	0.021
Slope(b)	0.018	0.026
%RSD	2.245	2.29
% Range of errors(95% Confidence limits) 0.05 significance level 0.01 significance level	2.235	2.406
	3.879	3.96

*Y= a + b c; Where Y= absorbance, c= concentration of RPR in µg/ml.

Assay:

Aliquots of standard drug solution in methanol (0.5 - 2.0ml, 100μg/ml for method M_1, 0.5-3.0 ml, 500μg/ml for method M₂) were placed in a series of 10ml calibrated tubes and volume of each test tube adjusted to 3.0ml with methanol. To each of these test tubes 1.0 ml of Vanillin (2.63x 10^-2M) or PDAB(6.31x 10^-3M) and 1.0 ml of concentrated sulphuric acid (14M) were added, while cooling under a tap with constant shaking and kept in water bath at 60ºc for 10min. cooled and diluted to the mark with methanol. The absorbance was measured at 560nm or 660nm for methods M₁andM₂ respectively (Fig.2and3 showing absorption spectra) against the reagent blank within 10 minutes. The amount of drug in a sample was computed from Beer’s law plot (Fig.4 and5).

Fig.2: Absorption spectra of RPR-VN

Fig.3: Absorption spectra of RPR-PDAB

Fig.4: Beer’s Law plot of RPR-VN

Fig.5: Beer’s Law plot of RPR-RPR-PDAB

RESULTS AND DISCUSSION:

Optimum operating conditions used in the procedure were established by adopting variation of one variable at a time (OVAT) method. The effect of various parameters such as time, volume and strength of reagents and acid solution and solvent for final dilution of the colored species were studied. The optical characteristics such as Beer’s law limits, Sandell’s sensitivity, molar extinction coefficient, percent relative standard deviation (calculated from the six measurements containing 3/4th of the amount of the upper Beer’s law limits) were calculated for all the methods and the results are summarized in Table-1. Regression characteristics like standard deviation of slope (S_b), standard deviation of intercept (S_a), standard error of estimation (S_e),% range of error (0.05 and 0.01 confidence limits) were calculated for both the methods and are shown in Table-1.

Commercial formulations containing RPR were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre-analyzed formulations at three different concentration levels. These results are summarized in Table-2.

The ingredients usually present in formulations of RPR did not interfere with the proposed analytical methods. Among the four aromatic aldehydes (vanillin, PDAC, PDAB and anisaldehydes) tried, all of them responded. But, Vanillin and PDAB were preferred as they were found to be better sensitivity in the assay of RPR. These methods can be extended for the routine assay of RPR formulations.

Chemistry of colored species:

In the present investigation, the presence of imino group in indole moiety (secondary amine) permits the development of visible spectrophotometric methods for its determination through the condensation reaction with aromatic aldehydes. The formation of colored species with these reagents may be assigned through above analogy as shown in Figure 6.

CONCLUSIONS:

The proposed methods for RPR determination have many advantages over other analytical methods due to its rapidity, lower cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are inexpensive and available in any analytical laboratory. These methods can be extended for the routine assay of RPR formulations.

Table 2: Analysis of RPR in pharmaceutical formulations by proposed and reference methods.

Method	*Formula-tions	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
			**Amount found ± SD	t	F
A	Batch-1	2	1.990±0.0015	0.41	3.06	1.993 ± 0.002	99.54 ± 0.075
	Batch-2	2	1.99±0.004	1.41	1.74	1.99± 0.003	99.39 ± 0.21
B	Batch-1	2	1.992±0.0012	0.41	3.06	1.993 ± 0.002	99.60 ± 0.06
	Batch-2	2	1.989±0.004	0.11	1.58	1.99± 0.003	99.496 ± 0 .19

* Different batches from two different companies (Batch-1 Ropin tablets of East west, Batch 2: Ropitor tablets of Torrent)

**Average ± Standard deviation of six determinations, the t- and F-values refer to comparison of the proposed method with reference method (UV). Theoretical values at 95% confidence limits t =2.57 and F = 5.05.

# Recovery of 10mg added to the pre-analyzed sample (average of three determinations). Reference method (reported UV method) using double distilled water (λ _max=249nm).

Fig.6: Probable scheme for method M₁andM₂

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Received on 06.04.2012 Accepted on 11.05.2012

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Asian J. Pharm. Ana. 2(2): April-June 2012; Page 41-45